EPU

User Manual
PN 1025707
Revision 2.14 • 25-JAN-2022
Contents
1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
1.1 Target audience for this user manual. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.2 System and software compatibility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2 Getting Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.1 Prepare for an EPU session. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.2 Start the EPU software. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.3 Verify that the microscope is ready for high quality data acquisition. . . . . . . . . . . . 7
2.4 Log in on Thermo Scientific Athena. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3 User Interface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.1 User interface panels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.2 Messages side panel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
3.3 Status side panel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
3.4 Histogram side panel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11
3.5 Image Information side panel. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 .
3.6 Image and plot display area. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 .

4 Home Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

4.1 Set up a new EPU Session based on Advised Preferences. . . . . . . . . . . . . . . . . .17.
4.2 Set up a new EPU Session based on a Saved Preference. . . . . . . . . . . . . . . . . . .17

5 Preparation Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18

5.1 Acquisition and Optics Settings task. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18 .
5.2 Atlas Optics Alignment task. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
5.3 Calibrate Image Shifts task. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 .
5.4 Calibrate I0 task. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58
5.5 Activate Phase Plate task. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 .

6 Auto Functions Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64

6.1 Alignments tasks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64 .
6.2 Auto-Functions (TEM) tasks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 .
6.3 Calibration tasks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
.

7 Atlas Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
.
7.1 Setup Session task. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 .
7.2 Screening task. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83

8 EPU Tab. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
.
8.1 Session Creation task. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .95 .
8.2 Session Setup task. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
.
8.3 Square Selection task. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107
..
8.4 Area Selection task for Lacey Carbon specimens. . . . . . . . . . . . . . . . . . . . . . . . .112 .
8.5 Hole Selection task for Quantifoil specimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 ..
8.6 Template Definition task for Quantifoil specimens. . . . . . . . . . . . . . . . . . . . . . . . .141 .
 8.7 Template Execution task for Quantifoil specimens. . . . . . . . . . . . . . . . . . . . . . . . 151 ..
8.8 Automated Acquisition task. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152
.

9 The EPU Multigrid Option. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .161

.
9.1 Create a queue with EPU Multigrid Sessions. . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
..
9.2 Edit an EPU Multigrid Session in the Queue. . . . . . . . . . . . . . . . . . . . . . . . . . . . .163
.
9.3 Remove an EPU Multigrid Session from the Queue. . . . . . . . . . . . . . . . . . . . . . .164 ..
9.4 Change the order of the EPU Multigrid Sessions in the Queue. . . . . . . . . . . . . . 164 ..
9.5 The Automated Acquisition task in the EPU Multigrid workflow. . . . . . . . . . . . . . 164 ..

10 Inspect the Acquired Images. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .168

..
10.1 View the images in Windows Explorer and Photo Viewer. . . . . . . . . . . . . . . . . .168 .
10.2 View the JPEG images. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
..
10.3 View the MRC, EMI, DMX and other microscope images. . . . . . . . . . . . . . . . . .168 .
10.4 View and post-process MRC images with Thermo Scientific Velox software. . . 169 ..

11 Detailed Preconditions for Successful EPU Usage. . . . . . . . . . . . . . . . . .170

..
11.1 Preconditions for the specimen and specimen holder. . . . . . . . . . . . . . . . . . . . .170 .
11.2 Preconditions for the microscope. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 ..
11.3 Use the Sherpa APM function to maintain the microscope alignments. . . . . . . .172 .
11.4 Preconditions for the microscope alignments. . . . . . . . . . . . . . . . . . . . . . . . . . . 172 ..
11.5 Aperture Alignments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 ..
11.6 Direct Alignments and Astigmatism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .182 .
11.7 Calibrations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
..

12 Troubleshooting: Symptoms and Solutions. . . . . . . . . . . . . . . . . . . . . . . .184

.
12.1 Troubleshooting: Atlas. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .184
.
12.2 Troubleshooting: GridSquare. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .186 .
12.3 Troubleshooting: Template Definition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 ..
12.4 Troubleshooting: Automated Acquisition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .194 .

A The MRC2014 Image Format. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .197

..
A.1 The Main Header and Extended Headers in an MRC file. . . . . . . . . . . . . . . . . . .197 .
A.2 The Extended Header specification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
..
A.3 Pixel sequence in the MRC2014 format. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205..

13 Copyright, Limited Rights and Revision History. . . . . . . . . . . . . . . . . . . .207

..

Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .209
.
Chapter | Introduction

1 Introduction
Cryo-electron microscopy (Cryo-EM) for high resolution three-dimensional single particle
reconstruction (3D-SPR) has reached near atomic resolution for structures of biological complexes of
all kinds.

3D SPA reconstruction of Apoferritin at a resolution of 2.14 Å.

Data acquired on a Krios G3i with Falcon 3EC camera in counting mode.
Reference information on EMDataBank (EMD4213, http://emsearch.rutgers.edu/atlas/
4213_downloads.html)

For the high resolution reconstruction of a particle, large amounts of well-aligned images must be
summed and averaged. Biological material is highly sensitive to electron radiation, so an extremely
low dose must be applied to prevent damage to the specimen. This can result in a low signal-to-noise
ratio in the recorded images. It would be a demanding and time consuming task for a microscopist to
acquire the images that the reconstruction software needs to complete its task with a satisfactory
result. Fortunately, automation software is available to drastically reduce the time and effort, and at
the same time improve the reliability of harvesting such large quantities of high quality data.
EPU is a Thermo Fisher Scientific software product for automated high quality data acquisition in a
Single Particle Analysis (SPA) workflow. SPA is an approach to 3D image creation, during which a
large number of vitrified, low-contrast complexes are imaged under low electron dose conditions.
After conformational classification and particle averaging, this results in a high resolution 3D
representation.
The acronym EPU is from the Latin "E Pluribus Unum" — Out of Many, One. This reflects the actual
pathway: a single 3D particle structure is extracted from numerous 2D images with multiple particles
per image. EPU automates the most time-consuming step in the SPA workflow: the acquisition of
useful images. The 3D reconstruction is done with various open source software applications.
EPU facilitates the following steps in the data acquisition procedure:
● Screening of multiple specimens to select the specimens and specimen areas with the highest
potential for high quality data acquisition.
● Automated or customized selection of the optimal acquisition areas on the specimen.
● High throughput, high resolution data collection.

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Chapter | Introduction

1.1 Target audience for this user manual

This manual is aimed at skilled users of Thermo Scientific Transmission Electron Microscopes.
In particular, the reader should be able to successfully perform the following:
● Evaluate the quality of acquired data.
● Identify the cause of accuracy and quality issues with the acquired data.
● Perform the necessary corrective actions to resolve the identified issues, provided that these
actions are available to the user. For higher level corrective actions the assistance of a Thermo
Fisher Scientific engineer may be required.

For online training materials to help you improve your Cryo-EM skills, visit the EM-learning.com
website .

For questions, remarks and support regarding the EPU software, please contact the EPU support
desk at [email protected] .

1.2 System and software compatibility

The Thermo Scientific EPU software is available for Thermo Scientific TEM systems and FEI TEM
systems that run up-to-date system software. For detailed system and software version compatibility
information, see the EPU Release Notes.
Note Not all features and functions in this manual are available on all systems and all supported microscope
software versions.

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Chapter | Getting Started

2 Getting Started

2.1 Prepare for an EPU session

Before starting an EPU session, make sure the following prerequisites are met and preparations are
completed.
1. Verify that an Alignments File and a FEG Register are loaded that match with the High Tension
and Extractor settings of the system.
2. Verify that all cameras are cooled and at a stable temperature.

For an extensive preconditions check, see Detailed Preconditions for Successful EPU Usage on
page 170 .
Detailed instructions for the individual alignments and calibrations are available in the online help of
the TEM User Interface.

2.2 Start the EPU software

1. Start EPU
Shortcuts to the EPU software can be found on the desktop and in the Windows Start menu.
2. A splash screen appears while EPU runs the startup checks.
If one or more checks fail, the pop-up displays the related messages.

● Solve the reported issue(s).

● Select Retry

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Chapter | Getting Started

2.3 Verify that the microscope is ready for high quality data acquisition
In the lower-left corner of the EPU user interface, the Microscope State, also called Traffic Light,
shows if the system is ready for high quality data acquisition. If a subsystem reports an error or is
otherwise not in an optimal status, then the status of that subsystem changes to red and the Recover
function becomes available to return the system to an all-green status.
When a subsystem is in a red state it is often still possible to use EPU, but the image quality may not
be optimal.

1. Verify that the Microscope State is green.

This indicates that the system is ready for high quality data acquisition.

2. If the Microscope State is not green, then:

a. (Optional) To display the reason(s) why a subsystem is not ready,
expand the corresponding subsystem section.
b. Select Recover
EPU requests the microscope software to recover the failing subsystems. The Recover
procedure is fully automated. During recovery, the EPU user interface is temporarily
disabled.

On Tundra systems, the automated Recover procedure includes the automated Daily Alignments
procedure.
● The Daily Alignments procedure is executed only when the preceding execution is expired. This
procedure can take up to 30 minutes. The Daily Alignments expire after 24 hours.
● For the best result, make sure that there is no specimen in the stage, or that the beam can pass
through a hole in the specimen.

2.4 Log in on Thermo Scientific Athena

The Thermo Scientific Athena software is part of the Data Management Platform (DMP)
functionalities. After logging in on Athena in EPU, the EPU software on the Microscope PC has a
connection to the following functionalities on the DMP Server:
● Athena for the automated upload of acquired data into a selected dataset.
● EPU Quality Monitor (EQM) for the real-time evaluation of the quality of the acquired data.
EQM requires a separate license.
● Smart EPU for various real-time optimizations. These optimizations are based on the data that
has been acquired so far, during the ongoing Automated Acquisition run. Initially, Smart EPU is
only available on Tundra systems.

To connect to Athena, follow the instructions below:

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Chapter | Getting Started

1. In the lower-right corner of the EPU user interface, select Athena Login

The red dot indicates that there is no active connection to Athena yet.
The Thermo Scientific Athena login screen appears.

2. Enter the username and password for Athena.

If the login is successful, the Athena connection indicator turns green and the username is
displayed.

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Chapter | User Interface

3 User Interface

3.1 User interface panels

The EPU user interface guides the user through all actions that are needed to prepare and execute
an Automated Acquisition run.

● Tab Selection
The tabs are typically worked through from left to right. Each tab provides a set of tasks.
● Task Selection
Tasks are typically executed from top to bottom. The set of available tasks depends on the
selected tab.
● Task Execution
The content of the Task Execution pane depends on the active task. It can display an input
dialog, one or multiple acquired images, or progress information for an ongoing function.
● Ribbon Bar
The Ribbon Bar offers a set of controls that are necessary or helpful for completing the active
task.
● Side Panels
The Side Panels pane contains a set of collapsible panels. The set of available side panels
depends on the active task and/or the selected image in the Task Execution pane.
● Status Bar
The Status Bar displays the Athena login and status, and the Traffic Light. The Status Bar is only
visible when Athena and/or the Traffic Light functionality are available.

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Chapter | User Interface

3.2 Messages side panel

The Messages panel shows a list of Error and Notification messages in chronological order.

By default, all Errors and Notifications are displayed.

Select Notifications to hide the Notification messages.

The same applies to the Error messages.

To clear messages that are no longer relevant:

● Select the cross at the right-side of each individual message.
● Right-click on any message and select Delete All Messages

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Chapter | User Interface

3.3 Status side panel

The Status panel displays various types of messages in chronological order, such as:
● Errors and Notifications.
● Progress messages for ongoing automated procedures.
● Intermediate and final results of automated procedures.
● Instructions and recommendations to the user.

3.4 Histogram side panel

If two or more images are present in the Task Execution panel, then the Histogram applies only to the
selected image. The selected image is recognizable by the highlighted image title and frame.

3.4.1 Histogram side panel

The Histogram side panel shows a histogram of image-pixel intensities for the selected image in the
Image Display.

The Histogram side panel offers the following functionalities:

Auto Filter:
● Ticked:
EPU automatically calculates the optimal contrast and brightness settings and applies these
values when a new image is acquired or selected.
● Cleared:
Manual adjustments of the contrast, brightness or gamma values are also applied to the next
acquisition.
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Chapter | User Interface

When ticked, it is possible to adjust contrast, brightness and gamma for the current image. To reset
contrast, brightness and gamma to their default values, clear Auto Filter and tick it again.
Black Level:
Drag the red line at the left-side of the main histogram to adjust the black level. Pixels with an
intensity below the black level value are displayed with zero intensity (black).
White Level:
Drag the red line at the right-side of the main histogram to adjust the white level. Pixels with an
intensity above the white level value are displayed with maximum intensity (white).
Gamma:
Drag the diagonal black line up or down to adjust the Gamma curve.
If the Black Level and/or White Level are adjusted, then the Gamma curve is scaled proportionally in
the range between the Black Level and White Level values.
Zoom:
In the lower histogram, drag a range to zoom in on a section of the spectrum.
Click outside the zoom range in the lower histogram to reset the zoom level.

3.5 Image Information side panel

The Image Information panel displays a small basic set of the image meta data.

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Chapter | User Interface

3.6 Image and plot display area

The availability of functionalities for the Image Display depends on the active task, the applied
Acquisition and Optics Preset and the selected image.
Zoom slider

Drag the slider with the mouse to change to the zoom level.

Color Enhancement

Applies a color mapping to the intensity values in the image. This makes it easier to recognize
intensity gradients and areas with similar intensity.

Show/hide the inset image (FFT)

By default, the Inset window displays the FFT of the image.

Filter the FFT image

The FFT filter optimizes the contrast and brightness of the FFT, so that for example Thon Rings are
shown clearer. The FFT filter does not change the acquired image data for which the FFT is
displayed.
Note This filter is not adjustable and has not been optimized for performance and low resource usage.
Activate the FFT filter only when necessary. Deactivate the FFT filter when there is no direct need to
display the FFT for an image.

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Chapter | User Interface

Swap the inset and main image

Swap the images in the Inset and the Main windows. If the Inset window is hidden, then the Main
window will toggle between the FFT and the normal camera image.

Zoom to fit

Adjust the zoom level, so the entire image fits in the image display frame.
Zoom to fit is also available in the right-click context menu of the image display.

Zoom to 100%

Adjusts the zoom level to 100%, so the image is displayed in actual size.
Zoom 1:1 is also available in the right-click context menu of the image display.

Show/hide the panning window

Show or hide the panning inset window.

3.6.1 Zoom in/out

1. Place the mouse cursor at the region of interest in the image display.
2. Scroll up or down with the mouse wheel.
The image will zoom in or out around the cursor location.

3.6.2 Navigate and pan in a zoomed image

To navigate and pan in an image, either drag the image with the mouse, or use the panning inset.
1. Select the panning inset

2. Drag the dark gray square across the panning inset

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Chapter | User Interface

3.6.3 The red crosshair

The red crosshair is commonly referred to as the stage position. Although the red crosshair moves
when the stage moves, it does not mark the stage position itself. The red crosshair marks the center
of the field of view if a new image were acquired.

3.6.4 Export an image to file

Images that are displayed in the Task Execution panel can be exported to file.
1. Right-click in the image to open the context menu.

The options in the context menu depend on the active task and image type.
2. Select either:
● Export image
Create a file of the image. The image is saved with the original resolution.
● Export image with overlay
Create a file of the image with scales, markers and other visual aids. The resolution of the
image file is the same as as displayed in EPU. This may be less detailed than the original
image.

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Chapter | Home Tab

4 Home Tab
Note The Home tab and all functionalities of the Home tab are only available on Tundra systems.

The main purpose of the Home tab is to quickly set up an EPU Session, based on pre-determined
settings and values that are stored as a Preference.
● The Advised Preferences contain generic, best-practice settings and values that are proposed by
EPU.
● A Saved Preference contains custom settings and values that have been defined and stored in
an earlier EPU Session.
The Home tab also provides fast access to the EPU User Manual (this document), the Athena portal,
and the OffloadData folder on the Storage Server.

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Chapter | Home Tab

4.1 Set up a new EPU Session based on Advised Preferences

If there are no (suitable) Saved Preferences, then follow the steps below to set up a new EPU
Session:

1. Select the Experiment Type:

● High Resolution: settings and values result in a small pixel size for accurate 3D
reconstructions.
● General Purpose: settings and values result in a medium pixel size.
● Screening: settings and values result in a larger field of view so that larger areas can be
assessed.
The Selected Preference panel displays the key characteristics of the selected Experiment Type.
2. Select the Grid Settings:
a. Select the Grid Type
b. Select the Hole Diameter
The exact hole diameter and spacing must be fine-tuned later in the EPU workflow, at the
EPU > Hole Selection task.
3. Select Start new session to create a new EPU Session.
EPU overwrites the settings and (calibrated) values of the current EPU Session with the settings
and values form the selected Experiment Type and grid.

The loaded settings and values are generic, best-practice for the selected selected Experiment Type
and grid. Continue with the EPU workflow to verify and adjust the settings and values where
necessary.

4.2 Set up a new EPU Session based on a Saved Preference

After the EPU workflow is completed, and it is verified that the preferences and settings result in the
desired data quality, then optimized preferences and settings can be stored as a Saved Preference.
This makes it much faster and easier to set up a new EPU Session for a similar experiment.
To set up a new EPU Session that is identical or similar to a preceding experiment:
1. Select the most suitable Saved Preference
The Selected Preference panel displays the key characteristics of the selected Saved
Preference.
2. Select Start new session to create a new EPU Session.
EPU overwrites the settings and (calibrated) values of the current EPU Session with the settings
and values from the selected Saved Preference.

Continue with the EPU workflow to verify and adjust the settings and values where necessary. It is
not possible to update the values in a stored Saved Preference. If adjustments are necessary in the
new experiment, and it is expected that there will be similar experiments in the future, then store the
adjusted values in a new Saved Preference.

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 Chapter | Preparation Tab

5 Preparation Tab
The Preparations tab provides a set of tasks and functionalities to set up the microscope and the
EPU application for successful automated acquisition.

5.1 Acquisition and Optics Settings task

For each step in the preparation and automated acquisition process, a Preset must be prepared that
fulfills a specific set of requirements. Each Preset consists of:
● Camera Settings.
● Advanced camera and/or exposure settings
(not for all camera types).
● Optics Settings.

5.1.1 Description of the Camera Settings

5.1.1.1 Camera Settings for all camera types

For the Thermo Scientific Falcon 3EC and Falcon 4(i) camera, the Exp. Time (s) is available in the
Exposure Settings.
Camera
Select the camera that is used for the selected Preset.

Binning
Select the sensor-to-image pixel grouping mode.
The options are:
● 1: each pixel in the acquired image corresponds to a single sensor pixel.
● 2: the signal of 4 sensor pixels (2x2) is integrated into a single image pixel.
● 4: the signal of 16 sensor pixels (4x4) is integrated into a single image pixel.
A higher Binning value:
● Does not affect the field of view.
● Decreases the image resolution.
● Increases the image acquisition speed for CCD cameras.
For CMOS cameras the image acquisition speed is practically independent of the Binning value.
CMOS cameras are:
● All FEI and Thermo Scientific Falcon and Ceta cameras.
● Gatan K2, K3 and OneView cameras.
● Increases the signal strength of the image pixels.
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Readout
Select the area section of the camera sensor that is used for image acquisition.
The options are:
● Full: on a 4096x4096 sensor, all the signal of all pixels is used.
● Half: on a 4096x4096 sensor, the signal of a 2048x2048 area around the center is used.
● Quarter: on a 4096x4096 sensor, the signal of a 1024x1024 area around the center is used.
A smaller Readout value:
● Decreases the field of view.
● Does not affect the image resolution.
● Does not affect the signal strength of the image pixels.

Exp. time (s)

For the Thermo Scientific Falcon 3EC and Falcon 4(i) camera, the Exp. Time (s) is available in the
Exposure Settings.
Specify the time during which the camera sensor is exposed to the electron beam.
A longer Exposure Time value:
● Does not affect the field of view.
● Does not affect the image resolution.
● Decreases the image acquisition speed.
Depending on the camera type and other settings, the frame rate is not necessarily affected.
● Increases the signal strength of the image pixels.

EPU validates the specified value. If necessary, EPU adjusts the specified value to the nearest valid
value and shows a message.

5.1.1.2 Exposure Settings for Thermo Scientific Falcon 3EC and Falcon 4(i) cameras

Mode
Select the integration mode.
When not in the LM magnifications range, the following options are available:
● Linear: the normal integrating mode.
For the Falcon 4(i) camera, the Linear mode is not available in the Data Acquisition preset.
● Counted: Electron Counting mode.
The Counted mode gives better performance of the detector, but requires low dose rates. The
Counted option delivers sub-pixel accuracy without increasing the image size. It describes the
detected electrons by a normalized pattern that is positioned with sub pixel accuracy.

Fractions

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Select whether or not to save Dose Fraction images.

Direct detectors read a lot of elemental frames that can be integrated into Dose Fractions. These
Dose Fractions can be saved as separate images, next to the integrated image. The total number of
frames depends on the exposure time and the internal frame rate of the camera.
The following options are available:
● No: Do not save Dose Fractions.
● Manual: Specify the number of Dose Fractions in Fractions (Nr).
● Auto: The Dose Rate is used to calculate the number of Dose Fractions. To ensure that the Dose
Fraction images can be aligned properly, every Dose Fraction has at least 1 e/px.
● Maximum: The maximum number of Dose Fractions is equal to the number of frames.
● EER: Electron Event Registration mode.
Only for the Thermo Scientific Falcon 4(i) camera.
In EER mode, the camera records the coordinates of the individual electrons that hit the Sensor
Package. The following data is stored on the Storage Server:
● An *.eer file with the coordinates and a Gain Reference image.
● An integrated image of the recorded specimen area.
In EER mode, the size of the acquired data is much smaller than in Counted mode. The camera
does not acquire Fractions, and the *.eer file is much smaller than an image file with same data
content.
To view and process *.eer files, specialist software is required.

Fraction settings are not applied when acquiring images for focusing and other preparatory actions.

Align
Select whether or not the camera frames are aligned before summing.
When Align option Yes is selected:
● The camera calculates the image shift between consecutive frames.
● The camera applies a matching correction to each frame before it is summed into an integrated
image or a Dose Fraction image.
The result of the Align function depends on the selected Fractions mode:
● Fractions is No: the frames are aligned before summing into an integrated image.
● Fractions is Manual or Auto: the frames are aligned before summing into a Dose Fraction image.
After that, the Dose Fractions are aligned before summing into the integrated image. An XML file
with the applied shifts is saved next to the Dose Fraction images.
● Fractions is Maximum: the Dose Fractions are aligned before summing into the integrated image,
and an XML file with the applied shifts is saved next to the Dose Fraction images.

Dose and Exp. Time

The values of the Dose and Exposure Time parameters are coupled via the measured Dose Rate.
When the Exposure Time value is changed, the measured Dose Rate is used to automatically
calculate the corresponding Dose value, and vice versa.
If the Dose Rate is known, then EPU automatically calculates Dose or Exposure Time. EPU uses the
leading parameter as the basis to calculate the value of the other parameter. In the image below,
Dose is the leading parameter.
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● The arrow points from Dose to Exposure Time.

● Exposure Time is grayed out.
To make Exposure Time leading, either:
● Select the arrow to reverse it.
● Select the Exp. Time input field.
● Select [+] or [–] to adjust the Exposure Time value.
Fractions (Nr)
● Manual: manually specify the number of Dose Fractions.
● Auto: EPU calculates the number of Dose Fractions.
● The average dose in each Dose Fraction must be at least 1 e/px. This requires that the the
Dose Rate is known.
● For Falcon 3EC cameras there are additional conditions.
See: The automatic Dose Fractions calculation for Falcon 3EC and Falcon 4(i) cameras on
page 22 .

Frames (Nr)
This is not a user setting, it is a calculated value. The number of frames depends on:
● The internal frame rate of the camera.
● The total Exposure time.

5.1.1.3 Exposure Settings for the Thermo Scientific Ceta-F camera

Fractions
● Enabled: acquire and store Dose Fractions.
● Disabled: acquire and store only integrated images.
Fractions can only be enabled for the Data Acquisition preset.

Frames (Nr)
This is not a user setting, it is a calculated value. The number of frames depends on:
● The internal frame rate of the camera.
● The total Exposure time.

Dose and Exp. Time

The values of the Dose and Exposure Time parameters are coupled via the measured Dose Rate.
When the Exposure Time value is changed, the measured Dose Rate is used to automatically
calculate the corresponding Dose value, and vice versa.

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If the Dose Rate is known, then EPU automatically calculates Dose or Exposure Time. EPU uses the
leading parameter as the basis to calculate the value of the other parameter. In the image below,
Dose is the leading parameter.

● The arrow points from Dose to Exposure Time.

● Exposure Time is grayed out.
To make Exposure Time leading, either:
● Select the arrow to reverse it.
● Select the Exp. Time input field.
● Select [+] or [–] to adjust the Exposure Time value.

5.1.1.4 Dose Rate for Thermo Scientific cameras

Measure
Select Measure to determine the Dose Rate that the camera sensor receives. The color bar
indicates if the measured dose rate is suitable for high quality data acquisition.
● Blue: the Dose Rate is too low. Images may not be usable for 3D reconstruction.
● Green: the Dose Rate is suitable for high quality data acquisition.
● Red: the Dose Rate is too high. The detector is over-exposed.
The measured Dose Rate is only valid for the current Optics Settings. When the Optics Settings are
changed, the Dose Rate must be measured again.

The acceptable Dose range in EPU can be different than the Dose range that is used in Velox. The
range in Velox is based on the technical range of the camera. The value in EPU is based on what a
typical life-science specimen can handle before it becomes severely damaged.

5.1.1.4.1 The automatic Dose Fractions calculation for Falcon 3EC and Falcon 4(i) cameras
The number of frames in each Dose Fraction image is determined as follows:
● Initially, the frames are distributed equally over the Dose Fractions.
● When Fractions is Auto, the average dose in each Dose Fraction must be at least 1 e/px.
This requires that the the Dose Rate is known.
● For the Falcon 3EC camera, when Align is Yes, the number of frames per Dose Fraction in
the initial distribution must be a multitude of 6.
For the Falcon 4(i) camera there is no additional condition when Align is Yes.
● Remaining frames are added to the last Dose Fraction.

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The rules above may lead to unexpected fractionation schemes. Run a test acquisition to check the
distribution. For the best results, the Dose Fractions should be as equidistant as possible.
If necessary, adjust the Dose or Exposure Time, or specify a different number of Dose Fractions to
reach a more balanced fractionation scheme.
In the example below, 216 frames are distributed in 20 Dose Fractions. This example does not meet
the optimal equidistant goal:
● Falcon 4(i), or Falcon 3EC with Align is No

● 216 / 20 = 10.8
Each Dose Fraction contains 10 frames.
● The remainder is 216 - (20 ´ 10) = 16 frames.
These are added to the last Dose Fraction, so that the total number of frames in the last
Dose Fraction is 26 frames.

● Falcon 3EC with Align is Yes

● 216 / 20 = 10.8
Each Dose Fraction contains 6 frames.
● The remainder is 216 - (20 ´ 6) = 96 frames.
These are added to the last Dose Fraction, so that the total number of frames in the last
Dose Fraction is 102 frames.

5.1.1.5 Advanced Camera Settings for Thermo Scientific Ceta cameras

Noise reduction
Select Yes to decrease the readout noise at low dose. Enabling Noise reduction decreases the
maximum frame rate by up to 50%.
Noise reduction is only available when Frames Summed is 1.

Frames Summed
Specify the number of frames that is summed during acquisition. A higher number of frames
increases the dynamic range in the acquired image.

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5.1.1.6 Advanced Camera Settings for Gatan K2 and K3 cameras

Mode
Select the integration mode.
When not in the LM magnifications range, the following options are available:
● Linear: the normal integrating mode.
The Linear mode is only used by the Gatan K2 camera.
The Gatan K3 camera always uses Counted mode or Counted/Super Resolution mode.
● Counted: use electron counting.
The Counted mode gives better performance of the detector, but requires low dose rates.
● Counted/Super Resolution:
This mode is only available in the Data Acquisition Preset, and when Fractions (Nr) is set to a
value higher than 1.
In Counted/Super Resolution mode, the position of an electron is assigned to a pixel to achieve
sub-pixel resolution. This information is saved only in the Dose Fraction images. Because of this
additional information, the Dose Fraction images will have double the width and height in pixels.

Number of frames
This is not a user setting, it is a calculated value. The number of frames depends on:
● The internal frame rate of the camera.
● The total Exposure time.

Fractions (Nr)
Specify the number of Dose Fraction images.
The specified value is only applied when acquiring images with the Data Acquisition Preset. When
acquiring a preview image, the Dose Fraction images are saved in the Preparation Preview
sub-folder of the camera root folder.

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5.1.2 Description of the Optics Settings

Optics Settings for microscopes with a C3 lens.
These are typically the High End systems with Titan software.

The beam diameter and magnification are used to calculate the illuminated area on the specimen.

Optics Settings for microscopes without a C3 lens.

These are typically the Mid Range systems with Talos software.

The beam diameter is displayed as an Intensity value. On a microscope with two condenser lenses it
is not possible to calculate the beam diameter or the illuminated area on the specimen.

Note Insert Slit and Slit Width are only available in EFTEM mode on a system with an energy filter.

Get
Imports the Optics Settings parameter values from the microscope.

Set
Applies the Optics Settings parameter values to the microscope.

Probe mode
The microscope software remembers the last used defocus value for MicroProbe and for NanoProbe
mode. When returning to a probe mode, the last used defocus value for that probe mode is
automatically restored. Because of this behavior, some of the Presets must use the same Probe
mode.

To prevent that the result of the Autofocus function is lost, the following Presets and Autofunctions
must use the same Probe Mode as the Data Acquisition Preset:
● Presets: Autofocus, Drift Measurement.
● Autofunctions: Eucentric height by beam tilt, Eucentric height by stage tilt, Autofocus

Magnification
The Magnification determines the field of view, and therefore also the dimensions of the imaged area
per pixel (or pixel size).

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Defocus
In most Presets it is useful to apply a small defocus for enhanced contrast. Too much defocus could
invalidate the alignments and calibrations.
EPU applies the specified defocus relative to the current focus, not relative to eucentric focus.
During the preparation of the Automated Acquisition run, the Defocus value in the Data Acquisition
Preset is used for all acquisitions with the Data Acquisition Preset. During the Automated Acquisition
run, EPU uses the Defocus values from the Defocus List.
Spot Size
The Spot Size determines the beam current. A higher Spot Size value corresponds to a lower beam
current, and therefore a lower Dose Rate.
Illuminated Area (Ill. Area)
Only available on microscopes with three condenser lenses.
If the beam diameter is larger than the camera field of view, then parts of the specimen that are not
imaged at that time are needlessly exposed to the electron beam. This may destroy valuable
specimen area, which is then lost for high quality data acquisition.
The ideal beam has the following properties:
● The beam is parallel.
● The beam diameter is just large enough to fully cover the camera field of view.
● The beam has no distortions or aberrations along the edges.
Intensity
Only on microscopes with two condenser lenses.
The Intensity value determines the spreading of the beam. On a system without a C3 lens, the beam
diameter can not be locked to a specific size. If the Probe, Magnification or Spotsize value changes,
then the Intensity value may require adjustment as well.
If the beam diameter is larger than the camera field of view, then parts of the specimen that are not
imaged at that time are needlessly exposed to the electron beam. This may destroy valuable
specimen area, which is then lost for high quality data acquisition.
The ideal beam has the following properties:
● The beam is parallel.
● The beam diameter is just large enough to fully cover the camera field of view.
● The beam has no distortions or aberrations along the edges.
Insert Slit
Only available in EFTEM mode on a system with an energy filter.
Select Yes to insert the slit.

Slit Width (eV)

Only available in EFTEM mode on a system with an energy filter.
Specify the electron energy bandwidth that can pass through the slit.

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5.1.2.1 Guidelines for the Optics Settings when using Phase Plates

A Volta Phase Plate is generated by exposing the Phase Plate to the beam. This means there are
limitations to the Optics Settings parameters of the Acquisition and Optics Presets when working with
a Phase Plate:
● The beam must be parallel, so that the unscattered beam focuses into a small spot on the Phase
Plate.
● The focus position on the phase plate has to stay within tight limits, which in turn puts tight limits
on the stability of the beam direction. For reference, the stability limits for imaging with a Phase
Plate are far stricter than those for coma free imaging.

In practice, the optics system of a microscope is not ideal. Changing the beam diameter may induce
a slight tilting of the beam, which may cause the beam to partially leave the Phase Plate. To prevent
this effect:
● Always use a parallel beam to illuminate the Phase Plate.
● For a selection of Presets, the Optics Settings must be partially or fully identical.
If a Phase Plate related restriction is applicable to a Preset, then this is described in the chapters
for that Preset.
It is possible to use a convergent beam when working on the microscope, as long as it does not hurt
the existing Phase Plate and does not activate a new one.

5.1.3 The recommended order to define the Acquisition and Optics Presets
The recommended order to define the Presets is not the same as their order in the Presets list:
1. Data Acquisition
2. Hole/EucentricHeight
3. Autofocus
4. Drift Measurement
5. Thon Ring
6. Zero Loss (only for EFTEM mode)
7. GridSquare
8. Atlas

In an Automated Acquisition run, the data acquisition step is the value-creating action. To get the
best quality images for 3D reconstruction, the Data Acquisition Preset must be optimized without
sacrifices to the other Presets.
Although the optics system is highly reproducible, it is always better to avoid changes that are not
strictly necessary. To achieve maximum stability, use the Data Acquisition Preset as the basis for all
other Presets that are used during the Automated Acquisition run.

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5.1.4 Define the Data Acquisition Preset

The Data Acquisition Preset is used to acquire the images that will be used for 3D reconstruction of
the particles in the specimen. The parameter values of the Data Acquisition Preset depend only on
the requirements for successful 3D reconstruction.

Parameters Typical Settings and Recommendations

Optics ● TEM Imaging Mode: Nanoprobe.

Settings Nanoprobe is well suited for a narrow parallel beam at high magnifications.
● Parallel beam.
● Intensity Zoom is Off.
● Magnification must match the required resolution.
● Illuminated Area:
● A small Illuminated Area prevents double exposure when the Acquisition Areas are
close together.
● A larger Illuminated Area is preferred when the Acquisition Areas are are widely
spaced and it is important for the beam to also hit some carbon.
● Spot Size must match the Illuminated Area to achieve the required Dose Rate.
● Defocus:
The Defocus value is only used during preparation for the Automated Acquisition run. The
data acquisitions during the run use the values that are specified in the Defocus List for
the Acquisition Areas.
When using Phase Plates, the Optics Settings must be defined with on-plane conditions. For
instructions, see:
● Guidelines for the Data Acquisition Preset when using Phase Plates on a microscope
with a C3 lens on page 31
● Guidelines for the Data Acquisition Preset when using Phase Plates on a microscope
without a C3 lens on page 34 .

Camera ● Binning: 1
Settings ● Readout: Full

Apertures ● C2: start with the 50 µm aperture.

● Objective:
When using Phase Plates, Make sure the dropdown list for the Objective aperture
mechanism contains at least five Phase Plates: PhP1 - PhP5. If present, it can be safely
assumed that the Phase Plate positions are defined accurately.

Use the procedure below to set the Data Acquisition Preset.

1. Select Preparation > Acquisition and Optics Settings

2. Select Preset Selection > Presets: Data Acquisition
3. Start a live image view
For Falcon and Ceta cameras, use Velox
For other cameras, use the TEM User Interface > CCD/TV Camera control panel and TIA,
or use Gatan Digital Micrograph

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a. Select the Camera that is used for this Preset.

b. Select Binning: 1
c. Select Readout area: Full
d. Start the live image view.
4. Move the specimen to an area that can be sacrificed for experimenting with the optics settings.
5. Select Search to start continuous acquisition.
6. Use the handpanels to adjust the optics setings.
Use the FluScreen and/or the FFT in TIA, Velox or Digital Micrograph to assess the image
quality.
7. Verify that the Dose Rate measurement is valid and stable.
For cameras that can measure the Dose Rate, monitor the Dose Rate in the acquisition software:
● For Falcon and Ceta-F cameras, use EPU or Velox.
● For Gatan cameras, use Digital Micrograph.
For cameras that do not report the Dose Rate, follow the instructions below:
a. Adjust Intensity and/or Spot Size, so that:
● The beam illuminates the entire FluScreen.
● The Screen Current is at 0.2 nA or higher.
If the FluScreen is not fully illuminated with sufficient intensity, then the Dose Rate value in
the TEM User Interface is not accurate.
b. If not visible yet, add Dose rate [e-/Å2s] to the TEM User Interface status panel
● Right-click in the status panel where you wish to display the Dose rate value.
● Select Dose rate > Dose rate [e-/Å2s]

c. Note the current Dose Rate value.

d. With the Magnification knob, increase the magnification by two index steps.

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e. Verify that the Dose Rate value is unchanged.

f. Decrease the magnification by two index steps, back to the initial value.
g. Verify that the Dose Rate value is unchanged.
8. Verify that the beam is parallel.
Depending on the system type, either:
● On a system with a C3 lens (typically High End systems with Titan software),
verify that Beam Settings control panel > Illumination is Parallel.

● On a system without a C3 lens (typically Mid Range systems with Talos software),
verify that the specimen and the Objective aperture are both focused.
If no Objective aperture is inserted, then:
● Select a small Objective aperture.
● Verify that the aperture is focused.
● Return the Objective mechanism to its initial position.
9. Select Optics Settings > Get to import the current optical settings from the microscope.
10. In Camera Settings:
a. Select the Camera
b. Select Binning and Readout
c. If the camera is not a Thermo Scientific Falcon 3EC or Falcon 4(i),
then specify the Exp. Time (s).
For Thermo Scientific Falcon 3EC and Falcon 4(i) cameras, this parameter is specified in
Exposure Settings, after the Dose Rate has been measured.
11. If a Thermo Scientific Falcon 3EC or Falcon 4(i) camera is used,
then select Dose Rate > Measure

If the measured Dose Rate is not in the green zone:

a. Adjust the illumination.
Either:
● Use the handpanels to adjust the Intensity and/or Spot Size, then select Optics
Settings > Get
● In the Optics Settings, adjust the Illuminated Area or Intensity, and/or Spot Size

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then select Set

b. Select Measure again to update the Dose Rate value.
If no Dose Rate value is known yet and Measure is skipped, then the Preview acquisition
includes a Dose Rate measurement.
12. Depending on the selected Camera, also specify the additional camera-specific parameters:
● Thermo Scientific Falcon 3EC / Falcon 4(i): Exposure Settings with the Exp. Time (s) and
Dose parameters.
● Thermo Scientific Ceta: Advanced Camera Settings
● Gatan K2 / K3: Advanced Camera Settings
13. Select Acquisition > Preview

5.1.4.1 Guidelines for the Data Acquisition Preset when using Phase Plates on a microscope
with a C3 lens

Microscopes with a C3 lens are typically the High End systems with Titan software.
5.1.4.1.1 Perform the Phase Plate Microprobe (uP) Alignment
On systems with Titan software, perform the Phase Plate Microprobe (uP) alignment procedure:

1. Select the TEM User Interface > Alignments control panel

2. Select Auto help to display detailed instructions for accurate execution of the alignments below.

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3. Select the Align PhasePlate > Phase Plate uP alignment.

In this alignment procedure:
a. Very accurately align the diffraction lens focus at the highest camera length.
This alignment ensures that the phase plate is exactly in focus.
b. Very accurately align the beam shift pivot points.
This alignment ensures a stable beam position relative to the phase plate, when an image-
beam shift is applied.

After the Phase Plate Microprobe (uP) alignment procedure is completed, continue with the Phase
Plate Nanoprobe (nP) alignment procedure.

5.1.4.1.2 Perform the Phase Plate Nanoprobe (nP) Alignment

Perform the Phase Plate Nanoprobe (nP) alignment procedure.
Note On systems with Titan software,
the Phase Plate nP Alignment must be preceded by the Phase Plate uP Alignment.

1. Select the TEM User Interface > Alignments control panel

2. Select Auto help to display detailed instructions for accurate execution of the alignments below.
3. Select the Align PhasePlate > Phase Plate nP alignment.
In this alignment procedure:
a. Very accurately align the diffraction lens focus at the highest camera length.
This alignment ensures that the phase plate is exactly in focus.
b. Very accurately align the beam shift pivot points.
This alignment ensures a stable beam position relative to the phase plate, when an image-
beam shift is applied.

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5.1.4.1.3 Verify the on-plane illumination on a microscope with a C3 lens

When the beam is focused onto the Phase Plate, the illumination is called on-plane. In all other
cases, the illumination is called off-plane.
For the desired illumination conditions, the on-plane conditions must be verified. On-plane
illumination of the phase plate implies parallel illumination of the specimen.
Follow the steps below to adjust the optics settings for on-plane illumination:
1. If the Data Acquisition Preset is already completed, then:
a. Select EPU > Preparation >
Acquisition and Optics Settings > Presets > Preset: Data Acquisition
b. Select Optics Settings > Set
2. If the Data Acquisition Preset is not defined yet,
then use the TEM User Interface and/or the Handpanels to:
a. Select the TEM imaging mode
b. Select the desired Magnification, Spot Size and Intensity for high quality data acquisition.
3. In the TEM User Interface > Beam Settings control panel, verify that Illumination is Parallel

4. Select Handpanels > Diffraction

5. With the Handpanels > Magnification knob, set the highest camera length.
6. Select Handpanels > Eucentric Focus
7. In the TEM User Interface > Phase plate control panel:

a. Select Active
b. Tick MF-Y Fine focus back-focal plane
8. With the Handpanels > Multifunction Y knob, focus the beam to the smallest possible spot on
the FluScreen.
9. Select Handpanels > Diffraction again to return to imaging mode.

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Note During the Automated Acquisition run, make sure that the Phase plate control panel is in Active status.

Note If the spot size and/or illuminated area are changed, the on-plane condition needs to be verified again.

5.1.4.2 Guidelines for the Data Acquisition Preset when using Phase Plates on a microscope
without a C3 lens

Microscopes without a C3 lens are typically the Mid Range systems with Talos software.
5.1.4.2.1 Perform the Phase Plate Nanoprobe (nP) Alignment
Perform the Phase Plate Nanoprobe (nP) alignment procedure.
Note On systems with Titan software,
the Phase Plate nP Alignment must be preceded by the Phase Plate uP Alignment.

1. Select the TEM User Interface > Alignments control panel

2. Select Auto help to display detailed instructions for accurate execution of the alignments below.
3. Select the Align PhasePlate > Phase Plate nP alignment.
In this alignment procedure:
a. Very accurately align the diffraction lens focus at the highest camera length.
This alignment ensures that the phase plate is exactly in focus.
b. Very accurately align the beam shift pivot points.
This alignment ensures a stable beam position relative to the phase plate, when an image-
beam shift is applied.

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5.1.4.2.2 Verify the on-plane illumination on a microscope without a C3 lens

Follow the steps below to achieve on-plane illumination:

1. If the Data Acquisition Preset is already completed, then:

a. Select EPU > Preparation >
Acquisition and Optics Settings > Presets > Preset: Data Acquisition
b. Select Optics Settings > Set
2. If the Data Acquisition Preset is not defined yet,
then use the TEM User Interface and/or the Handpanels to:
a. Select the TEM imaging mode
b. Select the desired Magnification, Spot Size and Intensity
3. In the TEM User Interface > Beam Settings control pane> Tune tab, verify that the beam is set
to Nanoprobe.
4. Insert the FluScreen
5. Verify that the beam is parallel:
a. Select Handpanels > Diffraction
b. With the Handpanels > Magnification knob, set an intermediate camera length.
c. On the FluScreen, verify that the beam is forming a probe.
6. With the Handpanels > Magnification knob, set the highest camera length.
7. Select Handpanels > Eucentric Focus
8. With the Handpanels > Intensity knob, narrow the beam to a probe.
9. Select Handpanels > Diffraction again to return to imaging mode.
Note If the Spot Size and/or Intensity are changed, then the on-plane condition must be verified again.

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5.1.5 Define the Hole/EucentricHeight Preset

The Hole/EucentricHeight Preset is used to perform the Auto-eucentric Height function during the
automated run.
On Quantifoil specimens, the Hole/EucentricHeight Preset is also used to:
● Find and center individual Foil Holes that have been selected at the Grid Square level.
● Specify the Template that defines the locations for Autofocus, Drift Measurement and one or
more Data Acquisition Areas.

The Hole/EucentricHeight Preset must meet the following requirements:

● For Quantifoil samples: the field of view must contain at least one complete Foil Hole.
The algorithm for marking and centering a hole is more reliable if the neighboring holes are also
partly visible.
● The field of view must contain an area of carbon foil for successful execution of the Auto-
eucentric function.
● The contrast and brightness must be good enough to clearly identify the edge of the carbon foil
around a hole.
● The Dose rate must be small enough to prevent damage to the Data Acquisition Area(s) in the
Foil Hole.

Parameters Typical Settings and Recommendations

Optics Settings ● TEM Imaging Mode: the same as the Data Acquisition Preset.
● Illuminated Area: typically ±20 µm.
On Quantifoil samples: twice as large as the Foil Hole interspacing.
● Magnification: low SA range (±3800X),
The Illuminated Area must not be much larger than the field of view.
● Defocus: 10-20 mm.
Defocus helps to enhance contrast, so it is easier to detect the edge of the carbon foil
around a hole.
● Parallel beam.
● Dose must be smaller than 0.1 [e- / Å2sec].
When using Phase Plates, the Hole/EucentricHeight Preset must be defined with an off-
plane illumination, so that an activated Phase Plate is not harmed.

Camera ● Binning: 1
Settings ● Readout: Full

Apertures Same as the Data Acquisition Preset.

Use the procedure below to set the Hole/EucentricHeight Preset.

1. Apply the Data Acquisition Preset values to the microscope:

a. Select the Preparation > Acquisition and Optics Settings task.
b. Select Preset Selection > Presets: Data Acquisition

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c. Select Optics Settings > Set

2. Select Preset Selection > Presets: Hole/EucentricHeight.
3. Start a live image view
For Falcon and Ceta cameras, use Velox
For other cameras, use the TEM User Interface > CCD/TV Camera control panel and TIA,
or use Gatan Digital Micrograph
a. Select the Camera that is used for this Preset.
b. Select Binning: 1
c. Select Readout area: Full
d. Start the live image view.
e. Select Binning: 1
4. Select Search to start continuous acquisition.
5. Move the specimen to an area that can be sacrificed for experimenting with the optics settings.
6. Use the handpanels to create an image that meets the field of view requirements.
This will typically be the case at a Magnification in the lower SA range, and an Illuminated
Area of 10–15 mm.
7. Adjust the illumination parameters to meet the contrast and brightness requirements.
8. Verify that the Dose rate matches the requirements.
Note Make sure that the beam covers the entire FluScreen, and that the Screen Current is at least 0.2 nA.

If the FluScreen is not fully illuminated with sufficient intensity, the Dose rate value in the
Microscope User InterfaceTEM User Interface is not accurate.

9. If the Dose Rate is higher than 0.1 [e- / Å2sec]:

a. Insert the FluScreen
b. Select a higher Spot Size number.
c. Use the Intensity knob to adjust the beam diameter.
Make sure that the entire FluScreen is illuminated and that the Screen Current does not drop
below 0.2 nA.
d. Retract the FluScreen again.
10. Verify that the beam is parallel.
Depending on the system type, either:
● On a system with a C3 lens (typically High End systems with Titan software),
verify that Beam Settings control panel > Illumination is Parallel.

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● On a system without a C3 lens (typically Mid Range systems with Talos software),
verify that the specimen and the Objective aperture are both focused.
If no Objective aperture is inserted, then:
● Select a small Objective aperture.
● Verify that the aperture is focused.
● Return the Objective mechanism to its initial position.
11. Acquire an image with the following camera settings:
● Bias/Gain correction: Bias/Gain
● The same Integration time, Binning and Readout area as used for the live image view.
12. Verify that the acquired image meets the requirements above.
If necessary:
a. Use the Handpanels to adjust Magnification, Intensity and/or Spot Size
b. Acquire a new image and verify it against the requirements above.
13. In EPU:
a. Select Optics Settings > Get to import the current optics values from the microscope.
b. In Camera Settings, select the same Camera, Binning, Readout and Exp. time (s) values
as used for the previously acquired image.
c. Select Acquisition > Preview

14. Verify that the image quality is identical to the previously acquired image.

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5.1.5.1 Guidelines for the Hole/EucentricHeight Preset when using Phase Plates

When using Phase Plates, the Hole/EucentricHeight Preset must be defined with an off-plane
illumination, so the currently activated Phase Plate is not harmed.
On Titan systems:
Use NanoProbe and verify that TEM User Interface > Beam Settings control panel > Illumination
is Parallel.

Above a certain illuminated area (~8 μm for a 50 μm C2 aperture) the beam changes from parallel to
spreading.
On Talos systems, verification of the off-plane condition is done in Diffraction mode.
To verify the off-plane condition:
1. Switch to MicroProbe
2. Set sufficient Defocus to enhance contrast.
3. Select Handpanels > Diffraction
4. With the Handpanels > Magnification knob, set the highest camera length.
5. Verify that the diffraction spot size is at least as large as the 40 mm circle on the FluScreen or
Flucam Viewer.

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5.1.6 Define the Autofocus Preset

The Autofocus Preset is used for:
● Calibration of the Autofocus function.
● Execution of the Autofocus step during the automated run.
The Autofocus function determines the amount of image shift while beam is tilted. After calibration,
the amount of image shift is a measure for the amount of defocus.
The Autofocus Preset must meet the following requirements:
● The Optics Settings for the Autofocus Preset must be as close to the Optics Settings of the Data
Acquisition as possible.
● The combination of beam diameter and field of view must be such, that the entire field of view is
illuminated at all times during execution of the Autofocus function.
Recommendations for the beam diameter and the field of view:

Sample flatness and stability Optics Settings > Camera Settings >
Illuminated Area or Intensity Readout

Better than average Same as the Data Acquisition Preset Half

Average Same as the Data Acquisition Preset Full

Worse than average Slightly larger than the Data Acquisition Preset Full

Parameters Typical Settings and Recommendations

Optics Settings ● Magnification: typically ± 45.000X

● Illuminated Area / Intensity: see recommendations above.
● Probe Mode: if the AFIS functionality is present and calibrated, then use the same
Probe Mode as for the Data Acquisition Preset.

Camera Settings ● See recommendations above.

● Binning: 1

Apertures Same as the Data Acquisition Preset.

Use the procedure below to define the Autofocus Preset.

1. Apply the Data Acquisition Preset values to the microscope:

a. Select the Preparation > Acquisition and Optics Settings task.
b. Select Preset Selection > Presets: Data Acquisition
c. Select Optics Settings > Set
2. Select Preset Selection > Presets: Autofocus
3. In Camera Settings, select the same Camera, Binning and Readout as used in the Data
Acquisition Preset.

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4. Depending on the selected camera, select the same camera-specific values as used in the Data
Acquisition Preset:
● Thermo Scientific Falcon 3EC and Falcon 4:
Duplicate the parameter values in the Exposure Settings section.
● Thermo Scientific Ceta and Gatan K2 / K3:
Duplicate the parameter values in the Advanced Camera Settings section.
Settings that are not used in the Autofocus Preset can be duplicated without negative
consequences. When a value is not applicable to the Autofocus Preset, it will be ignored.
5. Select Acquisition > Preview

The Autofocus Preset cannot be finalized without executing the Autofocus calibration and/or
Autofocus function. Depending on the flatness and stability of the sample, the Camera Settings
and/or the Optics Settings parameters may need adjustment. If adjustments are necessary, this will
be done during Autofocus calibration and/or during Stand-alone execution of the Autofocus function.

5.1.6.1 Guidelines for the Autofocus Preset when using Phase Plates

When using Phase Plates, changes in the Condenser Lens System must be avoided. This makes it
even more important to use the same Optics Settings parameter values as the Data Acquisition
Preset.
Since the Autofocus function acquires images with a tilted beam, it will create satellite spots on the
phase plate film. These spots are sufficiently far removed from the actual Volta area in the optical
center to have a negative effect on the quality of the data acquisitions.
If Phase Plates are used in combination with a Direct Detection camera in electron counting mode,
the beam intensity can be very low and exposure time for data acquisition can be very long.
Nevertheless, Autofocus will work with an integration time that is significantly shorter than the time
that is specified in the Data Acquisition preset. For the Autofocus function a very small dose is
sufficient to reliably perform cross-correlation based shift measurements. The equivalent of a couple
of dose fractions should be sufficient.

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5.1.7 Define the Drift Measurement Preset

The Drift Measurement Preset is used for:
● Stand-alone execution of the Drift Measurement function in the Auto Functions tab.
● Execution of the Drift Measurement function during the automated run.
The automated run postpones data acquisition until the drift speed has dropped below a
configurable threshold.

The Drift Measurement function tracks the position of recognizable features and patterns in
consecutive images, and compares the detected position shift with a specified threshold value.
The Drift Measurement Preset must meet the following requirements:
● The Optics Settings for the Drift Measurement Preset must be as close to the Optics Settings of
the Data Acquisition as possible.
● The exposure time must be small enough to minimize blur in the images that are used for the
Drift Measurement.
In a blurry image, it is hard to accurately identify recognizable features and patterns.
● The time interval between consecutive images must be short enough to allow for fast
measurements.
● If the Drift Threshold is set very low, the resolution of the acquired images must be high enough
to enable detection of very small shifts.
● For Quantifoil samples: the beam diameter must be small enough, so that the Drift Measurement
Area can be placed close to the Data Acquisition Area(s) without risking multiple exposure of the
Data Acquisition Area(s).

Parameters Typical Settings and Recommendations

Optics Settings If Phase Plates are used, then the Optics Settings for the Drift Measurement Preset must be
identical to the Data Acquisition Preset, including the on-plane illumination conditions.
If Phase Plates are not used, then the below recommendations are applicable.
● Probe Mode: if the AFIS functionality is present and calibrated, then use the same
Probe Mode as for the Data Acquisition Preset.
● TEM Imaging Mode: same as the Data Acquisition Preset.
● Magnification:
If the drift threshold is very low, it may be necessary to select a higher magnification
than is used for the Data Acquisition preset.
● Illuminated Area / Intensity for Quantifoil samples:
Decrease the beam diameter if possible, but make sure the entire field of view is
illuminated.

Camera ● Binning: 1
Settings Readout: Half.
This increases the readout speed.

Apertures Same as the Data Acquisition Preset.

Use the procedure below to define the Drift Measurement Preset.

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1. Apply the Data Acquisition Preset values to the microscope:

a. Select the Preparation > Acquisition and Optics Settings task.
b. Select Preset Selection > Presets: Data Acquisition
c. Select Optics Settings > Set
2. Select Preset Selection > Presets: Drift Measurement
3. In Camera Settings:
a. Select the same Camera as used in the Data Acquisition Preset.
b. Select Binning: 1
c. Select Readout: Half
4. Depending on the selected camera, select the same camera-specific values as used in the Data
Acquisition Preset:
● Thermo Scientific Falcon 3EC and Falcon 4:
Duplicate the parameter values in the Exposure Settings section.
● Thermo Scientific Ceta and Gatan K2 / K3:
Duplicate the parameter values in the Advanced Camera Settings section.
Settings that are not used in the Drift Measurement Preset can be duplicated without negative
consequences. When a value is not applicable to the Drift Measurement Preset, it will be
ignored.
5. Select Acquisition > Preview

The Drift Measurement Preset cannot be finalized without executing the Drift Measurement function.
Depending on the specified Drift Threshold and the measurement results, the Optics Settings and/or
Camera Settings parameters may need adjustment. If adjustments are necessary, this will be done
during Drift Measurement calibration and/or during Stand-alone execution of the Drift Measurement
function.

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5.1.8 Define the Thon Ring Preset

The Thon Ring Preset is used for:
● Stand-alone execution of the Autocoma function.
● Stand-alone execution of the Autostigmate function.
The Autocoma and Autostigmate functions are intended for optimization of the system alignments
before starting an automated run. Both functions are not executed during the automated run, which
means that also the Thon Ring Preset is not applied during the automated run.

The Thon Ring Preset must meet the following requirements:

● Thon Rings are clearly visible in the FFT of the acquired images.

The recommendations below are specified relative to the Data Acquisition Preset.

Parameters Typical Settings and Recommendations

Optics Settings ● When using Phase Plates, the Optics Settings for the Thon Ring Preset must be
identical to the Data Acquisition Preset to prevent accidental overexposure of an
activated Phase Plate.
● Spot Size:
● Do not use Spot Size 1 or 2.
● When using the survey camera: select Spot Size 3 or higher.
● When using the high sensitivity camera: check the dose rate to select a suitable
Spot Size.
● Illuminated Area / Intensity:
If desired, slightly decrease the beam diameter to increase the signal strength. When
the beam is too narrow, less Thon Rings will be visible.
● Defocus: typically between -1 mm and -3 mm.

Camera ● Binning: 2
Settings ● Readout: Full

Use the procedure below to set the Thon Ring Preset.

1. Apply the Data Acquisition Preset values to the microscope:

a. Select the Preparation > Acquisition and Optics Settings task.
b. Select Preset Selection > Presets: Data Acquisition
c. Select Optics Settings > Set
2. Select Preset Selection > Presets: Thon Ring
3. Select Optics Settings > Get
4. Prepare a live FFT view.
For Falcon and Ceta cameras, use Velox
For other cameras, use the TEM User Interface > CCD/TV Camera control panel and TIA,
or use Gatan Digital Micrograph
a. Select the same Camera as used in the Thon Ring Preset.
b. Use the following camera settings:

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● Binning: 2
● Readout area: Full
c. Start the live image acquisition
d. Move the specimen to an area with thin, uninterrupted, amorphous carbon foil that is not
close to a grid bar.
e. Display the FFT of the live image.
5. Assess the quality of the Thon Rings
If necessary adjust the optics parameters to improve the sharpness of the Thon Rings.
6. Verify that the beam is parallel.
Depending on the system type, either:
● On a system with a C3 lens (typically High End systems with Titan software),
verify that Beam Settings control panel > Illumination is Parallel.

● On a system without a C3 lens (typically Mid Range systems with Talos software),
verify that the specimen and the Objective aperture are both focused.
If no Objective aperture is inserted, then:
● Select a small Objective aperture.
● Verify that the aperture is focused.
● Return the Objective mechanism to its initial position.
7. Acquire an image with the following camera settings:
● Bias/Gain correction: Bias/Gain
● The same Integration time, Binning and Readout area as used for the live image view.
8. Verify that the acquired image meets the requirements above.
If necessary:
a. Use the Handpanels to adjust Magnification, Intensity and/or Spot Size
b. Acquire a new image and verify it against the requirements above.
9. In EPU:
a. Select Optics Settings > Get to import the current optics values from the microscope.
b. In Camera Settings, select the same Camera, Binning, Readout and Exp. time (s) values
as used for the previously acquired image.
c. Select Acquisition > Preview

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10. Verify that the image quality is identical to the previously acquired image.
11. In the Image Display, select Show/Hide Inset and/or Swap Inset and Main to display the FFT
for the acquired image.

12. Check the quality of the Thon Rings.

Select Filter FFT to improve the contrast and brightness of the FFT if the Thon Rings .

13. If necessary, adjust the Optics Settings.

Either:
● Use the handpanels to adjust the Intensity and/or Spot Size,
then select Optics Settings > Get
● In the Optics Settings, adjust the Illuminated Area or Intensity, and/or Spot Size
then select Set

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5.1.9 Define the Zero Loss Preset

The Zero Loss Preset is only available when the microscope is in EFTEM mode at the time EPU is
started. The Zero Loss Preset is used by the Auto Zero-Loss auto function. During an Automated
Acquisition run, EPU executes the Auto Zero-Loss function at the specified interval to maintain a
proper alignment of the energy slit in the filter with the Zero Loss Peak.
If Phase Plates are used, then the Optics Settings for the Zero Loss Preset must be identical to the
Data Acquisition Preset to prevent accidental overexposure of an activated Phase Plate.
If no Phase Plates are used, then it is still recommended to use the same Optics and Camera
Settings as the Data Acquisition Preset.

Parameters Typical Settings and Recommendations

Optics Settings Same as the Data Acquisition Preset.

Camera Settings Same as the Data Acquisition Preset.

Apertures Same as the Data Acquisition Preset.

Use the procedure below to define the Zero Loss Preset.

1. Apply the Data Acquisition Preset values to the microscope:

a. Select the Preparation > Acquisition and Optics Settings task.
b. Select Preset Selection > Presets: Data Acquisition
c. Select Optics Settings > Set
2. Select Preset Selection > Presets: Zero Loss
3. In Camera Settings, select the same Camera, Binning and Readout as used in the Data
Acquisition Preset.
4. Depending on the selected camera, select the same camera-specific values as used in the Data
Acquisition Preset:
● Thermo Scientific Falcon 3EC and Falcon 4:
Duplicate the parameter values in the Exposure Settings section.
● Thermo Scientific Ceta and Gatan K2 / K3:
Duplicate the parameter values in the Advanced Camera Settings section.
Settings that are not used in the Zero Loss Preset can be duplicated without negative
consequences. When a value is not applicable to the Zero Loss Preset, it will be ignored.
5. Select Acquisition > Preview

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5.1.10 Define the GridSquare Preset

The GridSquare Preset is used to find and select Target Areas that are suitable for data acquisition.
The GridSquare Preset must meet the following requirements:
● The field of view must contain an entire, centered Grid Square.
● The field of view must contain a part of each neighboring Grid Square.
This is needed to make sure that a Grid Square image still contains an entire Grid Square in
case the positioning of the specimen has drifted over time. As a rule of thumb, the field of view of
the Grid Square image must be approximately twice as large as a single Grid Square.
● The contrast and brightness must be good enough to assess the ice thickness.
● For Quantifoil samples, the contrast and brightness must be good enough to detect Foil Holes.

Parameters Typical Settings and Recommendations

Optics Settings ● TEM Imaging Mode: same as the Data Acquisition Preset.
● Magnification: LM 400X - LM 600X.
● Illuminated Area: typically ±200 mm.
● Parallel beam.
When using Phase Plates, do not focus the beam to a spot to prevent high doses on the
Phase Plate.

Camera Settings ● Binning: 1

● Readout: Full

Apertures Same as the Data Acquisition Preset.

Use the procedure below to set the GridSquare Preset.

1. Apply the Data Acquisition Preset values to the microscope:

a. Select the Preparation > Acquisition and Optics Settings task.
b. Select Preset Selection > Presets: Data Acquisition
c. Select Optics Settings > Set
2. Select Preset Selection > Presets: GridSquare
3. Start a live image view
For Falcon and Ceta cameras, use Velox
For other cameras, use the TEM User Interface > CCD/TV Camera control panel and TIA,
or use Gatan Digital Micrograph
a. Select the Camera that is used for this Preset.
b. Select Binning: 1
c. Select Readout area: Full
d. Start the live image view.
4. Select Search to start continuous acquisition.

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5. Use the handpanels to adjust the Magnification and/or Intensity to meet the requirements for
contrast and brightness, and for the field of view.
This will typically be the case at a Magnification of LM 400X - LM 600X with an Illuminated Area
of 150-200 mm.
6. Verify that the beam is parallel.
Depending on the system type, either:
● On a system with a C3 lens (typically High End systems with Titan software),
verify that Beam Settings control panel > Illumination is Parallel.

● On a system without a C3 lens (typically Mid Range systems with Talos software),
verify that the specimen and the Objective aperture are both focused.
If no Objective aperture is inserted, then:
● Select a small Objective aperture.
● Verify that the aperture is focused.
● Return the Objective mechanism to its initial position.
7. Acquire an image with the following camera settings:
● Bias/Gain correction: Bias/Gain
● The same Integration time, Binning and Readout area as used for the live image view.
8. Verify that the acquired image meets the requirements above.
If necessary:
a. Use the Handpanels to adjust Magnification, Intensity and/or Spot Size
b. Acquire a new image and verify it against the requirements above.
9. In EPU:
a. Select Optics Settings > Get to import the current optics values from the microscope.
b. In Camera Settings, select the same Camera, Binning, Readout and Exp. time (s) values
as used for the previously acquired image.
c. Select Acquisition > Preview

10. Verify that the image quality is identical to the previously acquired image.

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5.1.11 Define the Atlas Preset

An automated run starts by acquiring an Atlas. The Atlas is an overview of the specimen. The Atlas is
used to detect and select GridSquares for automatic data acquisition. It is assembled by stitching a
series of images (so-called Tiles) together.
For these purposes, the Atlas Preset must meet the following requirements:
● The field of view must cover the largest possible specimen area. The field of view must contain
an area of at least 3x3 entire Grid Squares.
The Grid Square detection algorithm becomes more reliable when more Grid Squares are
captured in the field of view.
● The field of view must be fully illuminated. This also means that the image does not contain any
cut-offs.
To fulfill this requirement it may be necessary to not only set the Atlas Preset, but also to perform
the Atlas Optical Alignment calibration.
● The grid bars of the carbon foil are clearly recognizable at the edges of each image.
● The contrast and brightness must be good enough to assess the ice thickness.
● The contrast and brightness must be good enough to detect broken carbon foil.

Parameters Typical Settings and Recommendations

Optics Settings ● TEM Imaging Mode: Microprobe

Microprobe is better suited for a wide parallel beam at lower magnifications.
● Magnification: low LM, typically LM 40X - LM 200X
● Illuminated Area: typically ±800 µm
● Parallel beam.
With defocus, a convergent beam may lead to an effective change of magnification.
When using Phase Plates, do not focus the beam to a spot to prevent high doses on the
Phase Plate.

Camera ● Binning: 1
Settings ● Readout: Full

Apertures ● C2: 150 µm

● All other aperture mechanisms:
If possible, use the same apertures as the Data Acquisition Preset.
If it is not possible to meet the illumination requirements with the same apertures as the
Data Acquisition Preset, then use the TEM User Interface > Apertures control panel to
select the required apertures before Atlas acquisition and to revert them after Atlas
acquisition.

Use the procedure below to define the Atlas Preset.

1. Select the Preparation > Acquisition and Optics Settings task.

2. Select Preset Selection > Presets: Atlas
3. In the TEM User Interface > Apertures control panel:
● Set Condenser 2 to the largest aperture.

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● Set Objective to the retracted position.

4. Start a live image view
For Falcon and Ceta cameras, use Velox
For other cameras, use the TEM User Interface > CCD/TV Camera control panel and TIA,
or use Gatan Digital Micrograph
a. Select the Camera that is used for this Preset.
b. Select Binning: 1
c. Select Readout area: Full
d. Start the live image view.
5. Select Magnification: LM 60X,
or a magnification close to this value.
6. With the handpanels, adjust the Intensity and/or Spot Size and/or Magnification, so the
requirements above are fulfilled.
7. If the system has a Cryobox and the image still has cut-offs at a magnification of LM 200X or
higher, then the center of the Cryobox aperture may have an offset relative to the system's
optical axis. To compensate for this offset, perform these steps:
a. Complete the Atlas Preset procedure as if there were no cut-offs.
The purpose of this procedure is no longer to fulfill all the requirements for the Atlas Preset.
Instead the goal is now to prepare an Atlas Preset that can be used as a starting point for the
Atlas Optical Alignment calibration. This means that the contrast and brightness
requirements still apply, but that any remaining cut-offs do not have to be removed by
adjusting the Intensity, Spot Size and Magnification.
b. Perform the Atlas Optical Alignment
The Atlas Optics Alignment determines the offset of the Cryobox aperture relative to the
system's optical axis. It then calculates the amount of beam-shift that is needed to let the
beam pass through the center of the Cryobox aperture.
See chapter Perform the Atlas Optics Alignment on page 53 .
c. Return to this Atlas Preset procedure at step 6 to adjust the Intensity and/or Spot Size and/or
Magnification.
It may be necessary to repeat steps 6 and 7 more than once.
8. Acquire an image with the following camera settings:
● Bias/Gain correction: Bias/Gain
● The same Integration time, Binning and Readout area as used for the live image view.
9. Verify that the acquired image meets the requirements above.
If necessary:
a. Use the Handpanels to adjust Magnification, Intensity and/or Spot Size
b. Acquire a new image and verify it against the requirements above.
10. In EPU:
a. Select Optics Settings > Get to import the current optics values from the microscope.
b. In Camera Settings, select the same Camera, Binning, Readout and Exp. time (s) values
as used for the previously acquired image.
c. Select Acquisition > Preview

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11. Verify that the image quality is identical to the previously acquired image.

5.1.12 Import and export the Acquisition and Optics Presets

With the Import and Export functions it is possible to archive the values for all current Presets, and to
load them again at a later time. This way, setting up a new Automated Acquisition run for a regularly
used specimen type can be done much easier and much faster.

Export
Writes the current parameter values for all Presets to a file (*.sxml) and save it to an appointed
drive and folder. The Presets file contains:
● The Optics Settings for all Presets.
● The Camera Settings for all Presets.
Depending on the selected camera the Advanced Camera Settings and/or Exposure Settings are
also included.

Import
Overwrite all current parameter values for all Presets with the values from the selected Presets file.
● There is no Undo function. It may be wise to export the current Presets to a file before importing
a different Presets file.
● Presets files that are created with previous software versions are supported with limitations.
When EPU can not import or convert a legacy Presets file it will display an error message.

Note A Presets file contains the parameter values for all Presets. It is not possible to export or import the
settings for a single Preset.

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5.2 Atlas Optics Alignment task

On systems with a Cryobox, a cut-off may become visible in low magnification images. This cut-off
appears when the center of the Cryobox aperture has an offset relative to the system's optical axis.
The Atlas Optics Alignment determines the offset of the Cryobox aperture relative to the system's
optical axis. It then calculates the amount of Image/Beam Shift that is needed to compensate for the
offset, so that the beam passes through the center of the Cryobox aperture. The Image/Beam Shift
value that results from the Atlas Optical Alignment calibration is only applied when the Atlas Preset is
used to acquire images. It does not affect other Presets.
After the Atlas Optics Alignments is completed, the Atlas Preset can typically use a lower
magnification. This means less images are needed to cover the entire specimen, which saves time.

5.2.1 Perform the Atlas Optics Alignment

To perform the Atlas Optics Alignment procedure, follow the steps below:

1. Select Atlas Optics Alignment > Acquire.

If the image does not meet the requirements for the Atlas Preset, then follow the instructions in
Define the Atlas Preset on page 50 . The requirement that no cut-offs must be visible does not
apply yet.
2. In the acquired image, check for cut-offs.
A Cryobox aperture cut-off is very similar to a C2 aperture or Objective aperture cut-off. It
appears as a dark area with a round edge.

The red dot represents the position of the system's optical axis relative to the center of the
Cryobox aperture.
3. If one or more cut-offs are present:
a. Estimate where the physical center of the Cryobox aperture is.
b. In the image, double-click on the estimated location.

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The red dot moves to the indicated location.

c. Select Acquire again.
d. Check again for cut-offs. If necessary, repeat steps a, b and c.
4. When the image is free from cut-offs, select Accept.

Note The Atlas Optics Alignment calibration is based on the Optics Settings of the Atlas Preset.
If the Optics Settings of the Atlas Preset are changed, then the calculated beam shift value may not be
accurate anymore, and the calibration must be renewed.

To renew the Atlas Optical Alignment calibration:

5. Select Reset Calibration.
6. Perform steps 4 - 6 again.

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5.3 Calibrate Image Shifts task

Even on a well-aligned system, a centered feature may shift away from the image center when a new
image is acquired at a different magnification. A good lens series alignment minimizes the amount of
shift, but a small amount of shift could be present due to the following factors:
● The alignment is done on the FluScreen or on a different camera than the camera that is used
for the image acquisition. Different cameras on the same system can have small offsets relative
to each other.
● Readjustment of the LM rotation center can cause a shift when switching between LM and HM
modes.
● The use of defocus during the Automated Acquisition run can cause a small shift due to
inaccuracies in the rotation center alignment.
● During lens series alignment, the normalization of the lenses may have been handled differently
than during Automated Acquisition.
For optimum performance of the Automated Acquisition run, any remaining shifts must compensated.
The Image Shift Calibration acquires images with each Preset. In each image, EPU requests to mark
the exact location of an easily recognizable feature. EPU uses the distance between the marked
locations to compensate for image center offsets between the Presets.
All shifts are relative to the Data Acquisition Preset, so reconstruction is always based on zero-shift
images.

5.3.1 When to reset and renew the Image Shift Calibration

The Image Shifts Calibration needs to be renewed:
● When the Optics Settings of a Preset are updated (magnification or defocus).
● When a different camera is selected in the Camera Settings of a Preset. The magnification
center may shift.
● When the Alignment file that was used to define the Presets is updated, or when a different
Alignment file is loaded.
● When the FEG Register that was used to define the Presets is updated, or when a different FEG
Register is loaded.
● When a feature does not stay centered after selecting a different Preset.

5.3.2 Prepare for Image Shift Calibration

To prepare for the Image Shift Calibration, a feature must be centered in the field of view that is
easily recognizable at all Preset magnifications. Follow the procedure below to find and center a
suitable feature. It is not necessary to center the feature in each acquired preview image.

1. Find a suitable feature and move it near the center of the field of view:
a. In the Preparation > Acquisition and Optics task,
select Preset Selection > Presets: Atlas

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b. Select Acquisition > Preview

c. Identify a unique and easily recognizable feature,

for example a distinctive edge or corner of a small asymmetric crack in the foil.
If necessary, zoom in to make it easier to identify a suitable feature.
d. Right-click on an the easily recognizable feature and select Move stage here
2. Select Preset Selection > Presets: GridSquare and select Acquisition > Preview
3. In the acquired image, right-click on the feature and select Move stage here
4. Select Preset Selection > Presets: Hole/EucentricHeight and select Acquisition > Preview
5. In the acquired image, right-click on the feature and select Move stage here
6. Select Preset Selection > Presets: Data Acquisition and select Acquisition > Preview
7. In the acquired image, verify that the selected feature is visible near the image center.

5.3.3 Perform the Image Shift Calibration

1. Make sure that the preparations for the Image Shift Calibration are completed.
For instructions, see: Prepare for Image Shift Calibration on page 55 .
2. If necessary, re-adjust the position of the easily recognizable feature so that it is centered on
the FluScreen.
3. Select Preparation > Calibrate Image Shifts task.
4. Select Image Shift Calibration > Start Calibration

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The calibration procedure acquires the first image and displays it on the left side of the Task
Execution panel.

5. Center the recognizable feature in the center of the image.

a. In the left-side image, double-click on the recognizable feature.
The red crosshair moves to the selected position. The red crosshair can be relocated as
often as necessary.
Optionally zoom in for better accuracy.
b. Select Re-acquire
EPU uses a backlash corrected stage move to center the marked feature and then acquires
a new image.
c. If the feature is not properly centered, select Re-acquire again.
On a well-aligned and calibrated system this should solve the offset.
Note that the CompuStage is not infinitely accurate. A very small offset may still exist at the
highest magnification. This is acceptable.
6. After the feature is properly centered, the shifts between the Presets can be determined:
a. Select Proceed
EPU acquires a new image using the next Preset, and displays it on the right-side of the
Task Execution panel.
b. In the new image on the right-side, double-click on the distinctive location of the
recognizable feature.
The red crosshair moves to the selected position.
● The red crosshair can be relocated as often as necessary.
● Zoom in for better accuracy.
c. Repeat step a and step b until the image shifts between all Presets have been calibrated.
For every shift:
● The image on the left-side serves as a reference.
● Use the new image on the right-side to mark the distinctive location on the recognizable
feature.
d. After the last shift is calibrated, the Status panel reports the successful completion of the
calibration.

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5.4 Calibrate I0 task

A Cryo-EM specimen consists of a solution that contains the particles of interest, which is flash-
frozen on a carbon foil. It forms a layer of vitreous ice that spans across the holes on the carbon foil.
The thickness and quality of the ice layer is a critical parameter for efficient, high quality data
acquisition:
● Ice that is too transparent is typically too thin and does not contain many particles.
● Ice that is not transparent enough is typically too thick, or is not vitreous.
Images from such ice layers typically have insufficient contrast.
When the beam passes through ice, the illumination intensity decreases. The ratio between the pre-
specimen illumination intensity (source intensity) and the post-specimen illumination intensity
(recorded intensity) is an indicator for the thickness and quality of the ice layer.
In an acquired image, the recorded intensity (counts) for each pixel is displayed as a gray scale
value. The Ice Quality Filter uses the recorded intensity to assess the thickness and quality of the ice
layer. This filter compares the average gray scale value of the candidate target areas with an upper
and a lower boundary value, and selects only the target areas that are inside the suitable range.
The source intensity can fluctuate a little over time, which transfers directly to the average gray scale
values. As a consequence, the absolute gray scale value is not a durable selection criterion for the
Ice Quality Filter. The I0 Calibration determines a reference value (I0) based on the counts in an
image that is acquired while the beam passes through an area of the specimen where no carbon foil
and no ice are present. Because there is no material between the source and the camera sensor, the
I0 reference value represents the source intensity. If an I0 reference value is available, then the upper
and lower boundaries of the Ice Quality Filter are defined relative to that I0 reference value. When the
I0 reference value changes, the Ice Quality Filter proportionally adjusts the boundaries for target area
selection.
To remain reliable, the I0 reference value must be measured regularly. The I0 reference value can be
refreshed manually at any time during the preparation of an Automated Acquisition run. EPU
automatically refreshes the I0 reference value during an Automated Acquisition run at regular
intervals. If the most recent I0 measurement is older than 2 hours, then EPU refreshes the I0
reference value automatically before target area selection in a new Grid Square. The I0 reference
images are saved in the EPU session directory.

The I0 Calibration is an optional task. If no I0 reference value is available, then the Ice Quality Filter
boundaries use fixed gray scale values. This is fine, as long as the source intensity is kept stable for
the duration of the experiment.
If the specimen is unloaded and reloaded after the Calibrate I0 task is performed, then the I0
reference value measurements during the Automated Acquisition run may be less reliable.

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5.4.1 Perform the I0 Calibration for the GridSquare Preset

In an Atlas, areas that are suitable for the I0 measurement can be found quite easy. It is also easier
to move the specimen to a suitable area.
To prepare and perform the I0 Calibration, follow the procedure below.

1. If an Atlas is available for the currently loaded specimen, then:

a. Select EPU > Square Selection
b. Right-click on a Grid Square with a large gap and select Move stage to grid square
2. Select Preparation > Calibrate I0
3. Select Acquisition Settings Selection > Acquisition Settings: GridSquare.

4. Select Preview to acquire an image at the current location.

5. Center the region that will be used for reference intensity (I0) measurement:
a. In the image, right-click in the center of a suitable area and select Move stage here.
b. Select Preview again to acquire a new image.
Note Do not center the I0 region by moving the specimen with the joystick.

The Move stage here function uses a backlash correction routine to ensure stage position
reproducibility. During the Automated Acquisition run the same backlash correction routine is
used to revisit the selected location for I0 calibration updates.
6. Set the diameter of the I0 region:
a. Double-click on the center of the I0 region.
A circle appears.
b. Increase the diameter of the circle, but make sure it stays at least 250 nm away from the
surrounding carbon foil.
The I0 measurement determines the average signal inside the circular boundary. A larger
diameter increases the reliability of the measurement.
7. Select Get Stage Position and Diameter

8. Either:
● Select Calibrate Current to measure I0 for the GridSquare Preset.
● Select Calibrate All to measure I0 for the GridSquare and the Hole/Eucentric Height
Presets.

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EPU acquires new images and measures the I0 values.

● The results are stored and are displayed in the Status side panel.
● The Status changes to "calibrated" with a datetime stamp.
To renew the I0 calibration it is not necessary to select Remove I0 Measurements before the
calibration is started.

5.4.2 Perform the I0 Calibration for the Hole/EucentricHeight Preset

The procedure to calibrate I0 for the Hole/EucentricHeight Preset is the same as for the GridSquare
Preset. For the Hole/EucentricHeight Preset, the same stage position and area diameter are used for
calibration and during the Automated Acquisition run.
Currently, I0 for the Hole/EucentricHeight Preset is only measured for reference. In the future it may
be used for Target Area analysis.

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5.5 Activate Phase Plate task

The use of a Phase Plate substantially enhances the low-to-medium frequencies when imaging weak
phase objects. This frequency enhancement results in a higher image contrast. A higher contrast
makes identifying and aligning features of interest easier and more reliable.
With an activated Phase Plate, image contrast is maintained under optical focus, so it should be
possible to acquire data that does not need any Contrast Transfer Function (CTF) correction.
Resolution is severely limited by focus accuracy. To avoid specimen damage the focus cannot be
adjusted on the region of interest. Therefore, the preferred strategy is to acquire data with a certain
amount of defocus and to apply a CTF correction.
For example, see: "Using the Volta phase plate with defocus for cryo-EM single particle analysis"
(Danev et al, eLife 2017;6:e23006) .

The duration of an Automated Acquisition run generally exceeds the lifespan of a Phase Plate area.
During an Automated Acquisition run procedure, EPU selects and activates a new Phase Plate at
regular intervals. A new Phase Plate must be activated to make sure that:
● The next data acquisition starts with a decent phase shift, so that the acquired image has
sufficient contrast for fast, reliable and accurate feature identification.
● The change in phase shift during the first acquisition stays within limits. At the beginning of the
Phase Plate development, phase shift changes rapidly. Activating the new Phase Plate before
acquiring an image stabilizes the phase shift.

To successfully activate new Phase Plates during an Automated Acquisition run, the following
parameters must be specified:
Activation time
For a successful EPU experiment, the Phase Plate should establish a phase shift range of 0.5π ±
0.3π. To achieve a phase shift of 0.5π, the Phase Plate typically needs to be irradiated with an
electron dose of 50 nC – 200 nC. The required activation time can be calculated from the electron
dose number:
t = dose / (beam current)
For example: with a 1 nA beam current, an activation time of 120 seconds is needed to reach 120nC.

Accelerate and Accelerate factor

● The Accelerate function temporarily selects the largest C2 aperture to increase the beam
current. After the phase plate activation is completed, the initial C2 aperture is selected again.
● The Accelerate factor is the area ratio between the initial C2 aperture and the largest C2
aperture.
The Accelerate function only changes the selected C2 aperture. The Optics settings are not affected.

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5.5.1 Determine the Phase Plate Activation Time

Note The Phase Plate activation time is best determined with the Sherpa > AutoCTF function.

For regular users it is possible to check the activation time in the Preparations tab > Activate Phase
Plate task. To do so, follow these steps:

1. Correct for astigmatism if necessary.

For instructions, see: Run the Autostigmate auto-function on page 69 .
2. In the TEM User Interface > Apertures control panel, select an Objective mechanism > Phase
Plate position.
3. Retract the FluScreen.
The beam will be blanked now.
4. Select the Preparation tab > Activate Phase Plate task.
5. Select Next Position to move the Objective aperture mechanism to a fresh area on the phase
plate.

6. Wait for the drift of the aperture mechanism to settle.

For a small move, this takes about 30 seconds.
Note After a large move with the aperture mechanism, for example a move to a different Phase Plate, it
may take up to 5 minutes for drift to settle.

7. Specify the Activation time (s) value.

8. (Optional) Select Accelerate: Yes
9. Select Start Activation.
An image is acquired and displayed in the top left section of the Task Execution panel.

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10. Wait for the Activation time to expire.

EPU acquires a new image and displays it in the top right section of the Task Execution panel.
The FFT of both images are displayed side-by-side in the bottom section of the Task Execution
panel, so they are easy to compare. If the Thon rings are shifted inward by half a period, then
Phase Plate activation is completed successfully.

Bottom left half: FFT of an image at the beginning of the activation.

Bottom right half: FFT of an image at end of the activation.

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6 Auto Functions Tab

The Auto Functions tab provides various (semi-)automated tasks. These tasks are organized in the
following categories:
● Alignments: execute an automated alignment.
Alignments tasks are only available on Tundra systems.
● Auto-Functions (TEM): execute the Auto Functions as stand-alone tasks, outside the context of
setting up an Automated Acquisition run.
For descriptions and instructions, see: Auto-Functions (TEM) tasks on page 65 .
● Calibrations: calibrate the Auto Functions.
For descriptions and instructions, see: Calibration tasks on page 75 .
The Auto Functions use the Acquisition and Optics Presets. Except for the Auto-eucentric by stage
tilt task, the Defocus value that is specified in the selected Preset is not applied during calibration or
stand-alone execution of the Auto Function.

6.1 Alignments tasks

The Alignments category contains only the Optimize Beam task.

6.1.1 Run the Optimize Beam task

The Optimize Beam task optimizes the beam, based on the Optics Settings of the Data Acquisition
preset and the currently loaded specimen. The optimizations include among others: automated coma
correction, automated stigmation, and an adjustment of the Intensity to ensure a parallel beam.

1. Select Auto Functions > Alignments > Optimize Beam

2. In Apertures:

a. Select the appropriate Objective aperture.

b. (Optional) Select Set to verify that the most suitable Objective aperture is selected.
3. Select Execution > Start

If the selected Objective aperture is not inserted yet, then the Optimize Beam procedure will
automatically insert the selected aperture.
4. Follow the instructions that are displayed.
The Optimize Beam procedure will request to navigate to an area of bare carbon foil. Then the
procedure executes various optimizations.
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After the optimizations are completed, the Optimize Beam procedure updates the Optics Settings in
the Data Acquisition preset.
Note The Optimize Beam task does not update the Optics Settings in Presets that should have the same
Optics Settings as the Data Acquisition preset.

6.2 Auto-Functions (TEM) tasks

The functions in the Auto-Functions (TEM) group of tasks can be executed outside of the workflow
context. To set up a properly performing Automated Acquisition run, it is not absolutely necessary to
run the Auto-Functions (TEM) tasks.

6.2.1 Run the Autofocus auto-function

The Autofocus function works in two steps.
First, the Autofocus function establishes focus. The Autofocus algorithm iterates to an initial defocus
value, typically –5 mm. By aiming for a small defocus:
● The images that are used to measure and adjust the current defocus have good contrast.
● The Cross-Correlation image shows a significant peak that is not at zero-shift.
The initial defocus value is specified in Auto Functions tab > Auto-Functions (TEM): Autofocus
task > Auto Function Settings section > Iterate to
In a stand-alone execution, the Autofocus function can also correct for astigmatism and/or for drift.
During an Automated Acquisition run these corrections are skipped.

After the algorithm established the initial defocus, the Autofocus function applies the final defocus
value. Which final defocus value is used depends on where in the EPU workflow the Autofocus
function is executed:
● In the Auto Functions tab > Auto-Functions (TEM): Autofocus task,
EPU uses the Auto Function Settings > Desired Defocus value.
● To acquire an image from an Acquisition Area during the Automated Acquisition run,
EPU uses the defocus value from the Defocus list. The Defocus list is specified in the EPU >
Area Selection task for Lacey Carbon specimens, or in the EPU > Template Definition task for
Quantifoil specimens.
● At any other time, EPU uses the defocus value from the active Preset.

To run the Autofocus function, follow the procedure below:

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1. Select Auto Functions > Auto-Functions (TEM) > Autofocus

2. Select the Preset Selection > Preset that will be used for the stand-alone execution of the
Autofocus function.
3. In the Auto Function Settings section:

a. Specify the final defocus value in Desired Defocus

b. Specify the initial defocus value in Iterate to
c. Select the Focus Method that will be used to change from the initial Iterate to value to the
final Desired Defocus value.
d. (Optional) Select Auto Stigmate: Yes to correct for astigmatism during the Autofocus
execution.
Including astigmatism during Autofocus works well on a stable area of the specimen such as
a piece of carbon film.
The Auto Stigmate action is not executed during an Automated Acquisition run. On CryoEM
specimens, the ice quality changes between consecutive exposures, which may cause
stigmation to fail or to give inaccurate results.
e. (Optional) Select Use Three Image Method: Yes to include drift correction. EPU will acquire
an extra image to measure drift.
4. Select Execution section> Start

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6.2.2 Run the 'Auto-eucentric by beam tilt' auto-function

The Auto-eucentric by beam tilt function is used to set the specimen to eucentric height. Before using
this function, the Eucentric correction calibration must be completed. See The Eucentric Correction
Calibration task on page 78 for background information and for instructions how to perform the
Eucentric correction calibration.
This method is more suited for higher magnifications. At low magnifications the method can be used
also, but the accuracy can be less than optimial.

To run the Auto-eucentric by beam tilt Auto Function, follow the procedure below:
1. Select Auto Functions > Auto-Functions (TEM) > Auto-eucentric by beam tilt

2. Select the Preset Selection > Presets with which the Auto Function will be executed.
3. In the Auto Function Settings section:

For a description of the parameters, see Run the Autofocus auto-function on page 65 .
a. Specify the Desired Defocus
b. Specify the Iterate to defocus value
c. (Optional) Select Use Three Image Method: Yes to include drift correction. EPU will acquire
an extra image to measure drift.
4. Select Execution > Start

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6.2.3 Run the 'Auto-eucentric by stage tilt' auto-function

The Auto-eucentric by stage tilt function is used to set the specimen to eucentric height. This method
is more suited for lower and mid range magnifications.
The Auto-eucentric by stage tilt auto-function is not available on Tundra systems.
The Auto-eucentric by stage tilt auto-function is not used during Automated Acquisition.

To run the Auto-eucentric by stage tilt auto-function, follow the procedure below:
1. Select Auto Functions > Auto-Functions (TEM) > Auto-eucentric by stage tilt

2. Select the Preset Selection > Presets that will be used to execute the auto-function.
The Auto Functions can be executed with any Preset.
3. In the Auto Function Settings ribbon:

a. Specify the Maximum Z Deviation

The default value is suitable for the majority of all experiments and specimens.
b. Specify the Final stage tilt
This is the maximum tilt angle at which the CompuStage Alpha tilt axis wobbles. The default
value is 15 degrees. Larger and smaller values can be entered, depending on the start
conditions.

4. Select Execution > Start

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6.2.4 Run the Autostigmate auto-function

The Autostigmate function uses Thon ring-based aberration corrections. This method corrects
astigmatism with higher accuracy than the Auto Stigmate option in the Autofocus function.

To run the Autostigmate Auto Function, follow the procedure below:

1. Verify that the Autofocus calibration is completed.
If not, see The Autofocus Calibration task on page 75 for instructions.
2. Move the specimen to an area of continuous amorphous carbon foil that is not close to a grid bar.
3. Select Auto Functions > Auto-Functions (TEM) > Autostigmate
4. Select Preset Selection > Presets: Thon Ring

5. Select Execution >Start

EPU acquires an image and then attempts to fit Thon Rings.

6. Wait for the Autostigmate function to complete.
The Autostigmate function does not have a Pause option.
The Autostigmate function is successful when Thon Rings can be properly fitted. If fitting fails, then a
white cross is drawn across the Thon Rings image.

Stigmation is successful Stigmation has failed.

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6.2.5 Run the Autocoma auto-function

Coma-free alignment is important since coma decreases the achievable reconstruction resolution. If
coma is not corrected, a beam shift or beam tilt will result in different objective astigmatism values
across an image.
Note For the best result, run the Autostigmate function before and after running the Autocoma function.

To run the Autocoma Auto Function, follow the procedure below:

1. Run the Autostigmate Auto Function as described in chapter Run the Autostigmate auto-
function on page 69 .
2. Move the specimen to an area of continuous amorphous carbon foil that is not close to a grid bar.
3. Verify that the Objective aperture is accurately centered.
If necessary, manually center the Objective aperture
4. Select Auto Functions > Auto-Functions (TEM) > Autocoma
5. Select Preset Selection > Presets: Thon Ring

6. Select Execution > Start

EPU acquires an image and then attempts to fit Thon Rings.

7. Wait for the Autocoma function to complete.
The Autocoma function does not have a Pause option.
8. Verify that the Objective aperture is still accurately centered.
During execution of the Autocoma, the Objective aperture may be automatically retracted and
inserted again.
If necessary, manually center the Objective aperture
9. Run the Autostigmate Auto Function again, as described in chapter Run the Autostigmate auto-
function on page 69 .

6.2.6 Run the Drift Stabilization auto-function

After a stage move, a certain amount of mechanical and thermal drift occurs.
● Friction in the stage mechanics needs to settle and relax.
● A small amount of heat is generated by the motors, which needs to dissipate and disappear.

To acquire images with the highest possible quality, data acquisition must be postponed until drift has
decreased to an acceptable level. The Drift stabilization task provides a good estimation for the
Delay after Stage Shift value that EPU uses during the Automated Acquisition run.
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● Quantifoil: Delay after Stage Shift is typically used to postpone the next action after a long move,
for example to a new Grid Square.
● Lacey Carbon: Stage Shift Delay is typically used to postpone high quality data acquisition after
the specimen is moved the next Acquisition Area.

The Drift Measurement function acquires a series of images, and then measures the image shift
between consecutive images to calculate the drift speed.
● If the drift speed decreases below a specified threshold value before it times out, then the Drift
stabilization function reports OK.
● If the drift speed times out before it reaches the specified threshold value, then the Drift
stabilization function reports a failure.

To run the Drift stabilization Auto Function, follow the procedure below:

1. Select Auto Functions > Auto-Functions (TEM) > Drift stabilization

2. Select Preset Selection > Presets: Drift Measurement
3. In Auto Function Settings:

a. Specify Max. Remaining Drift (nm/s)

This is the threshold value at which the Drift stabilization function reports a successful result.
b. Specify Time Out (s)
This is the time after which the Drift stabilization function reports a failure.
During the Automated Acquisition run, the Time Out is fixed at 600 seconds.
4. Prepare the CompuStage for a move at a similar speed and across a similar distance as during
the Automated Acquisition run:
a. With the Handpanel > Magnification knob, set the magnification to the same value as the
GridSquare Preset.
The maximum speed of the CompuStage depends on the current magnification. The
magnification that is specified in the Drift Measurement Preset will be not set to the
microscope until the Drift stabilization Auto Function is started.
b. Move the specimen to an area of continuous amorphous carbon foil that is not close to a grid
bar.
c. Select Add

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(Optional) enter a name to identify the stage position.

d. In the TEM User Interface > Stage2 control panel > Set tab, specify an X position and a Y
position at an appropriate distance from the current position.

● To mimic a large move, add 160 mm to both X and Y.

Specimens with a Grid Square width of 80 mm are commonly used. Adding two Grid
Square lengths assures there is a decent margin in the measured stabilization time.
● To mimic a short move, add 10 mm to both X and Y.

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On Lacey Carbon, an Acquisition Area spacing of 5 mm is fairly common, but this

depends on the beam diameter in the Data Acquisition Preset.
e. Select Go To and wait for the CompuStage to complete the move.
5. Select Stage2 control panel > Undo to move back to the previous position.
6. Wait for the move to complete, then immediately select Execution > Start

The Drift stabilization Auto Function will run until either the Time Out is exceeded or the specified
Max. Remaining Drift is reached. The drift measurement results are plotted in a graph. The Max.
Remaining Drift is indicated by the red line.

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6.2.7 Run the Auto Zero-Loss auto-function

The Auto Zero-Loss function maintains the alignment of the energy slit in the filter with the Zero Loss
Peak. This requires that the filter alignment must be accurate and recent.
Note The Auto Zero-Loss function is only available when the system is in EFTEM mode when EPU is started.

Follow the steps below to run the Auto Zero-Loss function:

1. Select Auto Functions > Auto-Functions (TEM) > Auto Zero-Loss

2. Select Preset Selection > Presets: Zero Loss

3. Select Start

4. Wait until the Auto Zero Loss function is completed successfully,

or:
● Select Pause to inspect the progress and intermediate result,
then select Resume to let the Auto Zero Loss function continue.
● Select Stop to abort the Auto Zero Loss function and revert the alignment value.

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6.3 Calibration tasks

6.3.1 The Autofocus Calibration task

6.3.1.1 Prepare for Autofocus Calibration

The Autofocus calibration calibrates the Autofocus function, and measures and corrects astigmatism.
Note The Autofocus calibration result is stored in the Windows Registry. It can only be completed
successfully by Thermo Fisher Scientific engineers, or after logging in with the Supervisor account.

For the best result, the Autofocus calibration requires an area on the specimen:
● Where no ice is present.
● Where only carbon foil with a decent amount of gold particles is visible.
If the currently loaded specimen does not meet all the above requirements, then please exchange
the specimen. For example, use the Combined Test Specimen (Agar S142) that is delivered with the
microscope. This is a holey carbon foil specimen with gold particles and graphitized carbon.
To prepare for the Autofocus calibration, use the Handpanels and the TEM User Interface to perform
the procedure below:

1. Select Right Handpanel > Eucentric Focus

2. Select the Handpanel User Button that is assigned to Reset Defocus
This is usually R2
3. Move the specimen to an area that meets the requirements listed above.
4. Manually set the specimen to eucentric height.
5. If necessary, use the TEM User Interface and the Handpanels to accurately correct for
astigmatism.
6. Accurately focus the specimen.

6.3.1.2 Perform the Autofocus Calibration for the Autofocus Preset

To perform the Autofocus calibration, follow the procedure below:

1. Select Auto Functions > Calibrations: Autofocus

2. Select Preset Selection > Preset: Autofocus

The Autofocus calibration ignores the Defocus value of the selected Preset.

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3. Select Execution > Start

4. Follow the instructions on screen.

EPU can request to manually adjust focus and correct astigmatism.
After completion of a manual adjustment, select Resume

The Autofocus calibration procedure acquires images with negative beam tilt and positive beam
tilt, and calculates their cross-correlation. The encircled bright spot in the Cross-Correlation
image corresponds to the shift between the beam tilt images.

5. In the Cross-Correlation image, estimate the length of the shift.

6. Compare the length of the shift to the width of the beam tilt images.
For accurate calibration results, the shift must be at least 10% of the beam tilt image width.
If the shift in the Cross-Correlation image is less than 10% of the image width:
a. Select Stop to abort the calibration procedure.
b. Estimate the length of the shift in the Cross-Correlation image.
c. Select Preparation > Acquisition and Optics Settings > Preset Selection > Presets:
Autofocus
d. Increase the Optics Settings > Magnification
e. Select Acquisition > Preview
f. Make sure the width of the acquired image is less than 10 times the length of the cross-
correlation shift.
If not, increase Magnification, select Preview and check again.
g. Return to the Auto Functions > Calibrations: Autofocus task and select Execution >
Start again.
7. At the end of the procedure, select Resume to accept and store the calibration results.
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8. Inspect the last Cross-Correlation image. If necessary, zoom in for a more accurate look.
a. Verify that the green arrows form a symmetric cross with the arms at 90° angles and of
approximately the same length. This indicates that there is no significant astigmatism.
If the arms are not at 90°, or are of different lengths, then correct the astigmatism and
perform the Autofocus calibration again.
b. Verify that the red circle marks a clear and bright spot. This indicates that the shift
between the beam tilt images is measured without problems.

6.3.1.3 Perform the Autofocus Calibration for other Presets

The Autofocus function is not only used in the Automated Acquisition run. It can also be executed as
a stand-alone Auto Function with any Preset.
The result of the Autofocus calibration is coupled to the Probe Mode in which it is executed. If both
Probe Modes are used in the Presets, then the Autofocus calibration must be performed with a
Preset that uses Nanoprobe mode and a Preset that uses Microprobe mode. The procedure is
exactly the same, except for the selected Preset.

6.3.1.4 How to improve the Autofocus Calibration result

If the green arrows in the final Cross-correlation image are not at 90 degree angles relative to each
other, the astigmatism has not been corrected completely.

Although the Autofocus function may work well enough for automated data acquisition, it may be
worthwhile to improve the stigmation and the accuracy of the calibration.

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1. Select Auto Functions > Auto Functions (TEM): Autofocus

2. In Auto Function Settings:

a. Specify Desired Defocus: 0.0

b. Select Auto Stigmate: Yes
3. Select Execution > Start to run the Autofocus function.

4. Repeat the calibration procedure.

6.3.2 The Eucentric Correction Calibration task

There are several methods to bring a specimen to eucentric height. Two common methods are:
● Via stage tilt, using the CompuStage Alpha tilt axis wobbler.
This is the most accurate method. It brings the specimen to the mechanically defined eucentric
height.
The stage tilt method is best suited for low to medium magnifications.
● Via beam tilt, using the beam tilt wobbler.
This method is better suited for high magnifications.

At higher magnifications, it can be difficult or even impossible to use the stage tilt method in an
automated routine, especially when the starting position is too far off eucentric height. Two reasons
are:
● The initial shift between images that are acquired at opposite Alpha tilt positions would be so
large that there is little or no overlap, so no meaningful cross-correlation can be calculated.
Without a meaningful cross-correlation, any adjustments to the CompuStage Z-position would be
guesswork.
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● Due to mechanical tolerances, the CompuStage is not infinitely accurate. At low magnifications,
the field of view of the camera is large enough to absorb these mechanical tolerances, especially
after running a backlash correction routine. As the magnification increases, the field of view of
the camera approaches the same order of magnitude as the tolerances of the CompuStage. This
means it is no longer certain that the region of interest on the specimen is returned to the field of
view after using the Alpha tilt wobbler.

The beam tilt method is the preferred method at higher magnifications, because it can use very small
tilt angles to limit initial image shift and does not suffer from mechanical tolerances.
To get the same result from both methods:
● The beam tilt pivot points must be accurately aligned at eucentric focus height.
● Eucentric focus height and the mechanical eucentric height must be exactly the same.

On an accurately aligned system, the stage tilt method and the beam tilt method result in the same Z-
position. In reality, the result of the beam tilt method can have a small offset relative to the
mechanically defined eucentric height. The purpose of the Eucentric correction calibration is to
measure this offset, so that the Auto-eucentric by beam tilt Auto Function can compensate for it.
The Eucentric correction calibration is optional. If there is a noticeable offset between the results of
the stage tilt method and the beam tilt method, then it is also possible to renew the microscope's lens
series alignments.

6.3.2.1 Prepare for the Eucentric Correction Calibration

1. Select Auto Functions > Auto-Functions (TEM) > Auto-eucentric by stage tilt
2. Select Preset Selection : Presets: Hole/EucentricHeight
3. Select Execution > Start

4. Wait until the Auto-eucentric by stage tilt function is completed.

Check the Status panel to monitor progress and intermediate results.
5. Use the Handpanels > Focus knob to accurately focus the specimen.

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6.3.2.2 Perform the Eucentric Correction Calibration

1. Select Auto Functions > Calibrations: Eucentric correction

2. Select Preset Selection > Presets: Hole/EucentricHeight.

3. Select Execution > Start
No manual actions are required during the calibration procedure.
4. Wait until the calibration procedure is completed.
Check the Status panel to monitor progress and intermediate results.

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7 Atlas Tab
An Atlas is an overview of a specimen. An Atlas is a collage of large area images (tiles) to form a
map of the specimen. At the edges of the tiles, small offsets can be visible. This is acceptable.

In EPU, the Atlas is used for:

● Screening:
Estimate the potential to yield high quality data during the Automated Acquisition run. The
success of an Automated Acquisition run depends among others on:
● Good ice quality and thickness, with low to no contamination.
● Good particle distribution.
The magnification of the Atlas Preset allows for an assessment of the ice quality and thickness. It
does not allow for a direct assessment of the presence and distribution of particles, although the
thickness of the ice layer is usually a good indicator for particle density.
● Navigation:
During the preparation and execution of an Automated Acquisition run, the Atlas is used to
identify Grid Squares and to move to their locations.

7.1 Setup Session task

7.1.1 Create a new Atlas Session
To setup a new Atlas session, follow the procedure below:

1. Select the Atlas tab > Session Setup task.

2. Select New Session

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3. Enter a Name for the session.

4. Select the Image format that will be used to store the acquired images.
● MRC: Electron microscopy image format. The MRC format includes an extensive set of
metadata about the microscope and the microscope settings.
See The MRC2014 Image Format on page 197 .
● TIFF: Raster image format: TIFF file format .
5. At Output folder, select [...] and navigate to the target folder.
In the specified folder, EPU creates a sub-folder with the Name of the Atlas session.
Note Do not rename or move the Output folder.

In this folder, EPU stores the following files:

● The Atlas session file ScreeningSession.dm
● For each specimen that is processed in the session: the images and metadata for the Atlas
and for all Tiles.
● On a system with an Autoloader, the Screening task can process multiple specimens.
● On a system without an Autoloader, the Atlas Acquisition task processes only the
specimen that is loaded on the stage.

6. (Optional) Tick Set as default storage folder.

If Set as default storage folder is ticked, then the specified folder is used as the default Output
folder for succeeding Atlas sessions.
7. Select Apply

7.1.2 Load an existing Atlas Session

Note If a specimen has already been (partly) processed in an Automated Acquisition run, then do not load an
existing Screening Session file for a new EPU session with that specimen.

The Screening Session file does not contain data about which Grid Squares have been processed
already in a preceding Automated Acquisition run. Processed areas may be too damaged to yield
new high quality data. Revisiting these areas is therefore not a productive use of system time.

To load an existing Atlas Screening Session, follow the steps below:

1. In the Atlas > Session Setup task, select Session Management > Load Session

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2. Navigate to the Output folder of the desired Screening Session and select the
ScreeningSession.dm file.

Note Do not load an Atlas.dm file.

7.2 Screening task

The Screening functionality acquires an Atlas and categorizes the Grid Squares. For the
categorization, EPU evaluates the size, brightness and contrast of the Grid Squares. These visual
properties loosely correlate to the physical properties that indicate the suitability of a Grid Square for
high quality data acquisition, such as:
● Ice thickness (average brightness of a Grid Square).
● Contaminations (local dark areas in the Grid Square).
● Cracks (sharply defined local bright areas in the Grid Square).
● Empty holes (local bright areas that coincide with foil holes).
On a system with an Autoloader the Atlas acquisition and Grid Square categorization can be done for
multiple specimens from a selection of Slot Positions.

7.2.1 The Screening task for systems with an Autoloader

7.2.1.1 Acquire Atlases for multiple specimens

To acquire Atlases for specimens from multiple Slot Positions, follow the steps below:

1. Select the Atlas > Screening task.

The Task Selection panel displays all Slot Positions in the Autoloader.
● Empty slot:

● Occupied slot:

2. For each occupied slot:

a. (Optional) Tick the Slot Position to schedule it for Atlas acquisition.

It is not possible to select an empty Slot Position.

b. (Optional) Edit the default description

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At the creation of a new Screening session, EPU copies the slot descriptions that are present
the TEM User Interface > Autoloader control panel. After the Screening session is created,
EPU and the TEM User Interface do not synchronize their slot descriptions.
3. In Atlas Settings:

a. If the currently loaded specimen is included in the set of selected Slot Positions,
then select the Start position. The selected Start Position only applies to the Slot Position of
the currently loaded specimen. For all other Slot Positions, Atlas acquisition starts at Close to
center. :
● Close to center
Atlas acquisition starts close to the center of the specimen.
● Close to current
Atlas acquisition starts close to the current stage position.
b. (Optional) Specify the Number of tiles to restrict the area that will be covered in the new
Atlas. The specified Number of tiles applies to all Slot Positions. It is not possible to specify a
different value per Slot Position.

4. Select Acquisition > Start to begin the screening procedure.

EPU prepares the system for Atlas acquisition.

● If the Objective Aperture Mechanism is enabled and inserted, then EPU retracts the aperture
before acquisition starts. After the acquisition is completed, the aperture is inserted again.
● If the Autoloader TMP is not running, then EPU will start it to shorten the exchange time
between Slot Positions. After the Screening acquisition is completed, EPU returns the
Autoloader TMP to its previous status.
5. Wait until all selected Slot Positions are processed.
For each included Slot Position, EPU loads the specimen and acquires an Atlas.
The progress bar displays the progress or the result of the Atlas acquisition.
● Acquiring

The green LED shows that the specimen from this Slot Position is currently loaded on the
CompuStage.
● Acquired

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● Failed

● Aborted

While the screening procedure is running, it is possible to:

● Schedule or unschedule Slot Positions for which Atlas acquisition has not started yet.
● Select any Slot Position to view the Atlas.
To select a Slot Position, click anywhere in the Slot Position, except for the checkbox and the
description.

The Slot Position is highlighted and the Atlas for the specimen appears in the Image Display.

Viewing the Atlas for a specimen does not affect an ongoing acquisition for a different
specimen.

7.2.1.2 Acquire a single Atlas from an unknown specimen on the stage

Sometimes the occupation status of the stage is not known. It is unknown if a specimen is currently
present on the stage, or it is unknown from which Slot Position the specimen on the stage was
loaded. This situation can occur when, for example, the cassette is undocked or when the Autoloader
is not initialized.
Under these circumstances, the Single Atlas functionality allows the acquisition of an Atlas from the
unknown specimen. If no specimen is present, then the Atlas will be blank.
The Single Atlas functionality is available in the Screening task as an additional (virtual) Slot Position,
below the regular Slot Positions. Unlike regular Slot Positions, the Single Atlas slot has its own
Acquire function. The Acquire function is only available when the occupation status of the stage is
unknown. An Atlas that is acquired with the Single Atlas functionality behaves the same and is
treated the same as an Atlas that is acquired for a regular Slot Position in a regular Screening
session.

To acquire an Atlas from an unknown specimen on the stage, follow the steps below:

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1. (Optional) Verify that a specimen is present on the stage:

a. In Preparation > Acquisition and Optics Settings,
select Preset Selection > Presets: Atlas or another Preset with a large illuminated area to
prevent that the specimen is exposed to a converged beam.
b. Select Optics Settings > Set
c. With the TEM User Interface and/or Hand Panels,
insert the FluScreen and open the Column Valves.
2. In Atlas > Screening > Atlas Settings:

a. Select the Start position:

● Close to center
Atlas acquisition starts close to the center of the specimen.
● Close to current
Atlas acquisition starts close to the current stage position.
b. (Optional) Specify the Number of tiles to restrict the area that will be covered in the new
Atlas.
c. At the bottom of the Autoloader Slot Positions, select Single Atlas > Acquire

If the Objective Aperture Mechanism is enabled and inserted, then EPU retracts the aperture
before the acquisition starts. After the acquisition is completed, the aperture is inserted
again.
d. Wait until the Single Atlas acquisition is completed.

7.2.1.3 Reset the Slot Position status

After a Slot Position is processed by the Screening task, it is possible to reset the status back to the
initial value. This means that the following information is erased or returned to its initial value:
● Slot Position status
● Atlas
● Selected Grid Square categories
The Reset Selected function is not available for the Slot Position from which the specimen is
currently loaded on the stage.

To reset the status of a Slot Position:

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1. Select the Slot Position

To select a Slot Position, click anywhere except for the checkbox or the description.

2. Select Reset Selected

The status, Atlas, name and selection Grid Square categories are erased, or are returned to their
initial values.

7.2.1.4 Load a specimen on the stage

1. Select Atlas > Screening to display all Slot Positions in the Autoloader.
2. Select the Slot Position at which the desired specimen is located.
To select a Slot Position, click anywhere except for the checkbox or the description.

The Slot Position is highlighted and the Atlas for the specimen appears in the Image Display.

3. Select Load sample

The status of the selected Slot Position changes to Loading

4. Wait until the loading procedure is completed and the LED is green

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If an Atlas exists for the selected Slot Position, then EPU automatically aligns the existing Atlas with
the specimen on the stage. Because there may be small inaccuracies between the aligned Atlas and
the physical position of the specimen on the stage, EPU displays a warning in the upper-left corner of
the Image Display.

If the specimen is unloaded and reloaded after the Calibrate I0 task is performed, then the I0
reference value measurements during the Automated Acquisition run may be less reliable.

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7.2.2 The Screening task for Tundra systems

7.2.2.1 Acquire an Atlas on a Tundra system

To acquire an Atlas on a Tundra system, follow the instructions below:

1. Select Atlas > Screening

2. In the Atlas settings section:

a. Select the Start position:

● Close to center
Atlas acquisition starts close to the center of the specimen.
● Close to current
Atlas acquisition starts close to the current stage position.
b. (Optional) Specify the Number of tiles to restrict the area that will be covered in the new
Atlas.
3. Select Acquisition > Start

EPU prepares the system for Atlas acquisition.

● If the C2 aperture mechanism is enabled, then EPU first selects the largest C2 aperture.
After the acquisition is completed EPU returns the aperture mechanism to its initial position.
● If the Objective aperture mechanism is enabled and inserted, then EPU first selects the
retracted position. After the acquisition is completed EPU returns the aperture mechanism to
its initial position.
4. Wait until EPU has acquired all images for the Atlas.
The progress bar displays the progress or the result of the Atlas acquisition.
● Acquiring

● Acquired

● Failed

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● Aborted

If the image quality is not good enough, or if stitching the tiles does not result in a good Atlas:
a. Select Stop
This can be done at any time during the Atlas acquisition and may take a few seconds.
b. Select the Preparation > Acquisition and Optics Settings task,
then select Preset Selection > Presets: Atlas
c. Adjust the Optics Settings and or Camera Settings
d. Select Atlas > Screening > Start again to acquire a new Atlas.
All tiles that were acquired before adjusting the Atlas Preset are discarded.

7.2.2.2 Exchange the specimen on a Tundra system

The procedure to unload the current specimen, and to load a new specimen is as follows:

1. Select Prepare Exchange

EPU closes the Column Valves.

2. Perform the exchange procedure for Tundra with the Transfer Device and the Cryo Loading
Station (CLS).
After the exchange procedure is started on the CLS, EPU locks the Column Valves and the stage
on the Tundra, and it is not possible to acquire images.
3. Insert the Transfer Device into the Tundra system.
4. In EPU > Atlas > Screening, select Load Sample
5. Return the Transfer Device to the CLS.
EPU unlocks the Column Valves and the stage.

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7.2.3 Acquire an Atlas for a single specimen on a system with removable holders
(side entry)
To acquire an Atlas on a system with a removable holder (a so-called side entry system), follow the
instructions below:

1. Select Atlas > Screening

2. In the Atlas settings section:

a. Select the Start position:

● Close to center
Atlas acquisition starts close to the center of the specimen.
● Close to current
Atlas acquisition starts close to the current stage position.
b. (Optional) Specify the Number of tiles to restrict the area that will be covered in the new
Atlas.
3. Select Acquisition > Start

EPU prepares the system for Atlas acquisition.

● If the C2 aperture mechanism is enabled, then EPU first selects the largest C2 aperture.
After the acquisition is completed EPU returns the aperture mechanism to its initial position.
● If the Objective aperture mechanism is enabled and inserted, then EPU first selects the
retracted position. After the acquisition is completed EPU returns the aperture mechanism to
its initial position.
4. Wait until EPU has acquired all images for the Atlas.
The progress bar displays the progress or the result of the Atlas acquisition.
● Acquiring

● Acquired

● Failed

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● Aborted

If the image quality is not good enough, or if stitching the tiles does not result in a good Atlas:
a. Select Stop
This can be done at any time during the Atlas acquisition and may take a few seconds.
b. Select the Preparation > Acquisition and Optics Settings task,
then select Preset Selection > Presets: Atlas
c. Adjust the Optics Settings and or Camera Settings
d. Select Atlas > Screening > Start again to acquire a new Atlas.
All tiles that were acquired before adjusting the Atlas Preset are discarded.
Note If the Optics Settings parameters of the Atlas Preset are changed, and the system has a Cryobox, then
the Atlas Optics Alignment must be performed again.

7.2.4 Pre-select the Grid Squares for the Automated Acquisition run
The Screening functionality assesses the quality of the individual Grid Squares. Grid Squares with
similar characteristics are assigned to the same category. Above the Atlas, a checkbox is visible for
each category. Each checkbox shows the number of Grid Squares in that category.

If present, the Broken Grid Squares category is always red. The other Grid Square categories have
randomly assigned colors. These colors do not indicate the suitability of the Grid Square category for
high quality data acquisition. The colors are help to mark the Grid Squares in each category.
EPU assumes that less than 50% of the Grid Squares is broken. If more than 50% of the Grid
Squares would be marked as broken, than the Broken category is omitted and all Grid Squares are
assigned to the non-broken categories.
In the Screening task, it is possible to make a pre-selection of suitable Grid Squares for the
Automated Acquisition run. If the Atlas from the Screening Session is used for the Automated
Acquisition run, then the EPU > Square Selection task loads the Grid Squares in the selected Grid
Square categories as the initial selection.

To pre-select Grid Squares for the EPU > Square Selection task:
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1. If the system has an Autoloader, then select a Slot Position with status Acquired.
To select a Slot Position, left-click anywhere, except for the checkbox or the description.

The Atlas for the specimen appears in the Image Display.

2. In the Image Display, above the Atlas:
● Tick the Grid Square categories that appear suitable for high quality data acquisition.
● Clear the Grid Square categories that do not appear suitable for high quality data
acquisition.

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8 EPU Tab
The EPU tab provides a set of tasks to start, prepare and execute Automated Acquisition sessions.
This chapter describes the functions and actions for an EPU experiment with a single specimen. The
single specimen workflow is supported on all system configurations.
If the system has an Autoloader and the EPU Multigrid license is registered and activated, then the
Multigrid functionality is available. With the Multigrid functionality it is possible to first prepare multiple
sessions, and then start the Automated Acquisition for the prepared Sessions in the queue. The
preparation tasks for each specimen in a Multigrid workflow are mostly the same as for the single
specimen workflow.
For instructions for the Multigrid workflow, see: The EPU Multigrid Option on page 161 .

The set of available tasks depends on the Grid type: Holey carbon or Lacey carbon.
The actions in each task depend on the Session type: Automated or Manual target selection.

Phase Task Session type

Automated Manual

Start Session The Session Creation task is generic for both Grid types
Creation and for both Session types.

Preparation Session Setup The Session Setup task is generic for both Grid types
and for both Session types.

Square The Square Selection task is generic for both Grid types
Selection and for both Session types.

Area Selection Once, Each individual selected Grid Square:

(Lacey) valid for all selected Grid Squares: ● Define and generate
● Define and generate an Acquisition Area pattern.
an Acquisition Area pattern. ● Set the filters for initial selection.
● Set the filters. ● Customize the selection manually.

Hole Selection Once, Each individual selected Grid Square:

(Holey) valid for all selected Grid Squares: ● Specify the Foil Hole diameter
● Specify the Foil Hole diameter and spacing.
and spacing. ● Find the Foil Holes.
● Find the Foil Holes. ● Set the filters for initial selection.
● Set the filters. ● Customize the selection manually.

Template How to define a Foil Hole Template does not depend on the Session type.
Definition
(Holey)

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Phase Task Session type

Automated Manual

Template How to validate the Foil Hole Template does not depend on the Session type.
Execution
(Holey)

Execution Automated The Automated Acquisition task is generic for both Grid types
Acquisition and for both Session types.

In a Manual session, the Area Selection or Hole Selection tasks offer Automated Preparation
functions to apply the manual selection settings from a prepared Grid Square to other Grid Squares.
This saves a large amount of time and effort that would otherwise be spent on manually repeating
the same selection settings for multiple Grid Squares.

8.1 Session Creation task

8.1.1 The EPU Session Preferences file
EPU offers the possibility store the settings and calibration results from a session in an EPU Session
Preferences file, so that they can be re-used to create a new session. The use of an EPU Session
Preferences file has the following consequences for the EPU workflow:
● The order in which the tasks must be completed is different than the regular EPU workflow.
● For many tasks it is sufficient to only verify the loaded settings.
The recommended workflow sequence for a New from preferences session is as follows:
1. Create an EPU Session with the New from preferences function.
2. Verify the Acquisition and Optics Presets.
Note If one or more presets are adjusted, then the related calibrations and other settings need to be
adjusted as well.

3. If no Atlas is available for the currently loaded specimen, then acquire an Atlas.
4. Continue with the tasks in the EPU tab.
For some tasks, the settings are pre-loaded from the EPU Session Preferences file.
The EPU Session Preferences file can contain the following values:

Tab Task Included Values

Preparation Acquisition and Optics Settings All Presets,

same as an Preparations > Preset Selection >
Export.

Atlas Optics Alignment All values

Calibrate Image Shifts All values

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Tab Task Included Values

Calibrate I0 All values

Activate Phase Plate None

Auto Functions Calibrations: None

Autofocus

Calibrations: None
Eucentric correction

Auto-Functions (TEM): Autofocus None

Auto-Functions (TEM): Auto-eucentric by None

beam tilt

Auto-Functions (TEM): Auto-eucentric by None

stage tilt

Auto-Functions (TEM): Autostigmate None

Auto-Functions (TEM): Autocoma None

Atlas Session Setup None

Atlas Acquisition None

Screening None

EPU Session Setup All values, except:

● Session name and Description
● Athena settings
● Output folder
● Email settings

Square Selection None

Area Selection All values and filter selections.

Hole Selection All values and filter selections.

Template Definition All values

Template Execution None

Automated Acquisition Phase Plate selections and values .

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8.1.2 Create a new EPU Session without an EPU Session Preferences file
To setup a new Automated Acquisition session without using a Session Preferences file, follow the
procedure below:

1. Select EPU > Session Creation > New Session

2. Select the preferred EPU Session creation method:

● Yes: create a new EPU Session that uses mostly the same settings and values as the
current EPU Session. For an overview of the copied settings and values, see The EPU
Session Preferences file on page 95 .
● No: create a new EPU Session without pre-selected settings and values.
● Cancel: no new EPU Session is created. The current EPU Session stays as it is.

EPU automatically moves on to the Session Setup task.

For instructions, see: Session Setup task on page 98 .

8.1.3 Create a new Session from an EPU Session Preferences file

To setup a new Automated Acquisition session based on a EPU Session Preferences file, follow the
procedure below.

1. Select EPU > Session creation > New from preferences

2. Browse to the desired *.epuSessionPreferences file and select Open

EPU automatically moves on to the Session Setup task.

For instructions, see: Session Setup task on page 98 .

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8.2 Session Setup task

In an EPU Multigrid workflow, the Session Setup task is named Session Queue.

8.2.1 Description of the Session Setup options

8.2.1.1 The Acquisition Mode for Quantifoil specimens

EPU has two methods to locate and center a position on the specimen:
● Optical centering with Image/Beam Shift:
The specimen remains stationary while the beam shifts to illuminate the feature of interest and
project it in the center of the camera field of view.
Although this is very fast, Image/Beam Shift may induce coma under sub-optimal conditions. The
amount of coma is typically not significant for small offsets, but for larger offsets this may reduce
the image quality.
● Mechanical centering with Stage Shift:
The beam remains stationary while the specimen moves, so that the feature of interest is located
in the center of the camera field of view.
Although the aberrations that are associated with Image/Beam Shift are absent, mechanical
centering is considerably slower than optical centering.

If certain conditions are met, then EPU can use optical centering also for relatively large offsets, and
thus replace most Stage Shifts by Image/Beam Shifts.
These conditions are:
● Aberration Free Image Shift (AFIS) is present, and is calibrated by a Thermo Fisher Scientific
engineer.
● A wide usable Image/Beam Shift range is available:
● The system does not have an Image Corrector.
An Image Corrector restricts the usable Image Shift range.
● Phase Plates are not used.
The use of Phase Plates is not compatible with the beam tilt angles that are needed for coma
correction.

For Lacey Carbon specimens, EPU automatically determines when to use Stage Shift and when to
use Image/Beam Shift to optimize accuracy and throughput.
For Quantifoil specimens, EPU > Session Setup offers a manual selection between two Acquisition
Mode options:
● Accurate Hole Centering
● Faster Acquisition
The Acquisition Modes use different methods to center and visit the Foil Holes for data acquisition.
The execution of the Foil Hole Template is the same for both Acquisition Modes.

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Accurate Hole Centering

In Accurate Hole Centering mode, EPU processes each individual Foil Hole independently from its
surrounding Foil Holes. EPU first locates the individual Foil Hole, and then accurately centers it in the
camera field of view. This procedure requires magnification changes and lens normalizations, and
uses small Stage Shifts to mechanically adjust the position of the specimen.
The Accurate Hole Centering procedure does not use Image/Beam Shift to center a Foil Hole. If the
Template Areas are within reach of the Maximum Image Shift, then Image/Beam Shift may still be
used to center the Drift Measurement, Autofocus and Acquisition Areas during execution of the Foil
Hole Template

Faster Acquisition
In Faster Acquisition mode, EPU processes the Foil Holes in groups.
The Faster Acquisition procedure uses Stage Shifts to center multiple Foil Holes at once as a group.
This reduces the number of Stage Shifts, magnification changes and lens normalizations.
● If the conditions for Faster Acquisition mode are fulfilled,
then EPU uses Image/Beam Shifts to visit the individual Foil Holes within the centered group.
This reduces the number of Stage Shifts even further.
● If the conditions for Faster Acquisition mode are not fulfilled,
then EPU uses Stage Shifts to visit the individual Foil Holes in the centered group.
See The Acquisition Mode for Quantifoil specimens on page 98 for the conditions for Faster
Acquisition mode.
If Image/Beam Shift is used to visit the individual Foil Holes, then the stage remains stationary. As a
result, the metadata of all acquired images in the centered group contains the same stage
coordinates. To separate and group the images per Foil Hole or per Acquisition Area, the file names
of the acquired images must be processed.
The format of the file names is:
FoilHole_[Hole ID]_Data_[Acquisition Area ID]_[date]_[time].mrc
For example: in FoilHole_31545690_Data_31547881_31547882_20210601_0819.mrc
● [Hole ID] is 31545690
● [Acquisition Area ID] is 31547881_31547882
● [date] is 20210601 in yyyymmdd format
● [time] is 0819 in 24-hour hhmm format

In Faster Acquisition mode, the Automated Acquisition run starts with an automatic calibration to
maximize centering accuracy. This calibration takes 2–3 minutes. In a longer automated run, the
calibration duration is easily recovered by the much faster Foil Hole processing method. For short
runs that acquire images from only a few Foil Holes, the Accurate Hole Centering is often faster.

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8.2.1.2 The pixel format for Gatan Dose Fraction images

The pixel format of the images that are acquired with the Gatan K2 and K3 cameras depends on the
Acquisition and Optics Presets > Advanced Camera Settings > Mode and the EPU > Session Setup
> Dose fraction output format. The pixel format has a direct impact on the image file sizes.

8.2.1.2.1 MRC image pixel format for Gatan K2 and K3 cameras

The pixel format specification below applies to:
● Gatan K3 cameras.
● Gatan K2 cameras on a system with Titan 3.6 or later, or Talos 2.6 or later.

Advanced Camera Settings > Dose fraction output format

Mode
Gain Non-Gain Non-Gain
Normalized Normalized Normalized Packed

Counted 8-bit unsigned integer 8-bit unsigned integer 4-bit unsigned integer
+ gain reference image + gain reference image
per Grid Square per Grid Ssquare

Counted / 8-bit unsigned integer 8-bit unsigned integer 4-bit unsigned integer
Super Resolution + gain reference image + gain reference image
per Grid Square per Grid Square

The pixel format specification below applies to Gatan K2 cameras on a system with Titan 3.5 or
earlier, or Talos 2.5 or earlier.

Advanced Camera Settings > Mode Dose fraction output format

Gain Non-Gain Non-Gain

Normalized Normalized Normalized Packed

Linear 32-bit float 16-bit unsigned integer not supported

+ dark reference image(s)
+ gain reference image(s)

Counted 32-bit float 16-bit unsigned integer 8-bit unsigned integer

+ gain reference image + gain reference image

Counted / 32-bit float 8-bit unsigned integer 4-bit unsigned integer

Super Resolution + gain reference image + gain reference image

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8.2.1.2.2 TIFF image pixel format for Gatan K2 and K3 cameras

The pixel format specification below applies to:
● Gatan K3 cameras
● Gatan K2 cameras on a system with Titan 3.6 or later, or Talos 2.6 or later.

Advanced Camera Dose fraction output format

Settings > Mode
Gain Non-Gain
Normalized Normalized

Counted 8-bit unsigned integer 8-bit unsigned integer

Counted / 8-bit unsigned integer 8-bit unsigned integer

Super Resolution

All Gatan Dose Fraction TIFF images use lossless LZW file compression.

8.2.1.3 The Athena settings

If the Microscope PC has a connection to the Athena software on the Data Management Platform
(DMP) Server, then EPU can store the acquired data in an Athena Dataset. Optionally, the Athena
Dataset can be related to a Grid, but this is not required for a successfull EPU session. To select the
Athena Dataset and to optionally link the Dataset to a Grid, the Session Setup > Athena Settings
must be defined.

The Athena Settings section is only visible when the Athena client software is installed on the
Microscope PC. If the Athena Settings are disabled, then first log in on Thermo Scientific Athena.
For instructions, see Log in on Thermo Scientific Athena on page 7 . For a successul Automated
Acquisition run it is not required to select an Athena Dataset.

Note It is not possible to change the Athena Settings during the Automated Acquisition run.

To select an Athena Dataset in the EPU, perform the actions below in the Session Setup task.

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1. In the Athena settings section, select Select

The Select Dataset dialog appears.

2. Select the Project for which the EPU session is executed.

The Experiments that are available in the Project appear.
3. Select the Experiment > select the Workflow > select the Step

4. If available, select the target Dataset

5. If the target Dataset is not available,
then select Add dataset and enter the new Dataset Name and optional Description

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Note It is not possible to change the Dataset selection for data that is already acquired.

If the Athena Settings are changed after data has already been acquired, then the new Athena
Settings apply only to the data that is acquired from that moment onwards.
6. (Optionally) Create a link between the Dataset and a specimen:
a. Select Select Grid

b. Select the Sample > select the Grid for which a relation with the Dataset must be created.
It is not possible to add a new Grid in EPU. If the desired Grid is not available,
then select Portal to open the Athena web portal to add a Grid to the Dataset.
7. If the Dataset is incorrectly linked to a Grid,
then select Reset Grid to remove the link.
Removal of the link does not affect the execution of the data acquisition and does not erase any
data that already been acquired.
8. Close the Select Dataset dialog(s).
The Session Setup form displays:
● The Workflow to which the select Dataset belongs.
● The selected Dataset.
● If selected, the Grid to which the selected Dataset is linked.
9. (Optional) If available, tick Enable Quality Monitor to use the EPU Quality Monitor (EQM)
functionality.

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8.2.2 Set up an EPU Session

1. Select the EPU > Session Setup task.
The Session form appears in the Task Execution panel.

If the EPU Session is created from the current EPU Session or from an EPU Session
Preferences file, then several values in the Sesion Setup form may be pre-loaded. All pre-loaded
values can be adjusted.
See The EPU Session Preferences file on page 95 for:
● An overview of the supported values and results.
● The changes in the EPU Workflow when using an EPU Session Preferences file.

2. In the General session settings section:

a. Enter a Session name
b. Select the Grid type
● Holey carbon: all foil holes are circular and have an identical diameter and spacing,
for example Quantifoil.
● Lacey carbon: the foil holes have irregular shapes and dimensions.
c. Select the Session Type
The Type setting defines how suitable areas are selected for Data Acquisition.
● Automated: suitable areas are automatically detected, assessed and selected by filters
and algorithms. For most sessions, the Automated session type results in the most
efficient acquisition of high quality data.

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● Manual: suitable areas must be hand-picked. Use this mode when the number of areas
from which high quality data must (or can) be acquired is very limited.
The Manual option is not available for EPU Sessions in an EPU Multigrid Queue.
It is not possible to use a mix of automated and manual area selection in a single Automated
Acquisition run. It is possible though to follow-up an automated session by a manual session
or vice versa, as long as the specimen remains loaded and the Atlas is not replaced or
renewed.
d. Select the Acquisition Mode
For a description of the available options, see The Acquisition Mode for Quantifoil specimens
on page 98 .
e. Tick or clear Use Phase Plates
Phase plates can only be used in a Session type: Manual session.
3. If the Athena Settings section is available, then optionally select a Workflow, Dataset and Grid.
See: The Athena settings on page 101 .
4. In the Output section:
a. Select the Image Format.
● MRC: Electron microscopy image format.
The MRC format includes an extensive set of meta-data about the image, the
microscope settings, and the used detector.
For a description of the Thermo Scientific meta-data, see The MRC2014 Image Format
on page 197 .
For a description of the generic meta-data, see MRC/CCP4 file format for images and
volumes .
● TIFF: Generic raster image format.
A description of the format can be found on the web, e.g. at TIFF file format .
Most 3D reconstruction software supports the MRC2014 format. If your reconstruction
software does not support the MRC format, select the TIFF format.
Note When using the Dose Fractions functionality that is available for Direct Detection cameras, the
TIFF image format cannot be used.

b. For systems with a Gatan K2 or K3 camera only: select the Dose fractions output format.
For a description of the options, see: The pixel format for Gatan Dose Fraction images on
page 100 .
c. At Output folder, select [...] and navigate to the target output folder.
All session data and acquired images are stored in the selected folder, except for Dose
Fraction images. Dose Fraction images are saved on the Storage Server (Falcon 3EC and
Falcon 4) or on the Gatan PC (Gatan K2 and K3).
Note Do not rename or move the EPU Session Output folder.

EPU loads the active EPU session at startup. If the folder path is changed, EPU cannot find
the session data. It is not possible to manually load an EPU session from a different location.
d. (Optional) Tick Default folder.
When ticked, the specified Output folder is the default location for succeeding sessions.
5. (Optional) In the Email section:

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a. Specify the Recipients

Addresses must be separated by commas, semi-colons or spaces.
b. (Optional) Select Test to confirm if the entered email addresses are valid and if the email
services are configured correctly.
The notification emails are sent via the email service components that are installed on the
Microscope PC and on the Support PC. If the test email is not delivered to at least one of the
specified addresses, contact Thermo Fisher Scientific.
EPU will send an email:
● When the Automated Acquisition run is completed.
● When the Automated Acquisition run terminates automatically before it is completed,
for example if the column valves are closed due to vacuum issues, or if there is no more
available disk space on the Storage Server or the Gatan PC.
If the Automated Acquisition run is stopped manually in the EPU user interface, then EPU will not
send emails.
The email settings can be updated after the Automated Acquisition run has started.
6. Select Apply

8.2.3 Limitations to loading or acquiring an Atlas after Session Setup

On a system without an Autoloader, EPU loads the most recently acquired Atlas as the basis for the
Automated Acquisition run. On a system with an Autoloader, the Atlas for the currently loaded
specimen is loaded from the Screening Session.
As long as no data has been acquired yet, it is possible to acquire a new Atlas or to load a different
Atlas Session file for the currently loaded specimen.
Note If a specimen has already been (partly) processed in an Automated Acquisition run, then do not load an
existing Screening Session file for a new EPU session with that specimen.

The Screening Session file does not contain data about which Grid Squares have been processed
already in a preceding Automated Acquisition run. Processed areas may be too damaged to yield
new high quality data. Revisiting these areas is therefore not a productive use of system time.
Once data acquisitions have taken place, the Automated Acquisition session relies on the underlying
Atlas to keep track of the processing progress and status and to maintain data consistency.

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8.3 Square Selection task

The purpose of the Square Selection task is to select only those Grid Squares that meet the
requirements for high quality data acquisition:
● The specimen area is thin enough to be electron translucent.
● The specimen area contains no broken carbon foil.
● For Cryo-EM samples:
● The holes in the carbon foil are filled with vitreous ice.
● The Grid Square contains no contamination.

The Square Selection task is the same for lacey and holey carbon grids.

8.3.1 Select Grid Squares for high quality data acquisition

Grid Square selection is a manual task. To select the Grid Squares that meet the requirements for
high quality data acquisition, follow the procedure below.

1. Select the EPU > Square Selection

The Atlas for the loaded specimen appears in the Task Execution panel.

In the Atlas image:

● Atlas Tiles have a yellow outline. The Atlas Tiles typically overlap a bit.
● Selected Grid Squares have a green outline and shading.
● If the Atlas is loaded from the Screening Session, then the default selection includes only
the Grid Squares of the selected categories.
For an explanation of the Grid Square categories, see Pre-select the Grid Squares for
the Automated Acquisition run on page 92 .
● If a new Atlas is acquired, the default selection includes all detected Grid Squares.

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● When hovering over a Grid Square, a tooltip displays additional information.

● If the specimen has been unloaded and reloaded after the Atlas was acquired, then EPU
automatically re-aligns the Atlas with the physical position and orientation of the specimen on
the stage. Because there may still be small inaccuracies, a warning is displayed in the upper-
left corner of the image display.

2. (Optional) Select Unselect All to start with an empty selection.

3. Customize the selection of Grid Squares from which data must be acquired.
● Use your own judgment to manually select or unselect individual Grid Squares.
Blue and orange Grid Squares are already (partly) processed in an Automated Acquisition
run. They cannot be selected or unselected anymore.
● Use the context menu:
Right-click on a Grid Square, then select Select or Unselect from the context menu.

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● Use the keyboard and mouse:

To select one or more Grid Squares, hold the Control key and select one or more gray
Grid Squares.
To unselect one or more Grid Squares, hold the Shift key and select one or more green
Grid Squares.

● Use the suggestions from the Grid Square Suggestion function.

Grid Square Suggestion is a machine learning algorithm that identifies Grid Squares that are
not selected yet, but that have similar properties as the existing selection. These best-match
Grid Squares are marked with a dotted green-and-white border.

To select a suggested Grid Square, either:

● Select it manually as described above.
● Select Smart Extend to add the best matching Grid Square from the suggested set.
The Grid Square Suggestion and Smart Extend functions are only available if the existing
selection includes at least one Grid Square, and there is at least one detected Grid Square
available that is not broken and not selected yet. For a good result, start with a selection of at
least three Grid Squares. A larger baseline will result in a better match.
4. (Optional) Create and select a new Grid Square on a location of choice.
a. Right click on the center of the new Grid Square location.
If necessary, zoom in for better accuracy.
b. Select Add new Grid Square here
EPU creates and selects a new Grid Square with the following properties:
● The center of the new Grid Square is at the position of the mouse cursor.
● The orientation of the new Grid Square is perpendicular to the image display.
The new Grid Square will not have the same orientation as the detected Grid Squares.
● The dimensions of the new Grid Square are according to Mesh-300 specifications.
If the newly created Grid Square overlaps with an already selected Grid Square, then both Grid
Squares remain selected.

8.3.2 Zoom in on an individual Atlas Tile image

In the Atlas overview image it can be difficult to assess the quality of a single Grid Square. For a
higher resolution image of an Atlas Tile:
Right-click on an Atlas Tile.

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1. Select Open tile.

2. Add individual Grid Squares to the selection, and/or remove individual Grid Squares from the
selection.
3. Right-click in the Atlas Tile image.
4. Select Close tile to return to the Atlas overview image again.

8.3.3 Change the processing order of the Grid Squares

The order in which the selected Grid Squares must be processed during the Automated Acquisition
run can be re-arranged. This makes it possible to prioritize Grid Squares with the best chance of
large quantities of high quality data. It is not possible to change the processing order during the
Automated Acquisition run or while the run is paused.
To view and change the processing order, follow the instructions below:

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1. In the Processing Order section of the Ribbon Bar, select Show.

The processing order appears in the Atlas overview image.

The processing order is determined on Atlas level, not per individual Atlas Tile.
2. Select Processing Order > Change
3. In the image display, select the Grid Square that must be processed first.
The selected Grid Square is moved to the number 1 position in the processing order. The other
Grid Squares in the Atlas automatically move one position down in the processing order.
4. Select the Grid Square that must be processed next.
This Grid Square is moved to the number 2 position in the overall processing order.
5. Continue selecting Grid Squares in the desired processing order.
After the Grid Squares with the best chances of high quality data yield are re-assigned to the
start o fthe processing order, then it is not necessary to re-assign all remaining Grid Squares.
6. Select Change again to leave the processing order change mode.

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8.4 Area Selection task for Lacey Carbon specimens

8.4.1 Define the Acquisition Area Pattern and Filter Settings for an Automated
Selection Session
If the Session type is Automated, then EPU uses the built-in filters and algorithms to select suitable
Acquisition Areas.
To configure the filter settings, follow the instructions below. This procedure must be executed for
only one of the selected Grid Squares. During the Automated Acquisition run, EPU uses the same
filter settings for all selected Grid Squares.

1. Acquire a representative Grid Square image:

a. Select the EPU tab > Square Selection task.
b. In the Image Display, identify a Grid Square that appears representative for the condition of
the specimen.
c. Right-click on the identified Grid Square and select Select Areas

EPU switches to the Area Selection task for the selected Grid Square.
d. If the specimen is already at eucentric height, or if there is a different reason to skip the Auto
Eucentric function, then select Acquire to only acquire a new Grid Square image.

If the specimen is not already at eucentric height,

then select Auto Eucentric

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If necessary, EPU first moves the specimen, so that the center of the selected Grid Square is
in the center of the field of view.
While the Auto Eucentric function runs, the Auto Eucentric button changes to Stop Auto
Eucentric. If selected, the Auto Eucentric function is aborted and the specimen returns to its
previous Z position.
The Auto Eucentric function includes the acquisition of a new Grid Square image.
2. If the particles all have a similar orientation in the ice layer:
a. Adjust the A-tilt angle of the specimen.
A different A-tilt axis can be applied for each Grid Square to acquire images from various
incident angles. EPU stores the A-tilt angle of the Grid Square during Hole Selection, and
applies the A-tilt angle during Automated Acquisition at the same Grid Square. This typically
results in a higher quality 3D reconstruction.
b. Select Acquire to update the Grid Square image.
On Tundra systems it is not possible to adjust the A-tilt angle of the stage.
3. (Optional) Modify the Defocus List
The Defocus List is applied to all Acquisition Areas on the specimen.
For instructions, see Define the Defocus List for a Lacey Carbon specimen on page 125 .
4. Generate a pattern of Acquisition Areas:
a. Specify the Spacing value.
This is the Center-to-Center distance of adjacent Acquisition Areas.

The Spacing value can not be smaller than the diameter of the Acquisition Area.
● On microscopes with a Three Lens Condenser system, the size of the Acquisition Area
is defined by the Illuminated Area which is specified in the Data Acquisition Preset.
● On microscopes with a Two Lens Condenser system, the Acquisition Area is defined as
the smallest circle that fully covers the camera field of view at the Data Acquisition
Preset magnification. The beam is typically slightly wider than the camera field of view.
b. Select Generate Pattern.
EPU draws hexagonal pattern of Acquisition Areas across the entire Grid Square image.

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Depending on the microscope type and configuration, the pattern contains circles and
rectangles. Rectangles mark the field of view of the camera. Circles mark the beam
diameter.
● On microscopes with a Three Lens Condenser system, the size of the Acquisition Area
is defined by the Illuminated Area which is specified in the Data Acquisition Preset.
● On microscopes with a Two Lens Condenser system, the Acquisition Area is defined as
the smallest circle that fully covers the camera field of view at the Data Acquisition
Preset magnification.
c. If the Acquisition Areas are too close to each other or too far apart,
then adjust Spacing and select Generate Pattern again.
5. Set or adjust the Ice Quality Filter:
a. Select Preset Ice Filter to reset the Ice Quality Filter boundaries.

b. In the Filter Ice Quality histogram:

● Drag the red lower boundary marker to exclude Acquisition Areas that are too dark.
● Drag the red upper boundary marker to exclude Acquisition Areas that are too bright.
After every change, the Ice Quality filter updates the selection of suitable Acquisition Areas
immediately. Depending on the number of areas in the Grid Square image, this may take
some time.

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6. (Optional) Select Remove Areas Close To Grid Bar to exclude Acquisition Areas near the edge
of the Grid Square.

If the result of the Remove Areas Close to Grid Bar filter is not satisfactory, then select the filter
button again to de-activate it. This will automatically generate a new pattern and apply the Ice
Quality filter.

8.4.2 Handpick the Acquisition Areas for a Manual Selection Session

If Session type is Manual, then it is possible adjust the Acquisition Area pattern and filter settings for
each individual selected Grid Square. With these settings, the Acquisition Areas are selected from
which data will be acquired. For each selected Acquisition Area, the X, Y and Z position of the area
center is stored. This selection can be customized in each individual Grid Square. During the
Automated Acquisition run, EPU does not re-evaluate the suitability of the selected Acquisition
Areas.

After the suitable Grid Squares are selected in the Square Selection task, the procedure to handpick
the target Acquisition Areas is as follows:
1. Select the Grid Squares from which data will be acquired:
a. Select the Square Selection task.
b. Select Processing Order > Show

c. (Optional) Change the Grid Square processing order.

For instructions, see Change the processing order of the Grid Squares on page 110 .
d. Right-click on Grid Square 1 and select Select Areas

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EPU switches to the Area Selection task for the selected Grid Square. The lower-left corner
of the Area Selection Image Display displays the processing order number of the Grid
Square and Z-position of the selected Grid Square.
2. Prepare the first selected Grid Square:
a. Select the Area Selection task.
If a Grid Square image is available, then the image display shows the selected Grid Square.
The lower-left corner of the image display shows the Stage Z-position for the displayed Grid
Square.
● White: the Eucentric Height value is not determined yet.
● Green: the Eucentric Height value is determined.
b. If the specimen is already at eucentric height, or if there is a different reason to skip the Auto
Eucentric function, then select Acquire to only acquire a new Grid Square image.

If the specimen is not already at eucentric height,

then select Auto Eucentric

If necessary, EPU first moves the specimen, so that the center of the selected Grid Square is
in the center of the field of view.
While the Auto Eucentric function runs, the Auto Eucentric button changes to Stop Auto
Eucentric. If selected, the Auto Eucentric function is aborted and the specimen returns to its
previous Z position.
The Auto Eucentric function includes the acquisition of a new Grid Square image.
c. Define the Acquisition Area pattern and filter settings.

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For instructions, see Define the Acquisition Area Pattern and Filter Settings for a Manual
Selection Session on page 118 .
d. (Optional) Customize the selection of target Acquisition Areas.
For instructions, see Customize the selection of Acquisition Areas on page 121 .
3. Prepare the other selected Grid Squares:
a. (Optional) Use the Automated Preparation functions to apply the Acquisition Area pattern
and filter settings of the current Grid Square to all other selected Grid Squares.
For instructions, see Automatically create a Manual Selection in all selected Grid Squares on
page 120, Automatically create a Manual Selection in all selected Grid Squares on page
135 .
b. Select Navigate > Next Square to move to the next Grid Square.

In the Square Selection image display, the white border moves to the next Grid Square. The
specimen does not move until Auto Eucentric or Acquire is selected.
c. If desired or necessary:
● Define or adjust the Acquisition Area pattern and filter settings.
For instructions, see Define the Acquisition Area Pattern and Filter Settings for a Manual
Selection Session on page 118 .
● Customize the selection of target Acquisition Areas.
For instructions, see Customize the selection of Acquisition Areas on page 121 .
4. (Optional) Inspect or adjust the Acquisition Area selection for a prepared Grid Square:
Either:
● Select Navigate > Previous Square until the desired Grid Square is reached.
In the Square Selection image display, the white border moves to the previous Grid Square.
The specimen does not move until Auto Eucentric or Acquire is selected.
● Select the Square Selection task
> Right-click on the desired Grid Square
> Select Select Areas
EPU displays the Grid Square image with the Acquisition Area selection for the selected Grid
Square.
Note Unless the current selection of Acquisition Areas must be cleared:
• Do not select Acquire
• Do not select Generate
• If Remove Areas Close to Grid Bar is active, then do not select Remove Areas Close to Grid Bar

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8.4.2.1 Define the Acquisition Area Pattern and Filter Settings for a Manual Selection Session

1. (Optional) Modify the Defocus List

The Defocus List is applied to all Acquisition Areas on the specimen.
For instructions, see Define the Defocus List for a Lacey Carbon specimen on page 125 .
2. Generate a pattern of Acquisition Areas:
a. Specify the Spacing value.
This is the Center-to-Center distance of adjacent Acquisition Areas.

The Spacing value can not be smaller than the diameter of the Acquisition Area.
● On microscopes with a Three Lens Condenser system, the size of the Acquisition Area
is defined by the Illuminated Area which is specified in the Data Acquisition Preset.
● On microscopes with a Two Lens Condenser system, the Acquisition Area is defined as
the smallest circle that fully covers the camera field of view at the Data Acquisition
Preset magnification. The beam is typically slightly wider than the camera field of view.
b. Select Generate Pattern.
EPU draws hexagonal pattern of Acquisition Areas across the entire Grid Square image.

Depending on the microscope type and configuration, the pattern contains circles and
rectangles. Rectangles mark the field of view of the camera. Circles mark the beam
diameter.
● On microscopes with a Three Lens Condenser system, the size of the Acquisition Area
is defined by the Illuminated Area which is specified in the Data Acquisition Preset.
● On microscopes with a Two Lens Condenser system, the Acquisition Area is defined as
the smallest circle that fully covers the camera field of view at the Data Acquisition
Preset magnification.
c. If the Acquisition Areas are too close to each other or too far apart,
then adjust Spacing and select Generate Pattern again.
3. Set or adjust the Ice Quality Filter:
a. Select Preset Ice Filter to reset the Ice Quality Filter boundaries.

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b. In the Filter Ice Quality histogram:

● Drag the red lower boundary marker to exclude Acquisition Areas that are too dark.
● Drag the red upper boundary marker to exclude Acquisition Areas that are too bright.
After every change, the Ice Quality filter updates the selection of suitable Acquisition Areas
immediately. Depending on the number of areas in the Grid Square image, this may take
some time.
4. (Optional) Select Remove Areas Close To Grid Bar to exclude Acquisition Areas near the edge
of the Grid Square.

If the result of the Remove Areas Close to Grid Bar filter is not satisfactory, then select the filter
button again to de-activate it. This will automatically generate a new pattern and apply the Ice
Quality filter.
Note When the Generate Pattern function is executed, all Acquisition Areas that match with the Ice
Quality Filter are automatically included in the target area selection.
Any previous customization of the target area selection is cleared.

5. (Optional) Customize the initial selection of target Acquisition Areas as described in Customize
the selection of Acquisition Areas on page 121 .

Note The Filter Ice Quality boundaries and the Remove Areas Close to Grid Bar status are not stored for
each Grid Square.

The settings of the Filter Ice Quality and the status of the Remove Areas Close to Grid Bar remain in
their last adjusted values. When a Grid Square is displayed for which the selection is already
completed, then the displayed filter settings may be different than the filter settings that were used to
select the Acquisition Areas in the displayed Grid Square.

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To apply the currently displayed filter settings to all Grid Squares, select Prepare All Squares or
Redo Preparation as described in Automatically create a Manual Selection in all selected Grid
Squares on page 120, Automatically create a Manual Selection in all selected Grid Squares on page
135 .

8.4.2.2 Automatically create a Manual Selection in all selected Grid Squares

The Automated Preparation functions significantly decrease the time and effort that is needed to
manually select the acquisition targets (Foil Holes or Acquisition Areas) for an Automated Acquisition
run. After the suitable Grid Squares are selected and the first Grid Square is prepared, Automated
Preparation offers the following functions:

Automated Preparation ribbon bar while no automated preparation is ongoing.

Automated Preparation ribbon bar while automated preparation is ongoing

● Prepare All Squares: EPU automatically performs the manual target selection procedure in all
selected Grid Squares:
● Move to the next selected Grid Square.
● Set the specimen to Eucentric height.
● Acquire a Grid Square image.
● Create the selection of acquisition targets, using the parameters and filter settings of the
current Grid Square.
If a Grid Square already contains a target selection, then no targets are added or removed.
Even if the parameters and filter settings need to be adjusted for each individual Grid Square,
then Prepare All Squares may still provide a good starting point for each Grid Square.
● Stop Preparation: Aborts the ongoing automated preparation.
The Eucentric Height values, Grid Square images and target selections that are completed so far
are not cleared or discarded.

If Prepare All Squares has been executed at least once in the EPU session, then the following
functions can be used:
● Redo Preparation:
Use Redo Preparation when the existing target selection must be renewed after the parameters
and/or filters are adjusted.
● In already prepared Grid Squares, Redo Preparation clears the target selection, then creates
a new selection. The Grid Square image and Eucentric Height value are maintained.

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● For not yet prepared Grid Squares the Redo Preparation function performs the same actions
as the initial Prepare All Squares execution.

● Select Prepare All Squares again to prepare newly selected Grid Squares, or to complete an
aborted preparation run. Prepare All Squares uses the current selection and filter values.
● Already prepared Grid Squares remain as they are. If the selection and filter values have
changed, then the existing selection is not changed.
● For not yet prepared Grid Squares the initial Prepare All Squares actions are performed.

8.4.2.3 Customize the selection of Acquisition Areas

Before starting the manual selection, check if the manual selection tools are available in the Ribbon
Bar.

If not, then select Session Setup > Session type: Manual.

Note The following steps must be repeated for each individual selected Grid Square.

1. Perform all the steps that are described in Define the Acquisition Area Pattern and Filter Settings
for an Automated Selection Session on page 112 .
Note Make sure the specimen is accurately set to eucentric height.

The positions of the selected areas are stored as (X,Y,Z) coordinates. If the specimen is not at
eucentric height during manual selection, the X and Y coordinates of the selected areas may
have an offset. This may cause failure of the Autofocus and/or Eucentric Correction functions
during the Automated Acquisition run, which may negatively impact the amount and quality of the
acquired data.
2. Create a selection of Acquisition Areas.
Either:
● Select the suitable areas in the generated pattern.
● Unselect the bad areas in the generated pattern.
● Select the bad areas in the generated pattern, then invert the selection.
● Create a selection of suitable areas without using a generated pattern.
Instructions for each of these selection procedures are described below.

If the Ice Quality Filter boundaries are changed after a manual selection has been made, then the
selected set is not updated. Selected areas that fall outside the new boundaries will not be removed
from the selection. Likewise, areas that are not part of the selection will not be added to selection if
their average gray scale value now falls inside the new boundaries.

8.4.2.3.1 Select the suitable areas in the generated pattern

When the number of bad areas is larger than the number of suitable areas:
1. Select Unselect All to clear the selected set.
2. Add the suitable areas to the selection:

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● To add a single area to the selected set, right-click on an area, then select Add from the
context menu.
● To add one or more separate areas to the selected set, hold the Control key and select the
individual suitable areas.
● To add multiple adjacent areas, select Selection Brush, hold the Control key and swipe
across the region of suitable areas.

8.4.2.3.2 Unselect the bad areas in the generated pattern

When most areas in the generated pattern are suitable for high quality data acquisition, it can be
more efficient to remove the ones that are not suitable:
1. Select Select All to add all areas to the selected set.
2. Remove the bad areas from the selection:
● To remove a single area from the selected set, right-click on an area and select Remove
from the context menu.
● To remove one or more separate areas from the selected set, hold the Shift key and select
the individual bad areas.
● To remove multiple adjacent areas, select Selection Brush and swipe across the region of
bad areas.

8.4.2.3.3 Select the bad areas in the generated pattern, then invert the selection
When the bad areas in the generated pattern are easier to identify than the suitable areas:
1. Select Unselect All to clear the selected set.
2. Select the bad areas as if they were added to the selection:
● To add a single area to the selected set, right-click on an area and select Add from the
context menu.
● To add one or more separate areas to the selected set, hold the Control key and select the
individual areas.
● To add multiple adjacent areas, select Selection Brush, hold the Control key and swipe
across the region of bad areas.
3. Select Invert

8.4.2.3.4 Add and remove acquisition areas with the Selection Brush
The instruction in Add and remove Foil Holes with the Selection Brush on page 137 are applicable for
a Quantifoil specimen. These instructions are also applicable for the generated pattern of Acquisition
Areas on a Lacey Carbon specimen.

8.4.2.3.5 Define a selection of Acquisition Areas without a generated pattern

When the generated pattern does not hit all the suitable areas in the Grid Square, it is possible to
freely create Acquisition Areas at any location.
1. Select Acquire to start with a fresh Grid Square image.
Do not select View Pattern.
2. Create Acquisition Areas.
Either:
● Right-click in the center of a suitable area,
then select Add from the context menu.

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● Hold the Control key,

then select the center of one or more suitable areas.

8.4.2.4 Inspect an Acquisition Area to assess suitability

To decide whether or not to include an Acquisition Area in the selection, a closer look may be needed
to assess particle content, ice thickness, charging, etcetera.
1. In the Grid Square image, right-click on the desired Acquisition Area
and either select:
● Move stage to location to center the Acquisition Area in which the right mouse button was
clicked.
● Move stage here to center the exact position at which the right mouse button was clicked.

2. Select TEM User Interface > CCD/TV Camera control panel > Acquire to acquire a single
image.
To avoid unnecessary exposure:
● Do not select Search.
● Do not use the FluScreen to view the specimen.
3. If necessary, change magnification and/or illumination, and acquire a new image.
Note Every exposure causes damage to the specimen.
To maintain the highest quality, try to keep the accumulated dosage to a minimum.

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8.4.3 Set the Autofocus and Drift Stabilization values

On a Lacey Carbon specimen there is no repetitive pattern of identically shaped Foil Holes. Because
of this, the Autofocus and Drift Measurement functions cannot be performed on predefined dedicated
areas.
● Autofocus:
After data has been acquired from an Acquisition Area, that Acquisition Area becomes the
Autofocus Area for the next Acquisition Area.
● Drift Measurement:
On Lacey Carbon specimens, the Drift Measurement function is not performed. Instead, a timed
delay is used to wait for drift to decrease after a stage move. Use the Auto Function tab > Drift
stabilization task to determine a realistic value for a short move. See Run the Drift Stabilization
auto-function on page 70 for instructions.

Set the Autofocus and Drift Stabilization values as follows:

1. Select or specify the Autofocus Area settings:

a. Select Recurrence:
● Never: Do not perform the Autofocus function at all.
● Always: Autofocus is performed at every Foil Hole.
● After Distance: Autofocus is performed only when the distance between the current Foil
Hole and the most recent Autofocus location is larger than a specified value.
● After Centering: Autofocus is performed after a cluster of Foil Holes is centered.
Only available when Session Setup > Acquisition Mode is Faster Acquisition.
b. If Recurrence is set to After Distance, then specify the Distance.
c. Select Focus using:
● Objective Lens: EPU adjusts the focal plane to the Z-position of the specimen.
● Stage Z axis: EPU adjusts the Z-position of the specimen to the focal plane.
2. In the Data Acquisition Settings section of the Ribbon Bar,
specify the Delay after Image Shift and Delay after Stage Shift:

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8.4.4 Define the Defocus List for a Lacey Carbon specimen

EPU acquires one image per Acquisition Area. By default, this image is acquired with the Defocus
value that is specified in the Data Acquisition Preset. To use a different defocus, specify one or more
defocus values in the Defocus List. During the Automated Acquisition run, EPU cycles through the
Defocus List to acquire images with different defocus values. On Lacey Carbon specimens, EPU
cycles through a single Defocus List that is applicable for all Acquisition Areas.
Follow the instructions below to specify or modify the Defocus List:

1. Select the Defocus list

2. Use the arrow keys or the mouse to move the cursor to the desired location in the list.
3. Specify the desired Defocus value(s)

Use a comma or a space to separate multiple values.

4. Select Enter or click outside the list to store the updated Defocus list.
EPU validates the list. If invalid values are present, then EPU corrects or removes the invalid
values.

It is not possible to modify the Defocus list while an Automated Acquisition run is ongoing or paused.
To modify the Defocus list, the run must first be stopped.

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8.5 Hole Selection task for Quantifoil specimens

8.5.1 Define the Foil Hole dimensions and Filter Settings for an Automated Selection
Session
If the Session type is Automated, then EPU uses the built-in filters and algorithms to select suitable
Foil Holes. At the start of each Grid Square, EPU evaluates the position and condition of the Foil
Holes, so that suitable Foil Holes are identified and selected just before data acquisition begins.
To configure the Hole Selection parameters and filter settings, follow the instructions below. This
procedure must be executed for only one of the selected Grid Squares. During the Automated
Acquisition run, EPU uses the same Hole Selection parameters and filter settings for all selected Grid
Squares.

1. Acquire a representative Grid Square image:

a. Select the EPU tab > Square Selection task.
b. In the Image Display, identify a Grid Square that appears representative for the condition of
the specimen.
c. Right-click on the Grid Square and select Select Holes

EPU switches to the Holes Selection task for the selected Grid Square.
d. If the specimen is already at eucentric height, or if there is a different reason to skip the Auto
Eucentric function, then select Acquire to only acquire a new Grid Square image.

If the specimen is not already at eucentric height,

then select Auto Eucentric

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If necessary, EPU first moves the specimen, so that the center of the selected Grid Square is
in the center of the field of view.
While the Auto Eucentric function runs, the Auto Eucentric button changes to Stop Auto
Eucentric. If selected, the Auto Eucentric function is aborted and the specimen returns to its
previous Z position.
The Auto Eucentric function includes the acquisition of a new Grid Square image.
2. If the particles all have a similar orientation in the ice layer:
a. Adjust the A-tilt angle of the specimen.
A different A-tilt axis can be applied for each Grid Square to acquire images from various
incident angles. EPU stores the A-tilt angle of the Grid Square during Hole Selection, and
applies the A-tilt angle during Automated Acquisition at the same Grid Square. This typically
results in a higher quality 3D reconstruction.
b. Select Acquire to update the Grid Square image.
On Tundra systems it is not possible to adjust the A-tilt angle of the stage.
3. Specify the Foil Hole diameter and center-to-center interspacing:
a. Select Measure Hole Size.

A measurement tool appears in the Grid Square image.

b. Zoom in on the location of the measurement tool.

c. Drag one of the yellow circles to the center of a Foil Hole.
d. Drag one of the green corner handles, so that the circle diameter accurately matches with
the diameter of the physical Foil Hole.
e. Drag the other yellow circle to the center of a nearest neighboring Foil Hole define the
interspacing.
4. Identify and select all Foil Holes in the Grid Square:

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a. If the Foil Holes appear darker than the surrounding carbon foil, then first select Allow Dark
Foil Holes.
By default, the Hole Detection algorithm searches for bright circles in a darker environment.
If the Allow Dark Foil Holes toggle in active, then the algorithm searches for dark circles in a
brighter environment.

Allow Dark Foil Holes is not active: Allow Dark Foil Holes is active:
EPU does not recognize the real Foil Holes. EPU finds the real Foil Holes.

b. Select Find Holes.

The Hole Detection algorithm searches for Foil Holes that match with the specified diameter
and interspacing, and generates a pattern of Foil Hole outlines in the Grid Square image.
c. Verify that the generated pattern matches with the physical Foil Holes in the Grid Square
image.
If necessary, adjust the diameter and interspacing and select Find Holes again.
5. Set or adjust the Ice Quality filter:
a. Select Preset Ice Filter to reset the Ice Quality filter boundaries.

b. In the Filter Ice Quality histogram:

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● Drag the red lower boundary marker to exclude Foil Holes that are too dark.
● Drag the red upper boundary marker to exclude Foil Holes that are too bright.
After every change, the Ice Quality filter updates the target Foil Holes selection.
6. (Optional) Select Remove Holes Close To Grid Bar to exclude Foil Holes near the edge of the
Grid Square.

Foil Holes that are near the Grid Bars are excluded from the selection.

If the result of the Remove Holes Close to Grid Bar filter is not satisfactory, then select the filter
button again to de-activate it. This will automatically run the Find Holes function and apply the Ice
Quality filter.

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8.5.2 Handpick the Foil Holes for a Manual Selection Session

If Session type is Manual, then it is possible adjust the Foil Hole dimensions and filter settings for
each individual selected Grid Square. With these settings, the Foil Holes are selected from which
data will be acquired. For each selected Foil Hole, the X, Y and Z position of the hole center is
stored. This selection can be customized in each individual Grid Square.
During the Automated Acquisition Run:
● EPU performs the Hole Position Refinement function. This function acquires a new just-in-time
Grid Square image and updates the stored coordinates for each target Foil Hole, so that the Foil
Holes can be centered with better accuracy for execution of the Foil Hole Template.
● EPU does not re-evaluate the suitability of the selected Foil Holes.

After the suitable Grid Squares are selected in the Square Selection task, the procedure to handpick
the target Foil Holes is as follows:
1. Select the Grid Squares from which data will be acquired:
a. Select the Square Selection task.
b. Select Processing Order > Show

c. (Optional) Change the Grid Square processing order.

For instructions, see Change the processing order of the Grid Squares on page 110 .
d. Right-click on Grid Square 1 and select Select Holes

EPU switches to the Hole Selection task for the selected Grid Square. The lower-left corner
of the Hole Selection Image Display displays the processing order number of the Grid
Square and Z-position of the selected Grid Square.
2. Prepare the first selected Grid Square:

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a. Select the Area Selection task.

If a Grid Square image is available, then the image display shows the selected Grid Square.
The lower-left corner of the image display shows the Stage Z-position for the displayed Grid
Square.
● White: the Eucentric Height value is not determined yet.
● Green: the Eucentric Height value is determined.
b. If the specimen is already at eucentric height, or if there is a different reason to skip the Auto
Eucentric function, then select Acquire to only acquire a new Grid Square image.

If the specimen is not already at eucentric height,

then select Auto Eucentric

If necessary, EPU first moves the specimen, so that the center of the selected Grid Square is
in the center of the field of view.
While the Auto Eucentric function runs, the Auto Eucentric button changes to Stop Auto
Eucentric. If selected, the Auto Eucentric function is aborted and the specimen returns to its
previous Z position.
The Auto Eucentric function includes the acquisition of a new Grid Square image.
c. Define the Foil Hole dimensions and the filter settings.
For instructions, see Define the Foil Hole dimensions and Filter Settings for a Manual
Selection Session on page 132 .
d. Customize the selection of target Foil Holes.
For instructions, see Customize the selection of Foil Holes on page 136 .
3. Prepare the other selected Grid Squares:
a. (Optional) Use the Automated Preparation functions to apply the Acquisition Area pattern
and filter settings of the current Grid Square to all other selected Grid Squares.
For instructions, see Automatically create a Manual Selection in all selected Grid Squares on
page 120, Automatically create a Manual Selection in all selected Grid Squares on page
135 .
b. Select Navigate > Next Square to move to the next Grid Square.

In the Square Selection image display, the white border moves to the next Grid Square. The
specimen does not move until Auto Eucentric or Acquire is selected.
c. Repeat steps a – e above until all Grid Squares are prepared.

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4. (Optional) Inspect or adjust the Acquisition Area selection for a prepared Grid Square:
Either:
● Select Navigate > Previous Square until the desired Grid Square is reached.
In the Square Selection image display, the white border moves to the previous Grid Square.
The specimen does not move until Auto Eucentric or Acquire is selected.
● Select the Square Selection task
> Right-click on the desired Grid Square
> Select Select Holes
EPU displays the Grid Square image for the selected Grid Square.
Note Unless the current selection of target Foil Holes must be cleared:
• Do not select Acquire
• Do not select Find Holes
• If Remove Holes Close to Grid Bar is active, then do not select Remove Holes Close to Grid Bar

8.5.2.1 Define the Foil Hole dimensions and Filter Settings for a Manual Selection Session

In a Manual Selection Session, the actions below must be repeated for each selected Grid Square.

1. Specify the Foil Hole diameter and center-to-center interspacing:

a. Select Measure Hole Size.

A measurement tool appears in the Grid Square image.

b. Zoom in on the location of the measurement tool.

c. Drag one of the yellow circles to the center of a Foil Hole.
d. Drag one of the green corner handles, so that the circle diameter accurately matches with
the diameter of the physical Foil Hole.
e. Drag the other yellow circle to the center of a nearest neighboring Foil Hole define the
interspacing.
2. Identify and select all Foil Holes in the Grid Square:

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a. If the Foil Holes appear darker than the surrounding carbon foil, then first select Allow Dark
Foil Holes.
By default, the Hole Detection algorithm searches for bright circles in a darker environment.
If the Allow Dark Foil Holes toggle in active, then the algorithm searches for dark circles in a
brighter environment.

Allow Dark Foil Holes is not active: Allow Dark Foil Holes is active:
EPU does not recognize the real Foil Holes. EPU finds the real Foil Holes.

b. Select Find Holes.

The Hole Detection algorithm searches for Foil Holes that match with the specified diameter
and interspacing, and generates a pattern of Foil Hole outlines in the Grid Square image.
c. Verify that the generated pattern matches with the physical Foil Holes in the Grid Square
image.
If necessary, adjust the diameter and interspacing and select Find Holes again.
3. Set or adjust the Ice Quality filter:
a. Select Preset Ice Filter to reset the Ice Quality filter boundaries.

b. In the Filter Ice Quality histogram:

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● Drag the red lower boundary marker to exclude Foil Holes that are too dark.
● Drag the red upper boundary marker to exclude Foil Holes that are too bright.
After every change, the Ice Quality filter updates the target Foil Holes selection.
4. (Optional) Select Remove Holes Close To Grid Bar to exclude Foil Holes near the edge of the
Grid Square.

Foil Holes that are near the Grid Bars are excluded from the selection.

If the result of the Remove Holes Close to Grid Bar filter is not satisfactory, then select the filter
button again to de-activate it. This will automatically run the Find Holes function and apply the Ice
Quality filter.
Note When the Find Holes function is performed, all Foil Holes that match with the Ice Quality Filter are
automatically included in the target Foil Hole selection.
Any previous customization of the target Foil Hole selection is cleared.

5. (Optional) Customize the initial selection of target Foil Holes as described in Customize the
selection of Foil Holes on page 136 .

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Note The Filter Ice Quality boundaries and the Remove Holes Close to Grid Bar status are not stored for
each Grid Square.

The settings of the Filter Ice Quality and the status of the Remove Holes Close to Grid Bar remain in
their last adjusted values. When a Grid Square is displayed for which the selection is already
completed, then the displayed filter settings may be different than the filter settings that were used to
select the Foil Holes in the displayed Grid Square.
To apply the currently displayed filter settings to all Grid Squares, select Prepare All Squares or
Redo Preparation as described in Automatically create a Manual Selection in all selected Grid
Squares on page 120, Automatically create a Manual Selection in all selected Grid Squares on page
135 .

8.5.2.2 Automatically create a Manual Selection in all selected Grid Squares

The Automated Preparation functions significantly decrease the time and effort that is needed to
manually select the acquisition targets (Foil Holes or Acquisition Areas) for an Automated Acquisition
run. After the suitable Grid Squares are selected and the first Grid Square is prepared, Automated
Preparation offers the following functions:

Automated Preparation ribbon bar while no automated preparation is ongoing.

Automated Preparation ribbon bar while automated preparation is ongoing

● Prepare All Squares: EPU automatically performs the manual target selection procedure in all
selected Grid Squares:
● Move to the next selected Grid Square.
● Set the specimen to Eucentric height.
● Acquire a Grid Square image.
● Create the selection of acquisition targets, using the parameters and filter settings of the
current Grid Square.
If a Grid Square already contains a target selection, then no targets are added or removed.
Even if the parameters and filter settings need to be adjusted for each individual Grid Square,
then Prepare All Squares may still provide a good starting point for each Grid Square.
● Stop Preparation: Aborts the ongoing automated preparation.
The Eucentric Height values, Grid Square images and target selections that are completed so far
are not cleared or discarded.

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If Prepare All Squares has been executed at least once in the EPU session, then the following
functions can be used:
● Redo Preparation:
Use Redo Preparation when the existing target selection must be renewed after the parameters
and/or filters are adjusted.
● In already prepared Grid Squares, Redo Preparation clears the target selection, then creates
a new selection. The Grid Square image and Eucentric Height value are maintained.
● For not yet prepared Grid Squares the Redo Preparation function performs the same actions
as the initial Prepare All Squares execution.

● Select Prepare All Squares again to prepare newly selected Grid Squares, or to complete an
aborted preparation run. Prepare All Squares uses the current selection and filter values.
● Already prepared Grid Squares remain as they are. If the selection and filter values have
changed, then the existing selection is not changed.
● For not yet prepared Grid Squares the initial Prepare All Squares actions are performed.

8.5.2.3 Customize the selection of Foil Holes

Before starting the manual selection, check if the manual selection tools are available in the Ribbon
Bar.

If not, then select Session Setup > Session type: Manual.

Note The following steps must be repeated for each individual selected Grid Square.

1. Perform all the steps that are described in Define the Acquisition Area Pattern and Filter Settings
for an Automated Selection Session on page 112 .
Note Make sure the specimen is accurately set to eucentric height.

The positions of the selected areas are stored as (X,Y,Z) coordinates. If the specimen is not at
eucentric height during manual selection, the X and Y coordinates of the selected areas may
have an offset. This may cause failure of the Autofocus and/or Eucentric Correction functions
during the Automated Acquisition run, which may negatively impact the amount and quality of the
acquired data.
2. Create a selection of Foil Holes.
Either:
● Select the suitable Foil Holes in the generated pattern.
● Unselect the bad Foil Holes in the generated pattern.
● Select the bad Foil Holes in the generated pattern, then invert the selection.
● Create a selection of suitable Foil Holes without using a generated pattern.
Instructions for each of these selection procedures are described below.

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If the Ice Quality Filter boundaries are changed after a manual selection has been made, then the
selected set is not updated. Selected areas that fall outside the new boundaries will not be removed
from the selection. Likewise, areas that are not part of the selection will not be added to selection if
their average gray scale value now falls inside the new boundaries.
8.5.2.3.1 Select the suitable Foil Holes in the generated pattern
When the number of bad Foil Holes is larger than the number of suitable Foil Holes:
1. Select Unselect All to clear the selected set.
2. Add the suitable Foil Holes to the selection:
● To add a single Foil Hole to the selected set, right-click on the Foil Hole and select Add from
the context menu.
● To add one or more separate Foil Holes to the selected set, hold the Control key and select
the individual suitable Foil Holes.
● To add multiple adjacent Foil Holes, select Selection Brush, hold the Control key and swipe
across the region of suitable Foil Holes.

8.5.2.3.2 Unselect the bad Foil Holes in the generated pattern

When most Foil Holes in the generated pattern are suitable for high quality data acquisition, it can be
more efficient to remove the ones that are not suitable:
1. Select Select All to add all Foil Holes to the selected set.
2. Remove the bad Foil Holes from the selection:
● To remove a single Foil Hole from the selected set, right-click on the Foil Hole and select
Remove from the context menu.
● To remove one or more separate Foil Holes from the selected set, hold the Shift key and
select the individual bad Foil Holes.
● To remove multiple adjacent Foil Holes, select Selection Brush and swipe across the region
of bad Foil Holes.

8.5.2.3.3 Select the bad Foil Holes in the generated pattern, then invert the selection
When the bad Foil Holes in the generated pattern are easier to identify than the suitable Foil Holes:
1. Select Unselect All to clear the selected set.
2. Select the bad Foil Holes as if they were added to the selection:
● To add a single Foil Hole to the selected set, right-click on an Foil Hole and select Add from
the context menu.
● To add one or more separate Foil Holes to the selected set, hold the Control key and select
the individual Foil Holes.
● To add multiple adjacent Foil Holes, select Selection Brush , hold the Control key and
swipe across the region of bad foil holeFoil Holes.
3. Select Invert.

8.5.2.3.4 Add and remove Foil Holes with the Selection Brush
1. From the Selection section of the the Ribbon Bar, select Selection Brush.

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2. Move the mouse to the Image Display.

The Selection Brush is displayed as a dark yellow circle.
3. (Optional) Change the Selection Brush size:
Hold down the Shift key and scroll with the mouse wheel
4. To remove Foil Holes from the selection:
a. Hold down the left mouse button
b. Drag the Selection Brush across the image.

Foil Holes that are touched by the brush have a purple highlight.
c. Release the left mouse button
The purple Foil Holes are removed from the selection.
5. To add Foil Holes to the selection:
a. Hold down the Control key.
b. Hold down the left mouse button
c. Drag the Selection Brush across the image.
d. Release the left mouse button
The purple Foil Holes are added to the selection.

8.5.2.3.5 Create a selection of Foil Holes without a generated pattern

When it not possible to let the Hole Detection algorithm generate a pattern that matches the
specimen, it is possible to freely create Foil Hole locations at any position on the specimen.
1. Select Acquire to start with a fresh Grid Square image.
Do not select Find Holes.
2. Create Foil Hole locations at any position.
Either:
● Right-click in the center of the new Foil Hole location,
then select Add from the context menu.
● Hold the Control key,
then select the center of one or more suitable Foil Holes.

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8.5.2.4 Inspect a Foil Hole to assess suitability

To decide whether or not to include a Foil Hole in the selection, a closer look may be needed to
assess particle content, ice thickness, charging, etcetera.
1. In the Grid Square image:
a. Right-click on the Foil Hole you wish to inspect.
b. Select one of the Move stage... options:
● Move stage to location: centers the Foil Hole in which the right mouse button was
clicked.
● Move stage here: centers the exact position at which the right mouse button was
clicked.
● Move stage here and acquire: centers the exact position at which the right mouse
button was clicked, and acquires a new Grid Square image.
To prevent clearing the current selection, this option is only available when no Foil Holes
have been selected.

2. Select TEM User Interface > CCD/TV Camera control panel > Acquire to acquire a single
image.
To avoid unnecessary exposure:
● Do not select Search.
● Do not use the FluScreen to view the specimen.
3. If necessary, change magnification and/or illumination, and acquire a new image.
Note Every exposure causes damage to the specimen.
To maintain the highest quality, try to keep the accumulated dosage to a minimum.

8.5.2.5 The impact of Hole Position Refinement on the 'Move stage to location' function

In a Session type: Manual session, the Hole Selection > Find Holes function identifies Foil Holes in a
Grid Square image, and stores the XYZ coordinates of each Foil Hole center. When the Move stage
to location function is used during preparation for the Automated Acquisition run, the red crosshair
in the Image Display will end up in the center of the Foil Hole.

Between the acquisition of a Grid Square image in the Hole Selection task and the time at which the
Automated Acquisition run reaches that same Grid Square, a small positioning offset may have
accumulated. To prevent skipped Foil Holes due to such offsets, the Automated Acquisition
procedure features an automatic Hole Position Refinement function. This function acquires a new
just-in-time Grid Square image and updates the stored coordinates for each Foil Hole, so that the Foil
Holes can be centered with better accuracy for execution of the Foil Hole Template.

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During the Automated Acquisition run it is possible to fine-tune the Foil Hole Template. To do so,
return to the Hole Selection task, use the Move stage to location function to center a Foil Hole,
and then select the Template Definition task to revise the Foil Hole Template.
In the Hole Selection task, the image display uses the initial Grid Square image, but the Move stage
to location function uses the updated Foil Hole coordinates. As a result, the crosshair in the image
display may end up at a small offset from the uncorrected Foil Hole center in the Grid Square image.

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8.6 Template Definition task for Quantifoil specimens

The purpose of the Template Definition task is to assign the areas in and around a Foil Hole where
the Autofocus function, the (optional) Drift Measurement function, and one or more Data Acquisitions
are executed. During the Automated Acquisition run, the Foil Hole template is executed at every
selected Foil Hole.
The Foil Hole image that is used to define the Foil Hole Template remains available in the Template
Definition task. If the template requires an adjustment at any later time, then it is not necessary to
acquire a new Foil Hole image.

8.6.1 Perform the Template Definition procedure

To set up a Foil Hole Template, follow the procedure below:

1. In the Hole Selection task,

right-click on an included Foil Hole and select Move stage to location
2. Select the Template Definition task.
3. Select Acquire to acquire a Hole/EucentricHeight image.

4. Select Find And Center Hole

5. Use the Template Definition functions to define the Foil Hole Template.

a. If the A-tilt is not 0 degrees, then select Show/Hide Tilt Axis

On Tundra systems, Show/Hide Tilt Axis is not available. The stage can not be tilted.
b. Automatically or manually create one or more Acquisition Areas as described in:
● Automatically generate a pattern of Acquisition Areas on page 142 .
● Manually define one or more Acquisition Areas on page 143 .
c. Define a Defocus List
as described in Define the Defocus List for one or more Acquisition Areas on page 144 .
d. Define an Autofocus Area
as described in Define the Autofocus Area on page 146 .
e. Optionally, define a Drift Measurement Area
as described in Define the Drift Measurement Area on page 147 .
f. Specify the shift and delay parameters

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as described in Specify the Maximum Image Shift and Delay Timers on page 148 .
When an Acquisition, Autofocus or Drift Measurement Area is selected in the image display, an
additional Ribbon Bar section appears to specify the values for the selected area. The Template
Definition section may change to a more compact layout to make room for the additional Ribbon Bar
sections.

Instead of creating a new Foil Hole Template for every new session, it is also possible to import the
Template Areas and their settings from an earlier session. See Import and export a Foil Hole
Template on page 149 .

8.6.2 Define one or more Acquisition Areas

8.6.2.1 Automatically generate a pattern of Acquisition Areas

EPU can automatically generate a pattern of Acquisition Areas in the Foil Hole Template.

Hexagonal pattern Rectangular pattern Circular pattern M X N Matrix

To generate a pattern of Acquisition Areas:

1. Select Auto > select the desired pattern

EPU adds as many Acquisition Areas as possible according to the selected pattern.
2. Define the Defocus List as described in Define the Defocus List for one or more Acquisition
Areas on page 144 .

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8.6.2.2 Manually define one or more Acquisition Areas

1. Select Template Definition > Add Acquisition Area

2. In the image display, select the location for the first Acquisition Area.
A green circle appears in the Foil Hole Template with its center at the selected position.

● On microscopes with a Three Lens Condenser system, the size of the Acquisition Area is
defined by the Illuminated Area which is specified in the Data Acquisition Preset.
● On microscopes with a Two Lens Condenser system, the Acquisition Area is defined as the
smallest circle that fully covers the camera field of view at the Data Acquisition Preset
magnification.
3. In the image display, select the Acquisition Area.
The Data Acquisition Area section appears in the Ribbon Bar.

4. Specify a Defocus List as explained in Define the Defocus List for one or more Acquisition
Areas on page 144 .
5. (Optional) Add more Acquisition Areas in the Foil Hole Template:
a. If the new Acquisition Area must use the same Defocus list as the first Acquisition Area,
then select the first Acquisition Area.
The Defocus List of the selected Acquisition Area will be copied to the new Acquisition
Area(s). Each Acquisition Area has its own Defocus List. Modifications to the Defocus List of
one Acquisition Area are not shared with the other Acquisition Areas.
b. Select Add Acquisition Area and select the location for the new Acquisition Area
6. (Optional) Adjust the location of an Acquisition Area by dragging it with the mouse.
7. (Optional) Modify the Defocus List for one or more individual Acquisition Areas.

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8.6.3 Define the Defocus List for one or more Acquisition Areas
EPU acquires one image per Acquisition Area. By default, this image is acquired with the Defocus
value that is specified in the Data Acquisition Preset. To use a different defocus, specify one or more
defocus values in the Defocus List. During the Automated Acquisition run, EPU cycles through the
Defocus List to acquire images with different defocus values. On Quantifoil specimens, the Foil Hole
Template can contain multiple Acquisition Areas. Each Acquisition Area has its own Defocus List.

The example below describes a Foil Hole Template with two Acquisition Areas:
● Acquisition Area A has a Defocus List with three values.
● Acquisition Area B has a Defocus List with two values.
As the Foil Holes are processed one after the other, EPU steps through the Defocus Lists for each of
the Acquisition Areas.

If a Foil Hole is skipped by EPU or by the user, then the defocus values that were planned for the
acquisitions on that Foil Hole are also skipped.

To add or remove Defocus values for the currently selected Acquisition Area, follow the instructions
below:

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1. In the image display, select an Acquisition Area

The Data Acquisition Area section appears in the Ribbon Bar.

2. Select the Defocus list

3. Add, modify, or remove values from the Defocus List.

Use a comma or a space to separate multiple values.

4. Select Enter or click outside the list to store the updated Defocus List.
EPU validates the list. If invalid values are present, then EPU corrects or removes the invalid
values.
5. (Optional) Select Copy defocus list to all areas to apply the Defocus List to all Acquisition
Areas in the Template.

After the Defocus List is applied to all Acquisition Areas, it is still possible to modify the Defocus
List for an individual Acquisition Area.

It is not possible to modify the Defocus List while an Automated Acquisition run is ongoing or paused.
To modify the Defocus List, the run must first be stopped.

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8.6.4 Define the Autofocus Area

The Foil Hole Template contains one Autofocus Area.

1. Add an Autofocus Area to the Foil template:

a. Select Template Definition > Add Autofocus Area
b. In the image display, select the location for the Autofocus Area.
● Select an area with only carbon foil, no ice and no particles.
● If the A-tilt is not 0 degrees, then select a location on the Tilt Axis.
A blue circle appears at the selected position.

● On microscopes with a Three Lens Condenser system, the size of the Autofocus Area is
defined by the Illuminated Area which is specified in the Autofocus Preset.
● On microscopes with a Two Lens Condenser system, the Autofocus Area is defined as
the smallest circle that fully covers the camera field of view at the Autofocus Preset
magnification.
2. In the image display, select the Autofocus Area
3. Select or specify the Autofocus Area settings:

a. Select Recurrence:
● Never: Do not perform the Autofocus function at all.
● Always: Autofocus is performed at every Foil Hole.
● After Distance: Autofocus is performed only when the distance between the current Foil
Hole and the most recent Autofocus location is larger than a specified value.
● After Centering: Autofocus is performed after a cluster of Foil Holes is centered.
Only available when Session Setup > Acquisition Mode is Faster Acquisition.
b. If Recurrence is set to After Distance, then specify the Distance.
c. Select Focus using:
● Objective Lens: EPU adjusts the focal plane to the Z-position of the specimen.
● Stage Z axis: EPU adjusts the Z-position of the specimen to the focal plane.
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8.6.5 Define the Drift Measurement Area

The Foil Hole Template contains at most one Drift Measurement Area. If a Drift Measurement Area is
present, then EPU executes the Drift stabilization function instead of counting down the Delay after
Stage Shift timer. The Drift stabilization function pauses the Automated Acquisition run as long as the
actual drift speed is higher than a specified threshold value. This threshold value is specified in Auto
Functions tab > Auto Functions (TEM): Drift stabilization task > Max. Remaining Drift

1. Add an Drift Measurement Area to the Foil Hole Template:

a. Select Template Definition > Add Drift Measurement Area
b. In the image display, select the location for the Drift Measurement Area.
A purple circle is placed in the Foil Hole Template with its center at the selected position.

The Drift Measurement Area is best placed outside the Foil Hole perimeter, on the carbon
foil. This way no valuable specimen material is exposed.
● On microscopes with a Three Lens Condenser system, the size of the Drift
Measurement Area is defined by the Illuminated Area which is specified in the Drift
Measurement Preset.
● On microscopes with a Two Lens Condenser system, the Drift Measurement Area is
defined as the smallest circle that fully covers the camera field of view at the Drift
Measurement Preset magnification.
2. In the image display, select the Drift Measurement Area.
3. In the Drift Measurement section of the Ribbon bar:

a. Specify the Recurrence.

● Always: the Drift Measurement function is executed after every CompuStage move.
In practice this means every time the Automated Acquisition run moves to the next Foil
Hole.
● Once per GridSquare: the Drift Measurement function is executed only after moving to
another Grid Square.
For long moves like this, the actual drift speed can vary more than for the relatively short
moves between Foil Holes within the same Grid Square. For the short hole-to-hole

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movements, the Delay after Stage Shift timer is used. If the Maximum Image Shift is set
smaller than the distance between the Acquisition Areas, then the Once per
GridSquare option is the only valid choice.
b. Specify the Drift Threshold.
This is the maximum allowed drift speed for data acquisition. If the specified threshold is not
achieved within 600 seconds, then the Foil Hole will be skipped and the Automated
Acquisition run continues with the next Foil Hole.

8.6.6 Specify the Maximum Image Shift and Delay Timers

The use of Image Shift to navigate between Template Areas is a trade-off between accuracy and
throughput. Image Shift is typically faster than moving the specimen with the stage, but it also
induces coma and astigmatism. The required resolution for 3D reconstruction limits the maximum
acceptable coma.
The Maximum Image Shift depends on:
● The presence and calibration status of Aberration Free Image Shift (AFIS).
AFIS drastically reduces coma at large Image Shift values.
● The presence of an Image Corrector.
An Image Corrector significantly improves image resolution near the optical axis, but it also limits
the available Image Shift range.
● The use of Phase Plates.
With Phase Plates, Image Shift must be limited so the beam does not shift outside the activated
Phase Plate area.
● The Session Setup > Acquisition mode.
When Session Setup > Acquisition Mode is Faster acquisition and the conditions for Foil Hole
navigation by Image Shift are met, then EPU locks the Maximum Image Shift value at a
predetermined value, and disables the Maximum Image Shift input field.

Follow the steps below to specify the Image Shift and delay values.

1. If not disabled, specify the Maximum Image Shift distance.

The Maximum Image Shift distance determines if a Template Area can be visited by applying an
Image Shift, or if it is necessary to move the stage.
2. Specify the Delay after Image Shift time.
The Delay after Image Shift timer postpones image acquisition until the image shift deflectors
have settled at their new values.
3. Specify the Minimum stage settling time.
The Minimum stage settling time postpones image acquisition until:
● The stage mechanics have relaxed and are settled at their new positions.
● Thermal expansion due to heat from the motors has decreased below an acceptable
threshold.
The countdown starts when the most recent stage move is completed. The Minimum stage
settling time is ignored when the Foil Hole Template contains a Drift Measurement Area. The
delay after a stage move is then no longer a fixed time.

The values for the delay timers can be determined with the Auto Functions > Drift stabilization
autofunction.

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8.6.6.1 Guidelines for the Drift Measurement Function and the Delay After Stage Shift Time

There are circumstances in which Drift Measurement function may not always be as accurate and
fast as desired. The appearance of the specimen can change due to the accumulating damage that
is caused by repetitive exposure. This may make it difficult to accurately determine the image shift
between consecutive images. In extreme cases, this could even cause the Drift Measurement
function to time out at 10 minutes.
Under these circumstances:
● Adding a Drift Measurement Area and selecting Recurrence: Always would significantly slow
down the Automated Acquisition run, and make the predictions in the Progress panel less
reliable. It may also result in a relatively large number of skipped Foil Holes due to failed Drift
Measurements.
● Adding a Drift Measurement Area and selecting Recurrence: Once per Grid Square decreases
the chance of occurrence and impact of a time out. At the same, the Automated Acquisition run
is not paused longer than necessary after moving on to the next Grid Square.
● If a predictable throughput is most important, it can be better to not add a Drift Measurement
Area to the Foil Hole Template, and use the Delay after Stage Shift timer for all moves.
Run the Auto Functions > Auto-Functions (TEM) > Drift stabilization autofunction to determine a
usable value for Delay after Stage Shift. For instructions, see Run the Drift Stabilization auto-function
on page 70 .

8.6.7 Enable the Smart Plugins in the Template Definition

Note Only available on systems with Smart EPU.

For the Template Definition, the following Smart Plugins are available:
● Stage Settling Time Predictions provides a stage settling time that is derived from the acquired
data. When a stage move is executed, EPU can request a stage settling time from Smart EPU.
The stage settling time that is provided by this Smart Plugin overrides the Template Definition >
Minimum stage settling time value.
This Smart Plugin can only be enabled when Session Setup > Acquisition mode is Accurate.
● Smart Focus Predictions provides a focus value that is derived from the acquired data. Instead of
executing the Autofocus auto function, EPU can request a focus value from Smart EPU. If the
Smart Plugin can provide a focus value, then EPU skips the execution of the Autofocus function
and uses the provided value.

8.6.8 Import and export a Foil Hole Template

With the Import and Export functions it is possible to archive the current Template Definition as a file,
and to load a Template Definition that has been created in an earlier EPU session. This way, setting
up an Automated Acquisition run for a regularly used grid type is much faster and easier.

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Export
Select Export to write the current Foil Hole Template to a file (*.epuTemplate).
The Template file contains:
● The type, location and all settings of all Template Areas.
The Template file does not contain the size of the Template Areas. The size is determined by the
pertaining Presets.
● The Maximum Image Shift, Delay after Image Shift and Delay after Stage Shift values.
● The Defocus list for the Acquisition Area(s).
The Template file also contains the Foil Hole diameter. The Foil Hole diameter is only used as a
reference value for the Import functionality.

Import
The Foil Hole diameter of the loaded specimen does not have to be the same as the Foil Hole
diameter in the Template file. EPU calculates the ratio of the Foil Hole diameters to proportionally
adjust the positions of the Template Areas. If the ratio is too large, then EPU shows a warning. In that
case, the Focus and/or Drift Area may be located outside the field of view of the Hole/Eucentric
Preset.
1. Select Import and select a previously stored Template file.
2. If necessary or desirable, reposition the Template Areas and/or adjust their settings.

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8.7 Template Execution task for Quantifoil specimens

The purpose of the Template Execution task is to validate the Foil Hole Template, including finding
and centering the Foil Hole that is nearest to the center of the camera field of view.

8.7.1 Validate the Foil Hole Template

To validate the Foil Hole Template, follow the procedure below. First perform this procedure at an
ideal Foil Hole. If the result is good, also perform the procedure at one or two Foil Holes that are not
ideal, but still good enough for high quality data acquisition.

1. Select a Foil Hole:

a. Select the EPU tab > Hole Selection task.
b. Select Acquire
c. Right-click on a Foil Hole and select Move stage to location
2. Prepare the starting conditions for Template Execution:
a. Select Hole Selection > Auto Eucentric
b. Select Auto Functions tab > Auto-Functions (TEM): Autofocus
c. Select the appropriate Handpanels > User Button to Reset Defocus
d. Insert the FluScreen
e. Move the specimen, so that the Foil Hole is still fully visible but not exactly centered in the
camera field of view.
This will test the Find And Center Hole function during Template Execution.
f. Change the Defocus a little.
This will test the Autofocus function during Template Execution.
3. Select the EPU tab > Template Execution task.
4. Select Preview to execute the Foil Hole Template,
including the Find and Center Hole and Autofocus functions.

5. Verify that the Preview procedure is executed successfully.

If the Template Execution procedure is not completed successfully, then:
a. Solve any issues.
b. Repeat this procedure from step 1.

The time it takes to complete a Preview run of the Foil Hole Template is a good estimate for the
Session Information panel > Exposures per hour. Keep in mind that the Foil Hole Template can
contain multiple Acquisition Areas.

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8.8 Automated Acquisition task

This section describes the Automated Acquisition task for a single specimen. For a description of this
task in the EPU Multigrid workflow, see: The Automated Acquisition task in the EPU Multigrid
workflow on page 164 .
To successfully complete an Automated Acquisition run, the following prerequisites must be fulfilled:
● The tasks in the Preparations tab and the Auto Functions tab must be completed.
● An Atlas of the specimen must be available.

8.8.1 How to plan for high microscope utilization

The Session Information panel indicates how efficient the available time is utilized.

To maximize utilization, follow these guidelines:

1. Specify the time and date for the End of tool time.
2. Specify the Exposures per hour.
This number is an estimate. It can be based on experience with similar Automated Acquisition
runs.
On Quantifoil specimens, the time that is needed to complete a Template Execution > Preview
also provides a good indication. Keep in mind that a Foil Hole Template can contain multiple
Acquisition Areas.
3. Check the Holes / Exposures numbers.
● Holes: the number of Foil Holes (Quantifoil) or Acquisition Areas (Lacey Carbon).
● For an Automated session, this number is an estimation. Among others, it is based on
the number of Grid Squares that is selected in the Square Selection task, and the
spacing between the Foil Holes or Acquisition Areas.
● For a Manual session it is the number of selected holes.
● Exposures: the number of Acquisition Areas.
● On Lacey Carbon specimens the number of exposures is the same as the number of
holes.
● On Quantifoil specimens, the number of holes is multiplied by the number of Acquisition
Areas in the Foil Hole Template.
The estimated number of exposures assumes that no Grid Squares are skipped and no holes
are skipped.

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4. Check the Total time.

This is the duration of the Automated Acquisition run. It is calculated by dividing the number of
exposures by the number of Exposures per hour.
The color indicates whether or not the available tool time is fully used, assuming that no Grid
Squares or holes are skipped:
● Red: poor utilization.
When the Total time is spent, there is still a significant amount of tool time left.
● Yellow: adequate utilization, there is room for improvement.
When the Total time is spent, there is a small amount of tool time left. If the Session type is
Manual, add a few more holes to the selection so all available tool time is used for data
acquisition.
● Green: maximum utilization.
When the Total time is spent, there is no tool time left.

8.8.2 Verify that there is sufficient free disk space and sufficient liquid nitrogen (LN2)
Before starting the Automated Acquisition run, check the following:
1. Make sure there is sufficient available disk space.
● On the Microscope PC.
Session related data and images are stored in the Session Setup > Output folder. In case
no more data can be stored in this location, EPU pauses the Automated Acquisition run and
requests another storage location. If this happens, select a new Output folder and resume
the Automated Acquisition run.
● On the Storage Server or Gatan PC.
Dose Fractions are stored on the Storage Server or Gatan PC. Data volumes of several
hundreds of Gigabytes per day are no exception. If there is no more space to store Dose
Fraction images, the Automated Acquisition run will stop.
2. Make sure the Liquid Nitrogen (LN2) dewars are full.
To avoid failure of an unattended run, make sure that the LN2 supply is sufficient to finish the
run. EPU frequently checks if a refill of the dewars is required and will request a refill if needed.
EPU cannot detect if the main LN2 supply runs out. If the LN2 is not refilled, the Automated
Acquisition run keeps going while the sample temperature slowly increases.

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8.8.3 Activate a new Phase Plate before an Automated Acquisition run

The instructions below are only applicable when EPU > Session Setup > Use Phase Plate is ticked.

To prepare a fresh Phase Plate, follow the instructions below:

1. In the Phase Plate section:

a. Specify Periodicity (exposures):

The number of acquisitions after which the Objective aperture mechanism moves to the next
Phase Plate position to start the activation of a new Phase Plate area. The interval is
typically between 50 and 80 exposures.
b. Specify Activation Time (s):
The time that is needed to activate a new Phase Plate.
c. If desired, select Accelerate: Yes
The Accelerate function temporarily moves the specimen to an area that has been exposed
already, and selects the largest C2 aperture to increase the beam current. After the phase
plate activation is completed, the C2 aperture mechanism and the specimen are returned to
their initial positions. The Accelerate function does not change any optics settings.
The Accelerate factor is the area ratio between the initial C2 aperture and the largest C2
aperture.
2. Retract the FluScreen.
The beam will be blanked now.
3. Select the Preparation tab > Activate Phase Plate task.
4. Select Next Position to move the Objective aperture mechanism to a fresh area on the phase
plate.

5. Wait for the drift of the aperture mechanism to settle.

For a small move, this takes about 30 seconds.
Note After a large move with the aperture mechanism, for example a move to a different Phase Plate, it
may take up to 5 minutes for drift to settle.

6. Specify the Activation time (s) value.

7. (Optional) Select Accelerate: Yes
8. Select Start Activation.
An image is acquired and displayed in the top left section of the Task Execution panel.

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9. Wait for the Activation time to expire.

EPU acquires a new image and displays it in the top right section of the Task Execution panel.
The FFT of both images are displayed side-by-side in the bottom section of the Task Execution
panel, so they are easy to compare. If the Thon rings are shifted inward by half a period, then
Phase Plate activation is completed successfully.

Bottom left half: FFT of an image at the beginning of the activation.

Bottom right half: FFT of an image at end of the activation.

8.8.4 Start the Automated Acquisition run

To start the Automated Acquisition, follow the procedure below:

1. Select Automated Acquisition task.

2. If the microscope is in EFTEM mode,
then specify the Auto Zero Loss settings:

a. Select Auto Zero Loss: Yes to enable periodic execution of the Auto Zero Loss function
during the Automated Acquisition run.
b. Specify the Periodicity
3. If the system has a Gatan filter with Gatan K3 camera,
then specify the Dark Reference Settings:

a. Select Dark reference acquisition: Yes to enable periodic acquisition of Dark Reference
images during the Automated Acquisition run.
b. Specify the Periodicity

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4. (Optional) If available, enable the Smart Plugins > Skip Gridsquare Prediction plugin.

This Smart Plugin uses the data that is acquired during the Automated Acquisition, and which is
evaluated by EPU Quality Monitor (EQM). If the quality of the acquired data is not good, then the
Skip Gridsquare Prediction plugin sends a recommendation to EPU to skip all remaining
acquisitions on the current Grid Square. The Skip Gridsquare Prediction plugin will never
recommend to skip the first Grid Square.
5. Select Start Run

The Start Run button changes into a Stop button.

Before the automated acquisition starts, EPU runs a preconditions check.
● If no issues are found, then the automated acquisition run starts.
● If issues are found, then EPU displays these in the Messages side panel, and the automated
acquisition run is not started.
6. Monitor the Automated Acquisition run during the first few data acquisitions.
7. If there are no irregularities, select the desired Options:

a. Select Close Col. Valves.

When active, EPU closes the Column Valves after the Automated Acquisition run is
completed.
b. On systems with a thermionic gun, select Switch Off Emission (not shown in the image
above).
When active, EPU switches off the emission, or brings the electron source to a safe standby
state after the Automated Acquisition run is completed.

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8.8.5 Monitor the progress of the Automated Acquisition run

EPU provides two to monitor the progress of an Automated Acquisition run:
● The EPU tab > Automated Acquisition task > Progress panel
The contents of the Progress panel are described below.
● The tooltip that appears when hovering over a Grid Square in the EPU tab > Square Selection
task

On a Lacey Carbon specimen, the counters apply to the number of defined and processed
Acquisition Areas.

The Progress panel displays the following information:

Exposures
● Number of completed data acquisitions.
If too many acquisitions are skipped, the number of exposures will appear yellow or red.
● Number of planned data acquisitions (set).
● Number of remaining data acquisitions (left).
Grid Square
● Number of processed Grid Squares.
The processing status of each Grid Square is also indicated by color coding in the Atlas.
● Number of planned Grid Squares.
Holes
Holey carbon only
● The number of processed Foil Holes.
● The number of planned holes.
● The number of skipped holes.
When too many holes are skipped, the counter will turn red.

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Exposure rate
The actual number of data acquisitions per hour, based on the statistics of the current session,
including the skipped areas.
Time remaining
The estimated remaining duration, based on the actual Exposure rate and the number of remaining
data acquisitions.
End time
The estimated end time of the run.
The color indicates whether or not the available tool time is fully used, assuming that no Grid
Squares or holes are skipped:
● Red: poor utilization.
When the Total time is spent, there is still a significant amount of tool time left.
● Yellow: adequate utilization, there is room for improvement.
When the Total time is spent, there is a small amount of tool time left. If the Session type is
Manual, add a few more holes to the selection so all available tool time is used for data
acquisition.
● Green: maximum utilization.
When the Total time is spent, there is no tool time left.
A progress bar at the bottom side of the Progress panel indicates the overall progress.

8.8.6 Pause and resume the Automated Acquisition run

There can be multiple reasons to pause an Automated Acquisition run, for example to refill an LN2
Dewar.
While a run is paused, it is not possible to adjust any settings, such as the Acquisition and Optics
Presets, the size and spacing of the Foil Holes on a Quantifoil specimen, or the spacing of the
Acquisition Area pattern on a Lacey Carbon specimen. If a setting needs to be adjusted, the run
must be stopped and restarted after the adjustments are done.

1. Select Pause

The Pause button changes into a Resume button.

2. Perform the activities for which the Automated Acquisition run needed to be paused.
3. Select Resume

The Resume button changes back into a Pause button.

When the Session Setup > Acquisition Mode is Faster acquisition, then EPU recalculates the
Foil Hole groups before data acquisition starts again.

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8.8.7 Skip a Grid Square, Foil Hole or Acquisition Area

During the Automated Acquisition run EPU performs various automated functions. Depending on the
results of the functions, EPU can decide to skip a Foil Hole or Acquisition Area, or even to skip an
entire Grid Square. The number of skipped locations is reported in the Progress panel.

It is also possible to manually skip a location.

● If the quality of the current Grid Square results in too many skipped Foil Holes or Acquisition
Areas, select Skip Gridsquare to move on to the next Grid Square.
● If the quality of the current Foil Hole or Acquisition Area causes a delay, or will surely result in
failure of an automated function, select Skip Foilhole or Skip Area.
When the Session Setup > Acquisition Mode is Faster acquisition then EPU automatically
recalculates the Foil Hole grouping.

8.8.8 Troubleshoot an ongoing Automated Acquisition run

If the automated run does not perform as expected, it is possible to adjust the settings:
1. Select Stop

The Stop button changes into a Start Run button, and the processing status of the Grid Squares
and the selected areas are stored in the EPU Session file.
2. Depending on the type of failures, adjust the Preparations tab > Acquisition and Optics
Presets values.
Note Be very careful! Some adjustments may invalidate certain calibrations. See the descriptions of the
Presets, Calibrations and Auto Functions to check if additional updates are necessary.

3. For Holey carbon grids:

● If execution of the Foil Hole Template fails often, then return to the Hole Selection task,
right-click on a Foil Hole in the Image Display and select Move stage to location to center a
Foil Hole. Then select the Template Definition task to adjust the Foil Hole Template.
If the Grid Square has been (partially) processed already, then the crosshair that marks the
stage position may not move the exact center of the Foil Hole. See The impact of Hole
Position Refinement on the 'Move stage to location' function on page 139 for an explanation
of such a possible offset.
● If the Find And Center Hole function fails often, use the Hole Selection task > Measure
Hole Size tool to verify the Foil Hole diameter and spacing.

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● In a Session type: Manual session, the Foil Hole center positions are already defined by
their XYZ-coordinates during the Hole Selection task, so an adjustment of the Foil Hole
spacing will have no effect.
To update the Foil Hole positions, return to the Square Selection task and Hole Selection
task to create a new selection of Foil Holes with more accurate coordinates.
● In a Session type: Automated session, an adjustment of the Foil Hole diameter and/or
spacing will have an impact on the Find And Center Hole function, but it may also have
an impact on the Find Holes detection algorithm. These functions use the Hole/
EucentricHeight Preset and the GridSquare Preset. If these Presets do not have a
parallel beam and/or do not use little to no defocus, then the hole diameters in the
acquired images may deviate significantly from the physical specimen.
4. (Optional) Change the processing status of one or more Grid Squares from Processed (blue)
back to Open (green).
In EPU > Square Selection, right-click on a processed Grid Square and select Reopen

5. Select Start Run

EPU reads the EPU Session file and starts the Automated Acquisition run from where it was
stopped earlier. Even if the EPU software is closed and opened again, the Automated Acquisition
run will start again from where it was stopped.

8.8.9 Phase Plate position logging

For each acquisition, the used Phase Plate positions are logged in the XML files that are associated
with that acquisition.
This logging facilitates Phase Plate mapping in the post-processing pipeline, where the measured
phase shift from the CTF estimation can be correlated with the used Phase Plate positions. The
Phase Plate position logging itself does not qualify the used positions as good or bad.

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9 The EPU Multigrid Option

Note EPU Multigrid is a licensed option and might require a paid upgrade to your system.
For more information, contact [email protected]

On a system with an Autoloader, the EPU Multigrid option makes it possible to prepare a queue of
individual EPU Sessions, and then start data acquisition for the entire prepared queue. It is possible
to prepare one EPU Session per specimen, but is is also possible to prepare multiple EPU Sessions
for a single specimen. If the Multigrid queue involves specimens from multiple Autoloader slot
positions, then EPU will automatically exchange the specimen between consecutive EPU Sessions.
The sections below explain how to create, edit and execute an EPU Multigrid experiment.

9.1 Create a queue with EPU Multigrid Sessions

To prepare an EPU Multigrid experiment, follow the steps below:

1. Perform the tasks in the Preparation and Auto Functions tabs.

There is no preference for a specific specimen to use for these tasks.
Note Do not perform the Preparation > Calibrate I0 task.

The Atlas Alignment function does not correct the coordinates of the I0 Calibration position.
Because the specimens are unloaded and reloaded after the I0 Calibration is performed, the I 0
reference value measurements during the Automated Acquisition run may be less reliable.
2. In the Preparation > Calibrate I0 task, select Remove I0 Measurements
If there is no calibrated value, then the Remove I0 Measurements button is not enabled.
3. In Atlas > Screening, acquire an Atlas for each specimen that will be included in the Multigrid
experiment.
4. Select New Queue

5. If a EPU Multigrid Session is currently active, then choose if the new EPU Multigrid Session uses
the preferences from the current EPU Multigrid Session.

● Yes: create a new EPU Multigrid queue and create an EPU Multigrid Session that uses
mostly the same settings and values as the currently active EPU Session. For an overview of
the copied settings and values, see The EPU Session Preferences file on page 95 .
● No: create a new EPU Multigrid queue and EPU Multigrid Session without pre-selected
settings and values.

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● Cancel: no new EPU Multigrid queue is created. The current EPU (Multigrid) Session stays
as it is.
EPU creates an EPU Multigrid Session for the specimen that is currently loaded on the stage,
and moves on to the Session Setup task.
6. Perform the Preparation tasks for the first EPU Session in the Multigrid queue.
If necessary, the settings and calibrations in the Preparation tab and/or Auto Functions tab can
be updated.
a. Perform the Session Queue task.
For instructions, see: Session Setup task on page 98 .
For an EPU Multigrid Session, the Session type is always Automated. Manual target
selection is not supported.
b. Perform the Square Selection task.
For instructions, see: Square Selection task on page 107 .
c. For a Holey carbon grid,
perform the Hole Selection, Template Definition and Template Execution tasks.
For instructions, see:
● Hole Selection task for Quantifoil specimens on page 126 .
● Template Definition task for Quantifoil specimens on page 141 .
● Template Execution task for Quantifoil specimens on page 151 .
d. For a Lacey Carbon grid,
perform the Area Selection task.
For instructions, see: Area Selection task for Lacey Carbon specimens on page 112 .
7. (Optional) Select Save preferences to store an *.epuSessionPreferences file.

Specify a filename that is easily recognizable, so that the stored preferences can be selected
later for a similar specimen. See The EPU Session Preferences file on page 95 for an overview
of the stored settings and calibrations.
8. (Optional) Specify Queue > Max Exposures to limit the number of acquisitions in this EPU
Multigrid Session.

The Max Exposures value can changed at any time during Preparation and Execution, also when
a different EPU Session is active.
9. Add one or more new EPU Multigrid Sessions to the queue.
For each new EPU Multigrid Session:
a. (Optional) Load a different specimen on the stage.
For instructions, see: Load a specimen on the stage on page 87 .

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It is also possible to create multiple EPU Multigrid Sessions for a single specimen, for
example for different sets of Grid Squares.
b. Select Add Session or Add from Preferences to create a new EPU Multigrid Session for
the specimen that is currently loaded on the stage.

c. Perform the Preparation tasks as described in step 3 above.

If necessary, the settings and calibrations in the Preparation tab and/or Auto Functions tab
can be updated.
d. (Optional) Select Save preferences to store an *.epuSessionPreferences file.
e. (Optional) Specify Queue > Max Exposures to limit the number of acquisitions.

9.2 Edit an EPU Multigrid Session in the Queue

To update the settings of an EPU Multigrid Session, follow the steps below:

1. Select the Queue > EPU Multigrid Session that needs to be edited.
To select a session in the Queue, double-click on it.
2. (Optional) Specify Queue > Max Exposures to limit the number of acquisitions in this EPU
Multigrid Session.

The Max Exposures value can changed at any time during Preparation and Execution, also when
a different EPU Session is active.
3. (Optional) In the Preparation tasks, change the desired settings and values.
If necessary, the settings and calibrations in the Preparation tab and/or Auto Functions tab can
be updated.
Note If the specimen of the selected EPU Multigrid Session not currently loaded on the stage,
then all actions that require a stage move or that require an image acquisition are not available.

The Slot number of the currently loaded specimen has a green highlight.
4. (Optional) Select Save preferences to store a new *.epuSessionPreferences file,
or to update an existing file that contains the preferences for this EPU Multigrid Session.

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9.3 Remove an EPU Multigrid Session from the Queue

Note In the current EPU version it is not possible to remove an EPU Multigrid Session from the queue.

To exclude an EPU Multigird Session from data acquisition, set Queue > Max Exposures to 0
(zero).

9.4 Change the order of the EPU Multigrid Sessions in the Queue
To change the order in which the EPU Multigrid Sessions will be processed during the Automated
Acquisition, drag and drop the EPU Multigrid Sessions to their desired position in the Queue.
It is not possible to change the order in the Queue while data acquisition is ongoing or paused. It is
only possible to change the order in the Queue before data acquisition starts, or after data
acquisition is stopped.

9.5 The Automated Acquisition task in the EPU Multigrid workflow

In the Multigrid workflow, the Automated Acquisition task offers functions to:
● Perform automated acquisition for the entire EPU Multigrid Queue.
● Perform automated acquisition for a single EPU Multigrid Session from the queue.

9.5.1 Perform Automated Acquisition for a single EPU Multigrid Session from the
Queue
To acquire data for only one of the prepared EPU Multigrid Sessions from the Queue, follow the
steps below:

1. In the Session Queue task,

select the Queue > EPU Multigrid Session for which data acquisition must be performed.
2. If the microscope is in EFTEM mode,
then specify the Auto Zero Loss settings:

a. Select Auto Zero Loss: Yes to enable periodic execution of the Auto Zero Loss function
during the Automated Acquisition run.
b. Specify the Periodicity

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3. If the system has a Gatan filter with Gatan K3 camera,

then specify the Dark Reference Settings:

a. Select Dark reference acquisition: Yes to enable periodic acquisition of Dark Reference
images during the Automated Acquisition run.
b. Specify the Periodicity
4. (Optional) If available, enable the Smart Plugins > Skip Gridsquare Prediction plugin.

This Smart Plugin uses the data that is acquired during the Automated Acquisition, and which is
evaluated by EPU Quality Monitor (EQM). If the quality of the acquired data is not good, then the
Skip Gridsquare Prediction plugin sends a recommendation to EPU to skip all remaining
acquisitions on the current Grid Square. The Skip Gridsquare Prediction plugin will never
recommend to skip the first Grid Square.
5. Select Start Run

The Start Run button changes into a Stop button.

Before the automated acquisition starts, EPU runs a preconditions check.
● If no issues are found, then the automated acquisition run starts.
● If issues are found, then EPU displays these in the Messages side panel, and the automated
acquisition run is not started.
6. Monitor the Automated Acquisition run during the first few data acquisitions.
7. If there are no irregularities, select the desired Options:

a. Select Close Col. Valves.

When active, EPU closes the Column Valves after the Automated Acquisition run is
completed.
b. On systems with a thermionic gun, select Switch Off Emission (not shown in the image
above).
When active, EPU switches off the emission, or brings the electron source to a safe standby
state after the Automated Acquisition run is completed.

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The actions and functions to pause, resume, stop and troubleshoot the Automated Acquisition run
are the same as in a single specimen experiment. For descriptions and instrucions, see: Automated
Acquisition task on page 152 .

9.5.2 Perform Automated Acquisition for all EPU Multigrid Sessions in the Queue
To successfully complete an Automated Acquisition run in an EPU Multigrid experiment, the following
prerequisites must be fulfilled for each specimen in the queue:
● The tasks in the Preparations tab and the Auto Functions tab must be completed.
● An Atlas must be available.

To perform an Automated Acquisition run for the entire Multigrid Queue, follow th esteps below:

1. If the microscope is in EFTEM mode,

then specify the Auto Zero Loss settings:

a. Select Auto Zero Loss: Yes to enable periodic execution of the Auto Zero Loss function
during the Automated Acquisition run.
b. Specify the Periodicity
2. (Optional) If available, enable the Smart Plugins > Skip Gridsquare Prediction plugin.

This Smart Plugin uses the data that is acquired during the Automated Acquisition, and which is
evaluated by EPU Quality Monitor (EQM). If the quality of the acquired data is not good, then the
Skip Gridsquare Prediction plugin sends a recommendation to EPU to skip all remaining
acquisitions on the current Grid Square. The Skip Gridsquare Prediction plugin will never
recommend to skip the first Grid Square.
3. (Optional) In the Queue, drag the EPU Multigrid Session for the currently loaded specimen to
the top of the list.
Although it is not strictly necessary, this will save time because the Automated Acquisition
doesn't have to start with a specimen exchange action.
4. Select Start Queue

The Start Queue button changes to a Stop button.

The Start Run button (for individual EPU Multigrid Sessions) changes to a Skip button.
Before the automated acquisition starts, EPU runs a preconditions check.
● If no issues are found, then the automated acquisition run starts.

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● If issues are found, then EPU displays these in the Messages side panel, and the automated
acquisition run is not started.
5. Monitor the Automated Acquisition run during the first few data acquisitions.
6. If there are no irregularities, select the desired Options:

a. Select Close Col. Valves.

When active, EPU closes the Column Valves after the Automated Acquisition run is
completed.
b. On systems with a thermionic gun, select Switch Off Emission (not shown in the image
above).
When active, EPU switches off the emission, or brings the electron source to a safe standby
state after the Automated Acquisition run is completed.
7. (Optional) Pause or skip or stop data acquisition.
● To pause and resume the currently ongoing EPU Multigrid Session,
see: Pause and resume the Automated Acquisition run on page 158 .
While acquisition is paused it is not possible to re-order the sessions in the Multigrid Queue.
● To abort data acquisition from the current Grid Square, Foil Hole or Acquisition Area,
see: Skip a Grid Square, Foil Hole or Acquisition Area on page 159 .
● Select Skip to abort data acquisition for the currently ongoing EPU Multigrid Session, and
move on to the next session in the Queue.

● Select Stop to abort data acquisition for the Mutligrid Queue.

The Stop button changes to a Start Queue button.

After data acquisition for the Queue is stopped, it is possible to troubleshoot or to change the
order of the Queue.

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10 Inspect the Acquired Images

10.1 View the images in Windows Explorer and Photo Viewer

Every image acquisition in EPU results in a number of stored files:
● All cameras:
● A high quality MRC or TIFF file.
● Additional MRC file(s) with the Dose Fraction images.
Only if the Intermediate Images functionality from a Direct Detection camera is used.
● An XML file with meta data.
● Thermo Scientific Falcon 4: EER data with an integrated image.
For EER data there are no commonly available viewers yet.
● For the Atlas, EPU stores a lower resolution JPEG file with compressed image data for each tile.

10.2 View the JPEG images

To view the JPEG files on any computer:
1. In Windows Explorer, go to the directory where the images are saved.

2. Select a JPEG image file.

Although TIFF images can be supported by Window's native image viewer, the TIFF format variation
that is used by the microscope may not be readable without installing a codec or an image viewer
with a broad TIFF format support.

10.3 View the MRC, EMI, DMX and other microscope images
The high resolution MRC files can be viewed and post-processed with most electron microscopy
post-processing applications.

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It is also possible to view the acquired images with the built-in Windows Photo Viewer:

1. Install the Thermo Scientific Imaging Codec Pack on the computer.

With the Imaging Codec Pack, Windows Explorer and Windows Photo Viewer support many
image formats that are commonly used by FEI and Thermo Scientific systems and software,
such as *.mrc files, *.emi files (TIA) and *.dmx files (Gatan Digital Micrograph).
2. Reboot the computer.
3. In Windows Explorer,
a. Go to the directory where the images are saved.
b. Open an image file in the default image viewer.
If an *.mrc file contains a stack of images, all the individual images can be viewed.

10.4 View and post-process MRC images with Thermo Scientific Velox
software
MRC images can be viewed and post-processed:
● On the Microscope PC with the Thermo Scientific Velox Online Processing software.
● On any other computer with the Thermo Scientific Velox Offline software.

To open an MRC image in Velox:

1. Open Velox Online Processing or Velox Offline.
2. Drag and drop the MRC image file in the Velox window.

See the Velox User Manual for detailed descriptions and instructions.
The Velox software does not provide 3D reconstruction functionalities.

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11 Detailed Preconditions for Successful EPU Usage

This chapter describes the preconditions that must be fulfilled for high quality data acquisition. Most
preconditions involve microscope alignments and calibrations.
Unless the alignment or calibration procedure describes otherwise, make sure all alignments and
calibrations are done:
● At a central stage position.
● At eucentric height.
● In focus.
● Preferably with a parallel beam.

The instructions and verifications in this chapter typically use the TEM User Interface > CCD/TV
Camera control panel and TIA for image acquisition and inspection. Depending on the camera type,
other applications may be more suitable to acquire and inspect images:
● Thermo Scientific Falcon 4(i): use Velox.
● Stand-alone Gatan cameras: use Gatan Digital Micrograph.

11.1 Preconditions for the specimen and specimen holder

Side entry holders must be cleaned and pumped down before insertion into the microscope.
After insertion into the microscope:
1. Manually adjust the eucentric height of the specimen, preferably at or near the center of the
specimen.
2. Roughly focus the specimen.

11.2 Preconditions for the microscope

The microscope must meet the following conditions:
● The column alignments must be completed.
● Magnification Calibrations for LM and SA ranges must be completed.
● High Tension is stable.
● For Thermionic instruments, the gun saturation (heating) must be optimized and the emission
chosen.
If necessary, optimize the gun settings via the Gun Tilt and Gun Shift Direct Alignments.
● FEG registers, Alignment files and calibrations should form a consistent set.
● Apertures are properly centered.
● Focus calibration must be completed.
This task is performed in EPU.
Note To work in both Nanoprobe and Microprobe modes, the focus calibration must be done separately
for both modes.

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● Direct alignments should be checked and, if necessary, adjusted in the modes that are used for
EPU.
These include, but may not be limited to the LM and SA range, Nanoprobe and Microprobe
modes.
● Bias/Dark and Gain Reference images are available and well averaged for all cameras.
● The camera is cooled and at a stable temperature.
● After any actions that may have introduced exceptionally strong drift, such as inserting a holder,
enough time should be allowed for settling.

11.2.1 Clear the Apertures > Options > React on Optical Mode Changes option

Note Do not use the React on optical mode changes function to change the apertures automatically.

Among others, the React on optical mode changes function automatically returns to the C2 aperture
that was selected the previous time that the optical mode was used. This may conflict with the C2
Aperture value in the Acquisition and Optics Presets.

11.2.2 Disable Beam Settings > Intensity Zoom

On systems with two condenser lenses, make sure that TEM User Interface > Beam Settings > Int.
Zoom is off when using magnifications below 10.000X.

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11.3 Use the Sherpa APM function to maintain the microscope

alignments
On Krios systems with Titan 2.13 or later, and on Glacios systems with Talos 1.13 software or later,
Sherpa APM is available to optimize and maintain the microscope alignments. Please see the
Sherpa User Manual for instructions.

11.4 Preconditions for the microscope alignments

High quality data acquisition relies on an accurate lens series alignment. The system alignments are
generally quite stable and do not require frequent adjustment. Only adjust the system alignments
when absolutely necessary.

11.4.1 Gun and Condenser

When switching between Acquisition and Optics Presets, the beam should ideally not change in
position. This is achieved by the Spot Size Dependent Gun Shift alignment (gun part) and the
Condenser (zoom) alignments.
Verify that the C2 aperture is centered before performing the tests described below.

11.4.1.1 Test the Gun Alignment

1. Select Spot Size: 2

2. Adjust the Intensity, so that the beam diameter matches the smallest circle on the FluScreen.
3. Center the beam on the FluScreen.
4. Verify the Gun Alignment for Spot Sizes 2 to 11:
a. Increase the Spot Size number by one step.
b. Verify that the beam stays centered.
If the verification in step 4 fails:
● Perform all Alignments control panel > Gun alignments.
● Repeat steps 1 to 4.

5. When Spot Size 11 is checked, verify the Gun Alignment in reverse sequence:
a. Decrease the Spot Size number by one step.
b. Verify that the beam stays centered.
If the verification in step 5 fails:
● Perform all Alignments control panel > Gun alignments.
● Repeat steps 1 to 5.

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11.4.1.2 Test the Condenser Alignment

1. Select the highest SA magnification.

2. Adjust the Intensity, so that the beam diameter matches the smallest circle on the FluScreen.
3. Center the beam.
4. In the TEM User Interface > Beam Settings control panel:
● Titan: enable Auto Zoom
● Talos:enable Intensity Zoom
5. Verify the Condenser Alignment for each SA magnification:
a. Decrease the magnification by one step.
b. Normalize all lenses.
c. Verify that the beam stays centered.
d. Verify that:
● Titan: the beam size does not change.
● Talos: the beam intensity does not change.
If the verifications in steps 5c and 5d fail, perform the Align HM-TEM > All SA Magnifications
alignment.
6. In the TEM User Interface > Beam Settings control panel:
● Titan: disable Auto Zoom
● Talos:disable Intensity Zoom

11.4.1.3 Test the Parallel Beam range on Titan Systems

Perform this test in the following optical modes:

● TEM nP
● TEM uP
● EFTEM nP (if a filter is present)
● EFTEM uP (if a filter is present)

1. Insert the FluScreen.

2. If the system is not in Diffraction mode, select Handpanels > Diffraction .
3. Select Camera Length: 800 mm
4. Select Handpanels > Eucentric Focus

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5. Decrease the Intensity to the smallest value at which the Beam Settings control panel >
Illumination is Parallel.

6. On the FluScreen, check that the diffraction pattern circles for gold are sharply defined.
7. Take note of the Area value.
8. Slowly increase the Intensity until the Beam Settings control panel > Illumination is no longer
Parallel.
9. Take note of the Area value.
10. Decrease the Intensity, so that the Area value is the average of the lower and upper Area
values that are noted in steps 7 and 9.
11. On the FluScreen, check that the diffraction pattern circles for gold are sharply defined.
If the diffraction pattern circles are not sharply defined at step 11, perform the Align HM-TEM >
Basic SA alignment.

11.4.1.4 Test the Condenser Zoom on Titan Systems

Perform this test in the following optical modes:

● TEM nP
● TEM uP

1. Select the lowest SA magnification.

2. With the Handpanels > Intensity knob, decrease the Beam Settings control panel >Area, so
that the Illumination changes from Parallel to Condensing.
3. Center the beam.
4. With the Intensity knob, slowly increase the illuminated area so that the Illumination is Parallel
again.
5. Verify that the beam is still centered.
6. With the Intensity knob, slowly increase the illuminated area further.
7. Check that the beam stays centered for as long as Illumination is Parallel.
If the check at step 7 fails, perform the Direct Alignments control panel > Condenser Zoom
alignment and repeat steps 1 to 7.

8. Center the beam on a feature of known size.

9. With the Intensity knob, adjust the beam diameter so that it matches the size of the feature.
10. Verify that the Area value corresponds with the known feature size.

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If the check at step 10 fails, perform the Direct Alignments control panel > Condenser Zoom
alignment and repeat steps 1 to 10.

11.4.1.5 Test the Condenser Focus Alignment

Perform this test in the following optical modes:

● TEM nP
● TEM uP

1. With the Handpanels > Intensity knob, condense the beam to the smallest achievable spot.
2. Verify that the Beam Settings control panel >Area, is 0±100 nm
If this check 2 fails:
a. Perform the Condenser > Condenser Preparation alignment.
b. Depending the used optical mode, either:
● Perform the Focus + calib nP alignment.
● Perform the Focus + calib nP alignment.

11.4.2 Deflectors
The image/beam shift calibration must have been done carefully:
● To keep the illumination centered when some image shift is added.
● To shift the image when you want to move the beam to another area of the specimen.
Both Image and Beam Shift pivot points must be aligned accurately so the illumination conditions
(coma, rotation center) and crossover shifts (GIF energy selection) do not change when applying
image (beam) shift.

11.4.2.1 Test the Image/Beam Shift Calibration

1. Select a high SA magnification

2. With the Handpanels > Intensity knob, adjust the beam diameter, so that it matches the largest
circle on the FluScreen.
3. In the TEM User Interface > Image Settings control panel, select MF knobs
4. With the Handpanels > MF-X and MF-Y knobs, shift the image roughly 50% of the beam
diameter in the positive and negative X and in Y directions.
5. Check that the beam stays centered.
6. Select an SA magnification of approximately 50 kX, so that the field of view spans
approximately 15 grid squares.
7. With the Handpanels > Intensity knob, adjust the beam diameter, so that it matches the largest
circle on the FluScreen.
8. In the TEM User Interface > Image Settings control panel, select MF knobs
9. With the Handpanels > MF-X and MF-Y knobs, shift the image roughly 50% of the beam
diameter in the positive and negative X and in Y directions.
10. Verify that the beam stays centered.

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If the check at step 5 and/or step 10 fails, perform the Calibrate HR-TEM > Image/Beam
Calibration alignment.

11.4.3 Projector

11.4.3.1 Test the Lens Series Magnification Center Alignment

The LM and HM lens series must be aligned accurately to ensure that a centered feature stays
centered when switching to another magnification. The alignment must be performed accurately on
the camera that is used for data acquisition.

1. With the Handpanels > Magnification knob, select the highest Acquisition and Optics
Presets magnification
2. In the TEM User Interface > Image Settings control panel, verify that no image shift is applied.
3. In the TEM User Interface > CCD/TV Camera control panel:
a. Select the Camera that is used for data acquisition.
b. Select Search
4. Move the specimen, so that a feature is visible in the image center that is also recognizable on
the camera at the Atlas Preset magnification.
5. Place a marker on the recognizable feature.
6. With the Handpanels > Magnification knob:
a. Step down through all Acquisition and Optics Presets magnifications and check if the feature
stays aligned with the marker.
b. Step up again through all Acquisition and Optics Presets magnifications and check if the
feature stays aligned with the marker.

This alignment is often done on the FluScreen, which might have a different magnification center.
Perform the Preparation tab > Calibrate Image Shifts task to compensate for magnification center
offsets.

11.4.3.2 Test the Eucentric Focus

The HM eucentric focus preset should not deviate more than a few microns from the true focus at
stage eucentric height.

1. If not visible yet, add the Defocus value to the TEM User Interface > Information Panels.
2. Accurately bring the specimen to eucentric height by using the stage wobbler.
3. Select a higher SA magnification (approx. 150kX).
4. Select Handpanels > Eucentric Focus.
5. Reset Defocus.
Either:
● Select the Handpanels > User button that is assigned to Reset Defocus.
This is often R2.
● Select TEM User Interface > Image Settings control panel > Reset Def.
6. Use the Handpanels > Focus knob to accurately focus the image.
7. Verify that the Information Panel > Defocus value is 0±500 nm.

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If the check at step 7 fails, either:

● Perform the Align HM-TEM > Basic SA alignment
● Perform the Auto Functions tab > Calibrations: Eucentric Correction task to compensate for
the eucentric offset

11.4.3.3 Test the Magnification-Dependent Focus

The lens series alignment should be parfocal as much as possible, so switching magnification does
not significantly affect (de-)focus. For the LM range and the lower magnifications of the HM series,
this alignment is often not performed with maximum achievable accuracy.

1. Insert the FluScreen.

2. Select a low LM magnification.
3. With the Handpanels > Intensity knob, condense the beam to the smallest achievable spot.
4. Select Handpanels > Wobbler.
5. With the Handpanels > Focus knob, adjust focus so that the spot does not move.
6. Reset Defocus.
Either:
● Select the Handpanels > User button that is assigned to Reset Defocus.
This is often R2.
● Select TEM User Interface > Image Settings control panel > Reset Def.
7. For all LM magnifications, check the magnification-dependent focus:
a. Increase the magnification one step.
b. Reset Defocus.
c. Adjust focus so that the spot does not move.
d. Verify that the defocus value is less than 1 mm.
If the check at step 7d fails, perform the nP calibrations and/or LM uP calibrations.
8. For the lower HM magnifications, check the magnification-dependent focus:
a. Increase the magnification one step.
b. Reset Defocus.
c. Adjust focus so that the spot does not move.
d. Verify that the defocus value is less than 2 um.
9. Select Handpanels > Wobbler to stop the beam wobbler.
10. For the lower HM magnifications, check the magnification-dependent focus:
a. Increase the magnification one step.
b. Reset Defocus.
c. Adjust focus so that the image has minimum contrast.
d. Verify that the defocus value is less than 2 um.
If the checks at step 8d and/or 10d fail, perform the nP calibrations and/or HM uP calibrations.

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11.4.3.4 Test the Magnification-Dependent Beam and Image Shift - TEM mode

1. Insert the FluScreen

2. Switch to TEM MicroProbe
3. Select the highest LM magnification
4. Move the specimen, so that an easily recognizable feature is located in the center of the
FluScreen.
5. With the Handpanels > Intensity knob, adjust the beam diameter, so that it covers
approximately 80% of the FluScreen.
6. Assign the Multifunction X and Y knobs to Image Shift
7. Use the Multifunction X and Y knobs to shift the recognizable feature away from the center, to a
location near the smallest circle on the FluScreen.
8. In the Flucam Viewer:
a. Place a marker on the recognizable feature
b. Place a circle around the circumference of the beam
9. Increase the magnification one step to the lowest SA magnification
10. In the Flucam Viewer:
a. Measure the distance between the recognizable feature position and the marker.
b. Measure the beam shift:
Measure the largest distance from the circle to the beam circumference.
The image shift and the beam shift from highest LM to lowest SA magnification must be less than
2 um
If the check at step 10 fails, perform the nP calibrations and/or LM uP calibrations, and repeat
steps 1 to 10.

11. Select the highest SA magnification

12. Move the specimen, so that an easily recognizable feature is located in the center of the
FluScreen
13. With the Handpanels > Intensity knob, adjust the beam diameter, so that it covers
approximately 80% of the FluScreen.
14. Assign the Multifunction X and Y knobs to Image Shift
15. Use the Multifunction X and Y knobs to shift the recognizable feature away from the center, to a
ocation near the smallest circle on the FluScreen.
16. In the Flucam Viewer:
a. Place a marker on the recognizable feature.
b. Place a circle around the beam.
17. Select the lowest HM magnification
18. In the Flucam Viewer:
a. Measure the distance between the recognizable feature position and the marker.
b. Measure the beam shift:
Measure the largest distance from the circle to the beam circumference.
The image shift and the beam shift from highest SA to lowest HM magnification must be less
than 50 nm

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If the check at step 18 fails, perform the nP calibrations and/or HM uP calibrations, and repeat
steps 1 to 18.

11.4.3.5 Test the Magnification-Dependent Beam and Image Shift - TEM mode

1. Insert the FluScreen

2. Switch to EFTEM MicroProbe
3. Select the highest LM magnification
4. Move the specimen, so that an easily recognizable feature is located in the center of the
FluScreen.
5. With the Handpanels > Intensity knob, adjust the beam diameter, so that it covers
approximately 80% of the FluScreen.
6. Assign the Multifunction X and Y knobs to Image Shift
7. Use the Multifunction X and Y knobs to shift the recognizable feature away from the center, to a
ocation near the smallest circle on the FluScreen.
8. In the Flucam Viewer:
a. Place a marker on the recognizable feature
b. Place a circle around the circumference of the beam
9. Increase the magnification one step to the lowest SA magnification
10. In the Flucam Viewer:
a. Measure the distance between the recognizable feature position and the marker.
b. Measure the beam shift:
Measure the largest distance from the circle to the beam circumference.
The image shift and the beam shift from highest LM to lowest SA magnification must be less than
2 um
If the check at step 10 fails, perform the nP calibrations and/or LM uP calibrations, and repeat
steps 1 to 10.

11. Select the highest SA magnification

12. Move the specimen, so that an easily recognizable feature is located in the center of the
FluScreen
13. With the Handpanels > Intensity knob, adjust the beam diameter, so that it covers
approximately 80% of the FluScreen.
14. Assign the Multifunction X and Y knobs to Image Shift
15. Use the Multifunction X and Y knobs to shift the recognizable feature away from the center, to a
ocation near the smallest circle on the FluScreen.
16. In the Flucam Viewer:
a. Place a marker on the recognizable feature.
b. Place a circle around the beam.
17. Select the lowest HM magnification
18. In the Flucam Viewer:
a. Measure the distance between the recognizable feature position and the marker.
b. Measure the beam shift:
Measure the largest distance from the circle to the beam circumference.
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The image shift and the beam shift from highest SA to lowest HM magnification must be less
than 50 nm
If the check at step 18 fails, perform the nP calibrations and/or HM uP calibrations, and repeat
steps 1 to 18.

11.5 Aperture Alignments

11.5.1 Check the C2 Apertures center positions on systems with a C3 lens
Three Lens Condenser systems include all Titan systems.
Not all C2 apertures are used for the acquisition of the Atlas or during the Automated Acquisition run.
Nonetheless, all C2 apertures must be properly centered in Microprobe and Nanoprobe mode. When
the condenser is in Two Lens Condenser mode (C3 off), a centered beam should expand
symmetrically without moving.

1. In the TEM User Interface > Beam Settings control panel:

a. Select Free Ctrl.

b. Select C3 off.
c. Select Microprobe.
2. Use the Handpanels> Multifunction knobs or Trackball to center the beam.
3. In the TEM User Interface > Apertures control panel:
a. Select the largest C1 aperture.
b. Select the largest C2 aperture.
c. If present, select the largest C3 aperture.
d. Retract the Objective and Selected Area aperture mechanisms.
4. Turn the Handpanels> Intensity knob on the handpanel in both directions, and:
● Check that the beam expands symmetrically.
● Check that the beam remains centered.
If the beam does not expand symmetrically or does not stay centered, adjust the C2 aperture
position.
5. Repeat step 4 for every C2 aperture.
6. Select TEM User Interface > Beam Settings > Nanoprobe.
7. Repeat steps 4 and 5.

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11.5.2 Check the C2 Apertures center positions on a system without a C3 lens

Two Lens Condenser systems include all Talos and Tecnai systems.
Not all C2 apertures are used for the acquisition of the Atlas or during the Automated Acquisition run.
Nonetheless, all C2 apertures must be properly centered in Microprobe and Nanoprobe mode. A
centered beam should expand symmetrically without moving.

1. Switch to Microprobe mode.

2. Use the Handpanels> Multifunction knobs or Trackball to center the beam.
3. In the TEM User Interface > Apertures control panel:
a. Select the largest C1 aperture.
b. Select the largest C2 aperture.
c. Retract the Objective and Selected Area aperture mechanisms.
4. Turn the Handpanels> Intensity knob on the handpanel in both directions, and:
● Check that the beam expands symmetrically.
● Check that the beam remains centered.
If the beam does not expand symmetrically or does not stay centered, adjust the C2 aperture
position.
5. Repeat step 4 for every C2 aperture.
6. Select TEM User Interface > Beam Settings > Nanoprobe.
7. Repeat steps 4 and 5.

11.5.3 Check the Objective Apertures center positions

Not all apertures are used for the acquisition of the Atlas and during the Automated Acquisition run.
Nonetheless, all Objective apertures must be properly centered in Microprobe and Nanoprobe mode.

1. Switch to HM diffraction mode.

2. Verify that all Objective apertures are accurately centered around the beam.
3. Switch to LM mode
4. Select a lower LM magnification.
5. Verify that all Objective apertures are accurately centered around the beam.
6. Select a lower SA magnification.
7. Verify that all Objective apertures are accurately centered around the beam.
Note Do not use the React on optical mode changes function to change the apertures automatically.

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Among others, the React on optical mode changes function automatically returns to the C2 aperture
that was selected the previous time that the optical mode was used. This may conflict with the C2
Aperture value in the Acquisition and Optics Presets.

11.6 Direct Alignments and Astigmatism

Direct alignments must be checked regularly.
The preconditions below describe the direct alignments for uncorrected systems. Systems with
image and/or probe correctors are not discussed here. Fine-tuning of the corrector eliminates coma,
astigmatism, and other aberrations.
● Beam Tilt Pivot Points
These should be checked at the accurate eucentric height and focus. When the beam is tilted,
the spot should not move. This is important for Autofocus and astigmation.
● Beam Shift
Center the beam when no user beam shift is applied.
Perform this Direct Alignment at the highest magnification that will be used during the Automated
Acquisition run.
● Rotation Center
When focusing, the central image features should stay on the optical axis and should not move.
Perform this Direct Alignment at the highest magnification that will be used during the Automated
Acquisition run.
● Coma-free Alignment X and Y
For acquiring high resolution images, it is important that the illumination is coma-free.
This procedure can only be done properly:
● On a thin carbon foil.
● With the camera in Search mode.
● With live FFT.
Image contrast and the FFT should not change when the beam is tilted.
Note Coma free overwrites rotation center, but the difference should not be critical.

● Astigmatism
In a simple FFT it is very difficult to distinguish astigmatism from beam tilt induced coma. Make
sure that the system is nearly coma-free before correcting astigmatism.
Use the objective stigmator to adjust astigmatism. This procedure can only be done properly:
● On a thin carbon foil.
● With the camera in Search mode.
● With live FFT.

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11.7 Calibrations
11.7.1 Magnification Calibrations
The Magnification Calibrations do not only contain information about the magnification, but also
about image rotation and how to transform shifts seen in the image to corresponding shift with stage
and image/beam deflectors. The Magnification Calibrations are generally very stable. They only need
to be renewed when the system alignments have changed significantly.
The calibrations must be present:
● For all cameras.
● For LM, SA and, if used, Mi ranges.
Use the TEM User Interface > Calibrations or Magnification Calibration control panel to perform
the Magnification Calibrations and follow the instructions in the control panel.

11.7.2 Focus Calibration

The Focus calibration is executed in the EPU > Auto Functions tab > Calibrations: Autofocus
task.
This calibration may need to be redone more frequently, as it may depend on changes in the direct
alignments. Redo this calibration if the Autofocus function does not converge nicely any more. Follow
the instructions in The Autofocus Calibration task on page 75 .

11.7.3 Eucentric Correction Calibration (Optional)

The Eucentric Correction Calibration is executed in the EPU > Auto Functions tab > Calibrations:
Eucentric Correction task. Follow the instructions in The Eucentric Correction Calibration task on
page 78 .

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Chapter | Troubleshooting: Symptoms and Solutions

12 Troubleshooting: Symptoms and Solutions

12.1 Troubleshooting: Atlas

Symptom Possible Cause Possible Solution

Atlas does not look consistent: The specimen may not be in focus. Refocus.
Large deviations in defocus will
● Tiles do not match.
cause the images to rotate and,
● Grid bars do not continue across since illumination is not parallel in
neighboring tiles. this step, the apparent
● Atlas contains a regular pattern of black magnification will also change.
areas.
If the specimen is focused, the Recalibrate.
calibrations are probably not valid
anymore and must be redone. (Has
there been a big change in
alignments or was another
alignment file loaded?)

Distortion can be inherent to Acquire an atlas at

images at very low magnification. increased magnification.

The view is partly blocked (e.g., by Acquire an atlas at

an aperture) at the selected increased magnification;
magnification. confirm with a preview (or
TIA) that the field–of–
view of a single tile is
unobstructed.

The system's beam and image shift Re–align the beam and
are not well–aligned, causing loss image shifts for LM mode
of illumination. using the TEM User
Interface (Alignments
Control, Calibrate LM–
Image/Beam calibration).

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Chapter | Troubleshooting: Symptoms and Solutions

Symptom Possible Cause Possible

Solution

“Move to” command does not center feature or The specimen may not be in focus. Refocus.
grid square. Large deviations in defocus will cause
the images to rotate and, since
Check this at the magnification at which the
illumination is not parallel in this step,
Atlas is acquired. Use the camera, not the
the apparent magnification will also
Flucam Viewer.
change.

If the specimen is focused, the Recalibrate.

calibrations are probably not valid
anymore and must be redone. (Has
there been a big change in alignments
or was another alignment file loaded?)

Distortion can be inherent to images at Acquire an Atlas

very low magnification. at increased
magnification.

The Image Shift calibration is not Accurately

performed accurately. perform the Image
Shift Calibration.

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12.2 Troubleshooting: GridSquare

Symptom Possible Cause Possible Solution

“Move to” command (in Atlas) does not Some image shift is applied (check in the Reset it.
center the Grid Square. 'Image Settings' control panel in the TEM
User Interface).
Atlas looks OK, but navigation to the grid
square is inaccurate.
If you see the same problem at Atlas Focus and/or redo the
Grid square is not centered when
magnification (see above), specimen was calibrations.
acquiring an image in the Location / Area
not focused when taking the atlas (see
Selection view.
above) or calibrations are not valid
anymore.

Rotation center is off (any change in Adjust rotation center.

focus can result in image shifts). Usually,
to enhance contrast you will have applied
a defocus of a few mm during image
acquisition.

Nonparallel beam or excessive defocus is Work with a parallel

used. (This results in small centering beam and do not use
errors.) excessive defocus.
There may always be
some small
discrepancy because
positions are calculated
and not all stage
characteristics may be
taken into account.

Imperfect LM lens series alignment and Repeat EPU image

EPU image shift calibration. When shift calibration or when
changing magnifications, a feature should shifts are large, adjust
stay centered (especially true when the LM lens series
switching between atlas and grid square alignment.
magnifications).
Test:
● Select the Preparation tab.
● Select the Acquisition and Optics
Settings task.
● Use the Acquire buttons for easy
switching with correct lens
normalizations and image shift
corrections.

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Chapter | Troubleshooting: Symptoms and Solutions

Symptom Possible Cause Possible Solution

“Move Stage to” command does not center Foil Hole or Too much defocus Make sure to work with no
feature. can change image or just a few mm defocus.
rotation. A non- (Focus specimen correctly
If you see this while staying at the magnification used for
parallel beam will by using the wobbler and
imaging the grid square, check the causes and solutions in
lead to additional specify the wanted defocus
this table.
change in in the optics settings of
If you see this problem only in the Template Definition view magnification. EPU.) If possible, use a
(at higher magnification), see “Move Stage to …” parallel beam.
command in Location Selection / Area Selection view does
not center foil holeFoil Hole or feature in Template Mechanical play in Make sure that before
Definition view.” stage. taking the grid square
image, the grid was
centered with a Move
Stage… command in the
atlas (compensates for any
backlash problems).

Calibrations are no Redo calibrations.

longer valid
(microscope
alignments have
changed?).

Note Since the feature positions are calculated and not all characteristics of the stage are known exactly, a
feature will not be centered 100% accurately.
A micron deviation is not uncommon.

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Chapter | Troubleshooting: Symptoms and Solutions

12.3 Troubleshooting: Template Definition

Symptom Possible Cause Possible

Solution

“Move Stage to …” command in Location Some image shift is applied (check this in Reset it.
Selection / Area Selection view does not 'Image Settings' control panel in the TEM
center Foil Hole or feature in Template User Interface).
Definition view.
If you see this problem also at the Bad alignment of LM lens series against HM Repeat EPU
magnification at which the grid square is lens series and bad EPU image shift image shift
imaged, see information under “Grid Square calibrations. calibration or
(Location Selection/ Area Selection View):”. when the shifts
Test:
are large, adjust
When switching between Location Selection LM lens series
view and Template Definition view alignment.
magnifications, a feature should stay
centered in the acquired images.
● Select the Preparation tab in EPU.
● Select the Acquisition and Optics
Settings task.
● Use the Acquire buttons for easy
switching with correct lens normalizations
and image shift corrections.

Symptom Possible Cause Possible Solution

“Move Stage here...” command in Mechanical play in stage. Try at least twice to make sure
Template Definition view does that a backlash correction was
not center feature. performed.

Working far from eucentric focus will Make sure the sample is at
render the magnification calibrations eucentric height, focused, rotation
invalid. Large defocus will slightly center is well corrected, and the
rotate the image and (with a defocus chosen in the Optics
nonparallel beam) magnification settings of EPU should not
changes. exceed 20 um.

Calibrations are invalid (maybe the Redo calibrations

microscope alignment changed?).

Stage may have a hardware problem Call Service

and does not move correctly over
these smaller distances

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Chapter | Troubleshooting: Symptoms and Solutions

Symptom Possible Cause Possible Solution

Yellow circle does not Correct Quantifoil type was not selected at Set the size correctly in this view.
match Foil Hole size. start of session or the size of the holes
deviates from specification.

Size of the holes deviates from specification. Set the size correctly in this view.

Measure Foil Holes/Find Foil Holes in the Return to Location Selection and
Location Selection view was not performed. do it.

Specimen is not at eucentric height for the Bring specimen to eucentric

current sample position and not focused. height and focus it.

Too large defocus is used and beam is not Select a defocus selected in the
parallel. (The apparent magnification of the Template Optics settings of EPU
image may change under these of less than 20 μm. If possible,
circumstances.) choose a parallel beam.

The Measure Foil Hole step was inaccurate. Repeat this step (Location
Selection view) with a slight
change in the size of the yellow
glasses.

Calibrations are invalid (maybe the Redo calibrations

microscope alignment changed?).

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Chapter | Troubleshooting: Symptoms and Solutions

Symptom Possible Cause Possible Solution

“Find Foil Hole” Does the yellow circle match the hole size? -
does not succeed.
If not, see above, Yellow circle does not
match Foil Hole size.

Measure Foil Holes/Find Foil Holes in the Switch to the Location Selection view and
Location Selection view not done. (The perform Measure Foil Holes and Find Foil
“find Foil Hole” algorithm uses information HoleFoil Holes.
about hole size and spacing.)

The circle size is wrong. (The algorithm of Repeat the Measure Foil Holes step
finding holes may be quite sensitive to the (Location Selection view). Change the
exact hole size.) size of the yellow glasses slightly.

Magnification is too high. Use a magnification in which neighboring

Foil Holes are at least partly visible. This
will increase the stability of the algorithm
since the spacing and orientation of the
foil holeFoil Hole pattern can be exploited.

Magnification is too low. (The current Increase the magnification such that only
algorithm does not work with too many Foil the Foil Hole and its nearest neighbors
Holes visible.) are visible on the acquired image.

The current specimen location contains too Find a cleaner sample area.
many features (crystalline ice, etc.) that
cause the algorithm to fail.

Contrast is too low. Add or increase the defocus in the optics

settings. (Make sure to start from focused
specimen, specimen at approximately
eucentric height).

Image quality too bad (noise). The human Take image with higher dose.
eye is very good at integrating over
features, so noise may easily be
underestimated at this magnification

Foil Holes appear dark when the Allow If the Foil Hole appears darker than the
Dark Foil Holes option is not selected. foil itself, select the option Allow Dark Foil
HoleFoil Holes (in ribbon of Location
Selection view).

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Chapter | Troubleshooting: Symptoms and Solutions

Symptom Possible Cause Possible

Solution

Foil Hole centering fails (or hole is not correctly The Foil Hole was not fully Reduce the
centered). visible initially and the fitted magnification.
circle degenerated to an
Tracing this problem is a combination of tracing problems
ellipse. In this case the
in:
centering will not be perfect.
● “Find Foil Hole”: see “'Find Foil HoleFoil Hole' does
not succeed”.
Can be tested separately.
● “Move stage here”: see “'Move Stage here...'
command in Template Definition view does not
center feature”.
Can be tested separately.

Symptom Possible Cause Possible Solution

Pattern acquisition Foil Hole cannot be recentered. (Pattern See above, Foil Hole centering fails
fails. acquisition requires the Foil Hole to be centered (or hole is not correctly centered).
within 300 nm.)
Test:
Click Find and Center Foil Hole button.

Auto Focus fails. See below, “Pattern acquisition: Auto

Focus fails.”.

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Chapter | Troubleshooting: Symptoms and Solutions

Symptom Possible Cause Possible Solution

Pattern Specimen is too far off from eucentric Adjust eucentric height
acquisition: height and focus. Usually during an
Auto Focus fails. automatic run of EPU, after every move to
a new grid square, the eucentric height is
adjusted. During the setup phase, you
moved to a grid square, but may not have
adjusted eucentric height

Cause: Auto focus does not converge Use the Direct Alignments panel to align the
because the beam is visible in the images. beam shift at the auto focus or data
There maybe a couple of reasons for this. acquisition magnification.

Test: Note: If the beam moves when you change

Acquire a single image with the auto focus Intensity of illuminated area, then you have to
presets (from the Preparation tab). If the align apertures or condenser system as well.
beam edge is visible on this image, then If the beam edge is not visible in the test,
the beam shift was not aligned. then it will become visible only when the
beam is tilted or shifted.

Beam tilt pivot point misaligned. Use Direct Alignments panel to align the
beam tilt pivot point.
Tip: Adjust eucentric height and focus first.
There is a little problem in that the pivot point
is well aligned only at a certain focus setting.
You may have to allow for some inaccuracy
by choosing a slightly larger beam diameter
in the focus optics setting since in the
automatic data acquisition you will not always
start close to focus.

The image/beam shift calibration is not Perform the Calibrate HM-TEM > Image/
done. Beam calibration (use the 'Alignments' control
panel in the TEM User Interface).
Test:
When you add some image shift in the
Image Settings control cluster, do you then
see the beam edge moving into the field of
view?

Auto focus does not converge, although the Redo focus calibration
beam edge is not visible; invalid focus
calibration.
Test with the standalone Auto Focus
routine.

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Chapter | Troubleshooting: Symptoms and Solutions

Symptom Possible Cause Possible Solution

Pattern acquisition: Acquire a single image with the Data Use the Direct Alignments panel to
beam edge visible in Acquisition presets (Preparation tab). align the beam shift data acquisition
data acquisition magnification.
If the beam edge is visible on this image, this
means Beam shift was not aligned correctly in Note: If the beam moves when you
the first place. change intensity of illuminated area,
align apertures or condenser system
as well.

When you add some image shift in the Image Perform HM-TEM > Image/Beam
Settings control cluster, do you then see the calibration ('Alignments' control panel
beam edge moving into the field of view? If so of the TEM User Interface).
Image/beam shift calibration is not done.

Symptom Possible Cause Possible Solution

Pattern acquisition: you use Auto Focus by Stage Z Adjustment, maybe the Try again. This time the Z
acquisition areas are first time Z had to be adjusted a lot, which leads to adjustment should be
not placed correctly. displacements in X and Y. small.

If you use Auto Focus by Change of Objective Lens Use the Direct Alignments
Current, focusing may shift the feature (hole) when the panel to align the rotation
rotation center is not well aligned. center.

EPU image shift calibration no longer valid or not done. Redo the EPU image shift
calibrations. If the shift is
Use the Preparation tab to acquire images with the Hole/
very large, redo the
EucentricHeight preset and Data Acquisition preset.
microscope HM image shift
Does a given feature stay centered? If yes, then the lens
alignment first.
series alignment is not well done and the deviations are
not captured by the EPU image shift calibration.

Image beam shift calibration not valid. (If you use image/ Perform HM-TEM > Image
beam shift only to place the acquisition areas, placement Beam calibration
should generally be quite accurate.) ('Alignments' control panel
of TEM User Interface).

Magnification calibration not valid. Redo the magnification

calibrations.
Test:
Check the validity of the calibrations by using the Move
Stage Here… feature. However, in these stage
movements, some inaccuracy may have to be accepted
due to the mechanics of the stage.

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Chapter | Troubleshooting: Symptoms and Solutions

12.4 Troubleshooting: Automated Acquisition

Symptom Possible Cause Possible Solution

Grid squares The eucentric height procedure is failing. Increasing the number of counts per
are all skipped. exposure may help if the images are
The performance of the auto-eucentric height
very noisy. The focus calibration may
procedure can be tested standalone in the Auto
have to be redone.
Functions tab. During automated data acquisition,
the procedure is performed with the Hole/
EucentricHeight optical preset (a different defocus
may be applied). The procedure used is the one that
is based on a focus measurement and, therefore, a
valid focus calibration is needed.

No holes are found. A grid square image is acquired, Redo the “Measure Foil Holes”
and auto eucentric height succeeds, but then no Foil procedure that should have been
Holes were found. Measure Foil Holes procedure done during the setup phase
was not set up correctly. (Location Selection task).

No Foil Holes are selected. A grid square image is Redefine the filters (go to the
acquired, holes are found, but none is selected by Location/Area Selection). If the
the ice thickness filters. intensity changes due to an instability
of the gun, use the I0 calibration in
EPU in addition. It will periodically
measure the intensity in a hole and
correct the ice filters accordingly.

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Chapter | Troubleshooting: Symptoms and Solutions

Symptom Possible Cause Possible Solution

Too many Foil They are not found in the grid Repeat setup steps
Holes: square image in the first place
target areas are because step(s) were omitted in
skipped. the setup phase.

They do not pass the ice filtering. Go to the Location/Area Selection view and adjust the
Either the illumination conditions ice filter settings
or acquisition settings have
changed after the filters have been
selected or the filters were tuned
on a grid square with too thin/thick
ice.

Foil Holes were selected in the For troubleshooting, see “'Find Foil Hole' does not
grid square image, but could not succeed.” and “Foil Hole centering fails (or hole is not
be found or centered at the Foil correctly centered)”.
Hole magnification. A Foil Hole is
skipped if it could not be centered
within certain accuracy

Auto focus fails. A Foil Hole is For trouble shooting, see “Pattern acquisition: Auto
skipped if the focus could not be Focus fails.”.
determined.
During the automatic data acquisition, auto focus may
also fail if the lens series focus presets are (very) ill-
defined. The routine is based on the measurement of
beam tilt-induced image shifts that are proportional to
the amount of defocus (it works like the wobbler on the
hand panel).
For valid measurements, the images must overlap by
at least 60%, which means that the range that the auto
focus can cover is limited, especially when run at high
magnification. Initially, eucentric height and focus are
adjusted at the magnification used for centering the
Foil Hole (before an image of the grid square is
acquired), but later the auto focus routine is run at a
much higher magnification typical for data acquisition.
If the magnification switch results in a large focus
change, auto focus may fail. In this case, correct the
objective lens presets of your lens series (part of the
lens series alignment).

Symptom Possible Cause Possible Solution

Acquisition areas are See “Pattern acquisition: acquisition areas are not placed correctly"
not placed correctly.
When you have chosen Z adjustment as the method of Little to nothing can be
auto focus, Z adjustments will always induce some X and done to fix this.
Y movements of the specimen. Big adjustments can be a
consequence of a non-flat or tilted specimen ().

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Chapter | Troubleshooting: Symptoms and Solutions

Symptom Possible Cause and Solution

Beam is
shifted in
Autofocus
routine.

For high magnifications (> ~75 kX) and small beam sizes as shown in the image above, it can
happen during the Autofocus routine that the beam seems slightly shifted, resulting in a not
completely illuminated detector.
This is usually due to one or both of the following causes:
● Beam tilt pivot points not set correctly
● A too large value in Autofunctions > Autofocus > Iterate to:.
To resolve:
● Make sure you are at eucentric height (run Autofunctions > Eucentric Height or set
manually).
● Select Eucentric focus on the hand panel; a well aligned TEM will now be in focus.
If not, carefully focus manually or use Autofunctions > Autofocus at a lower magnification.
● Select Reset defocus.
Defocus value in the TEM User Interface is now '0'.
● Perform Direct Alignments > beam tilt pivot points.
● Go to the desired optical settings for Autofocus.
● Check the number in Iterate to: in Autofunctions > Autofocus.
● Manually change defocus to this number and check the movement of the beam.
● If it is too large (as show in the image above), try the following (in the order shown below):
● Find the largest defocus value for which the beam stays on the detector and reduce
Iterate to: to the number found,
● Reduce the camera area to Half or Quarter,
● Increase the beam diameter.

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Chapter | The MRC2014 Image Format

A The MRC2014 Image Format

A.1 The Main Header and Extended Headers in an MRC file

MRC files have a generic Main Header and an optional Extended Header.
● The Main Header contains generic image information, such as the image dimensions and the
pixel format. For the specification of the main header,
see MRC/CCP4 File Format for Images and Volumes .
● The Extended Header contains application specific metadata.
For Thermo Scientific and FEI products, the extended header contains information about:
● The microscope state at acquisition time, such as magnification, accelerating voltage, stage
position, beam shift and many other relevant parameters.
● Image acquisition information, such as binning and exposure time.

Among others, the Main Header contains the following fields:

● NZ: the number of frames in the MRC file.
● NSYMBT: the reserved size for the Extended Header.
● EXTTYP: the format of the Extended Header: FEI1 or FEI2.
The FEI2 format is an extended version of the FEI1 format.
For every frame in the file, the Extended Header contains one Metadata Block. The first element of
each block contains the Metadata Block size. All Metadata Blocks in the Extended Header have the
same size and contain the same fields. The sum of the Metadata Block sizes fits within the reserved
size for the Extended Header.

A.2 The Extended Header specification

The FEI1 and FEI2 Extended Header formats allow for the addition of new fields without breaking
compatibility. When a new field is added, the Metadata Size and Metadata Version fields are
updated. Image reading and processing software can use the Metadata Size value from the first
Metadata Block to index the blocks for the other frames in the MRC file.
With the introduction of the FEI2 format, the format of the FEI1 Extended Header is frozen. For MRC
files with an FEI1 Extended Header, image reading and processing software can assume the values
of the Metadata Size and Metadata Version fields are 768 bytes and version number 0.

The tables below specify the content of the FEI1 and FEI2 Extended Headers for the MRC2014 file
format. In these tables, the Format and 'IsPresent' flag columns have to the following values:
● Format:

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Chapter | The MRC2014 Image Format

● Bool: Boolean of 1 byte (0 = false, other value = true).

● Int32: Signed integer of 4 bytes.
● Int64: Signed integer of 8 bytes (only used in FEI2 Extended Header).
● UInt32: Unsigned integer of 4 bytes.
● Float64: Floating point number of 8 bytes.
● IsPresent:
UInt32 value that is used as a 32-bit / little-endian bitmask. If a metadata field is set, then the
value of the IsPresent bit in the bitmask is 1.

A.2.1 FEI1 Extended Header specification

Image, System and Application

Name Offset Offset Format IsPresent Description

(dec) (hex)

Metadata size 0 0x0000 Int32 NA Metadata size [bytes]

All Metadata Blocks in the file have the
same size.
● FEI1: 768 bytes
● FEI2: updated for each version.

Metadata 4 0x0004 Int32 NA Version ID of the metadata format.

version All Metadata Blocks in the file have the
same format.
● FEI1: 0
● FEI2: initial value: 2
The value is updated for each new
version.

Bitmask 1 8 0x0008 UInt32 NA Individual bits indicate which metadata

fields are set.

Timestamp 12 0x000C Float64 Bitmask 1 – #0 Time when the image was taken. The used
format is the DATE data type that is used
in OLE automation by Microsoft:
Microsoft OLE DATE data type
specification

Microscope 20 0x0014 16 chars Bitmask 1 – #1 Identifier for microscope type

type (Krios, Talos, Titan, Metrios, etc.)

D-Number 36 0x0024 16 chars Bitmask 1 – #2 Microscope identifier

Application 52 0x0034 16 chars Bitmask 1 – #3 Application name

Application 68 0x0044 16 chars Bitmask 1 – #4

version

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Gun

Name Offset (dec) Offset (hex) Format 'Is Present' flag Description

HT 84 0x0054 Float64 Bitmask 1 – #5 High tension [Volt]

Dose 92 0x005C Float64 Bitmask 1 – #6 Dose [electrons/m²]

Stage

Name Offset Offset (hex) Format 'Is Present' flag Description

(dec)

Alpha tilt 100 0x0064 Float64 Bitmask 1 – #7 Holder Alpha tilt along axis [degr.]

Beta tilt 108 0x006C Float64 Bitmask 1 – #8 Holder Beta tilt along axis [degr.]

X-Stage 116 0x0074 Float64 Bitmask 1 – #9 Stage X position [m]

Y-Stage 124 0x007C Float64 Bitmask 1 – #10 Stage Y position [m]

Z-Stage 132 0x0084 Float64 Bitmask 1 – #11 Stage Z position [m]

Tilt axis angle 140 0x008C Float64 Bitmask 1 – #12 Angle of tilt axis in image [degr.]

Dual axis 148 0x0094 Float64 Bitmask 1 – #13 Measured rotation angle after b flip
rotation [degr.]
(Tomography only)

Pixel Size

Name Offset (dec) Offset (hex) Format 'Is Present' flag Description

Pixel size X 156 0x009C Float64 Bitmask 1 – #14 Pixel size X [m]

Pixel size Y 164 0x00A4 Float64 Bitmask 1 – #15 Pixel size Y [m]

Optics

Name Offset Offset Format 'Is Present' flag Description

(dec) (hex)

Defocus 220 0x00DC Float64 Bitmask 1 – #22 Defocus [m]

STEM Defocus 228 0x00E4 Float64 Bitmask 1 – #23 STEM defocus [m]

Applied defocus 236 0x00EC Float64 Bitmask 1 – #24 Relative defocus applied by
application [m]

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Chapter | The MRC2014 Image Format

Instrument mode 244 0x00F4 Int32 Bitmask 1 – #25 ● 1: TEM

● 2: STEM

Projection mode 248 0x00F8 Int32 Bitmask 1 – #26 ● 1: Diffraction

● 2: Imaging

Objective lens 252 0x00FC 16 chars Bitmask 1 – #27 ● LM

mode ● HM
● Lorentz

High magnification 268 0x010C 16 chars Bitmask 1 – #28 ● Mi

mode ● SA
● Mh

Probe mode 284 0x011C Int32 Bitmask 1 – #29 ● 1: NanoProbe

● 2: MicroProbe

EFTEM On 288 0x0120 Bool Bitmask 1 – #30 TRUE when the magnifications are
adapted to the energy filter

Magnification 289 0x0121 Float64 Bitmask 1 – #31 Nominal magnification

Bitmask 2 297 0x0129 UInt32 NA Individual bits indicate which

metadata fields are set.

Camera length 301 0x012D Float64 Bitmask 2 – #0 Nominal camera length [m]

Spot index 309 0x0135 Int32 Bitmask 2 – #1 -

Illuminated area 313 0x0139 Float64 Bitmask 2 – #2 ● TEM: beam diameter in

meters
● STEM: not used
● Undefined on 2 lens
condenser systems

Intensity 321 0x0141 Float64 Bitmask 2 – #3 Uncalibrated measure of beam

diameter on 2 lens condenser
systems

Convergence angle 329 0x0149 Float64 Bitmask 2 – #4 [degr.]

Undefined on 2 lens condenser
systems

Illumination mode 337 0x0151 16 chars Bitmask 2 – #5 ● None

● Parallel
● Probe
● Free
● Undefined on 2 lens
condenser systems

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Chapter | The MRC2014 Image Format

Wide convergence 353 0x0161 Bool Bitmask 2 – #6 Undefined on 2 lens condenser

angle range systems

EFTEM Imaging

Name Offset (dec) Offset (hex) Format 'Is Present' flag Description

Slit inserted 354 0x0162 Bool Bitmask 2 – #7 -

Slit width 355 0x0163 Float64 Bitmask 2 – #8 Slit width [eV]

Acceleration voltage offset 363 0x016B Float64 Bitmask 2 – #9 [Volt]

Drift tube voltage 371 0x0173 Float64 Bitmask 2 – #10 [Volt]

Energy shift 379 0x017B Float64 Bitmask 2 – #11 [eV]

Image Shifts

Name Offset Offset Format 'Is Present' flag Description

(dec) (hex)

Shift offset 387 0x0183 Float64 Bitmask 2 – #12 Corrective image or beam shift relative to
X exposure preset (in logical units)
● TEM: pure image shift
Shift offset 395 0x018B Float64 Bitmask 2 – #13
Y ● STEM: image-beamshift-

Shift X 403 0x0193 Float64 Bitmask 2 – #14 Applied shift due to optimized position and
tracking (in logical units)
Shift Y 411 0x019B Float64 Bitmask 2 – #15 ● TEM: image beam shift

● STEM: beam shift-

Camera

Name Offset Offset (hex) Format 'Is Present' flag Description

(dec)

Integration time 419 0x01A3 Float64 Bitmask 2 – #16 Camera or dose fraction
exposure time

Binning Width 427 0x01AB Int32 Bitmask 2 – #17 -

Binning Height 431 0x01AF Int32 Bitmask 2 – #18 -

Camera name 435 0x01B3 16 chars Bitmask 2 – #19 Name of the camera

Readout area left 451 0x01C3 Int32 Bitmask 2 – #20 -

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Readout area top 455 0x01C7 Int32 Bitmask 2 – #21 -

Readout area right 459 0x01CB Int32 Bitmask 2 – #22 -

Readout area bottom 463 0x01CF Int32 Bitmask 2 – #23 -

Ceta noise reduction 467 0x01D3 Bool Bitmask 2 – #24 -

Ceta frames summed 468 0x01D4 Int32 Bitmask 2 – #25 Number of frames
summed for dynamic
range

Direct detector electron 472 0x01D8 Bool Bitmask 2 – #26 -

counting

Direct detector align frames 473 0x01D9 Bool Bitmask 2 – #27 -

Camera param reserved 0 474 0x01DA Int32 Bitmask 2 – #28 -

Camera param reserved 1 478 0x01DE Int32 Bitmask 2 – #29 -

Camera param reserved 2 482 0x01E2 Int32 Bitmask 2 – #30 -

Camera param reserved 3 486 0x01E6 Int32 Bitmask 2 – #31 -

Bitmask 3 490 0x01EA UInt32 NA Individual bits indicate

which metadata fields
are set.

Camera param reserved 4 494 0x01EE Int32 Bitmask 3 – #0 -

Camera param reserved 5 498 0x01F2 Int32 Bitmask 3 – #1 -

Camera param reserved 6 502 0x01F6 Int32 Bitmask 3 – #2 -

Camera param reserved 7 506 0x01FA Int32 Bitmask 3 – #3 -

Camera param reserved 8 510 0x01FE Int32 Bitmask 3 – #4 -

Camera param reserved 9 514 0x0202 Int32 Bitmask 3 – #5 -

Phase Plate 518 0x0206 Bool Bitmask 3 – #6 Indicates whether

phase plate was used
for data acquisition

STEM

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Chapter | The MRC2014 Image Format

Name Offset (dec) Offset (hex) Format 'Is Present' flag Description

STEM Detector name 519 0x0207 16 chars Bitmask 3 – #7 -

Gain 535 0x0217 Float64 Bitmask 3 – #8 -

Offset 543 0x021F Float64 Bitmask 3 – #9 -

STEM param reserved 0 551 0x0227 Int32 Bitmask 3 – #10 -

STEM param reserved 1 555 0x022B Int32 Bitmask 3 – #11 -

STEM param reserved 2 559 0x022F Int32 Bitmask 3 – #12 -

STEM param reserved 3 563 0x0233 Int32 Bitmask 3 – #13 -

STEM param reserved 4 567 0x0237 Int32 Bitmask 3 – #14 -

Scan settings

Name Offset (dec) Offset (hex) Format 'Is Present' flag Description

Dwell time 571 0x023B Float64 Bitmask 3 – #15 Dwell time per pixel [sec]

Frame time 579 0x0243 Float64 Bitmask 3 – #16 Frame time [sec]
(currently it will not be used)

Scan size left 587 0x024B Int32 Bitmask 3 – #17 -

Scan size top 591 0x024F Int32 Bitmask 3 – #18 -

Scan size right 595 0x0253 Int32 Bitmask 3 – #19 -

Scan size bottom 599 0x0257 Int32 Bitmask 3 – #20 -

Full scan FOV X 603 0x025B Float64 Bitmask 3 – #21 Field of view [m]

Full scan FOV Y 611 0x0263 Float64 Bitmask 3 – #22 -

EDX Elemental maps

Name Offset (dec) Offset (hex) Format 'Is Present' flag Description

Element 619 0x026B 16 chars Bitmask 3 – #23 -

Energy interval lower 635 0x027B Float64 Bitmask 3 – #24 -

Energy interval higher 643 0x0283 Float64 Bitmask 3 – #25 -

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Chapter | The MRC2014 Image Format

Method 651 0x028B Int32 Bitmask 3 – #26 -

Dose fractions

Name Offset (dec) Offset (hex) Format 'Is Present' flag Description

Is dose fraction 655 0x028F Bool Bitmask 3 – #27 -

Fraction number 656 0x0290 Int32 Bitmask 3 – #28 -

Start frame 660 0x0294 Int32 Bitmask 3 – #29 -

End frame 664 0x0298 Int32 Bitmask 3 – #30 -

Reconstruction

Name Offset (dec) Offset (hex) Format 'Is Present' flag Description

Input stack filename 668 0x029C 80 chars Bitmask 3 – #31 -

Bitmask 4 748 0x02EC UInt32 NA Individual bits indicate which

metadata fields are set.

Alpha tilt min 752 0x02F0 Float64 Bitmask 4 – #0

Alpha tilt max 760 0x02F8 Float64 Bitmask 4 – #1

A.2.2 FEI2 Version 2 Extension to the Extended Header specification

Name Offset Offset Format IsPresent Description

(dec) (hex)

Scan rotation 768 0x0300 Float64 Bitmask 4 – #2 Rotation of the scan pattern in STEM
mode [radians]

Diffraction pattern 776 0x0308 Float64 Bitmask 4 – #3 Rotation of the diffraction pattern in
rotation diffraction mode [radians]

Image rotation 784 0x0310 Float64 Bitmask 4 – #4 Rotation of the image in imaging mode
[radians]

Scan mode 792 0x0318 Int32 Bitmask 4 – #5 ● 0: Other

enumeration ● 1: Raster
● 2: Serpentine raster

Acquisition time 796 0x031C Int64 Bitmask 4 – #6 Microseconds since

stamp 1970-01-01T00:00:00Z at which the
image was acquired

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Detector 804 0x0324 16 chars Bitmask 4 – #7 Commercial name of the detector or

commercial name camera

Start tilt angle 820 0x0334 Float64 Bitmask 4 – #8 Start tilt angle of a tomography series
[degr.]

End tilt angle 828 0x033C Float64 Bitmask 4 – #9 End tilt angle of a tomography series
[degr.]

Tilt per image 836 0x0344 Float64 Bitmask 4 – #10 Tilt increment per image in a
tomography series [degr.]

Tilt speed 844 0x034C Float64 Bitmask 4 – #11 Tilt speed in a tomography series
[degr./sec]

Beam center X 852 0x0354 Int32 Bitmask 4 – #12 Beam center X on image [pixels]
pixel

Beam center Y 856 0x0358 Int32 Bitmask 4 – #13 Beam center Y on image [pixels]
pixel

CFEG flash 860 0x035C Int64 Bitmask 4 – #14 Microseconds since

timestamp 1970-01-01T00:00:00Z of the most
recent CFEG flashing

Phase plate 868 0x0364 Int32 Bitmask 4 – #15 Position index of the phase plate
position index aperture

Objective aperture 872 0x0368 16 chars Bitmask 4 – #16 Name of the inserted objective aperture
name

A.3 Pixel sequence in the MRC2014 format

In the MRC2014 files, the image pixel data is stored as rows from top to bottom, where each row is
stored from left to right.

Most image viewers and image processing applications use the same pixel position sequence as the
MRC file. Some image viewing and processing applications such as IMOD and Fiji/ImageJ use a
pixel position sequence. In these applications, the image display may be mirrored and/or rotated.

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A.3.1 The MRC image pixel data encoding for Thermo Scientific Ceta cameras
If the image is acquired with a Ceta camera, then the MRC image pixel data encoding depends on
the presence of the Ceta Speed Enhancement (Ceta-2).

Camera MRC Pixel Data

Ceta without Speed Enhancement 32-bit floating point

Ceta with Speed Enhancement 16-bit signed integer

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Chapter | Copyright, Limited Rights and Revision History

13 Copyright, Limited Rights and Revision History

Copyright
The information and materials contained herein are confidential and proprietary to FEI Company, part
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basis. They cannot be duplicated or disseminated for any third party without the express consent of
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Revision Table

Revision Date Description of Changes

A-L EPU 1.X User Manual revisions

M OCT-2018 Update for EPU 2.0 - 2.1

N JAN-2019 Update for EPU 2.2

O APR-2019 Update for EPU 2.3

P OCT-2019 Update for EPU 2.4 - 2.5

Q JAN-2020 Update for EPU 2.6

2.7 APR-2020 Update for EPU 2.7

2.8 JUL-2020 Update for EPU 2.8

2.9 OCT-2020 Update for EPU 2.9

2.10 JAN-2021 Update for EPU 2.10

2.11 APR-2021 Update for EPU 2.11

2.12 JUL-2021 Update for EPU 2.12

2.13 OCT-2021 Update for EPU 2.13

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Chapter | Copyright, Limited Rights and Revision History

Revision Date Description of Changes

2.14 JAN-2022 Update for EPU 2.14

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Chapter | Index

Index
Change the processing order of the Grid Squares
A
110
Acquire Atlases for multiple specimens 83
Check the C2 Apertures center positions on a system
Acquire a single Atlas from an unknown specimen on without a C3 lens 181
the stage 85
Check the C2 Apertures center positions on systems
Acquire an Atlas for a single specimen on a system with a C3 lens 180
with removable holders (side entry) 91
Check the Objective Apertures center positions
Acquire an Atlas on a Tundra system 89 181
Acquisition and Optics Settings task 18 Clear the Apertures > Options > React on Optical
Activate Phase Plate task 61 Mode Changes option 171
Activate a new Phase Plate before an Automated Copyright, Limited Rights and Revision History 207
Acquisition run 154 Create a new Atlas Session 81
Add and remove Foil Holes with the Selection Brush Create a new EPU Session without an EPU Session
137 Preferences file 97
Add and remove acquisition areas with the Selection Create a new Session from an EPU Session
Brush 122 Preferences file 97
Advanced Camera Settings for Gatan K2 and K3 Create a queue with EPU Multigrid Sessions 161
cameras 24
Create a selection of Foil Holes without a generated
Advanced Camera Settings for Thermo Scientific pattern 138
Ceta cameras 23
Customize the selection of Acquisition Areas 121
Alignments tasks 64
Customize the selection of Foil Holes 136
Aperture Alignments 180
Area Selection task for Lacey Carbon specimens D
112
Define a selection of Acquisition Areas without a
Atlas Optics Alignment task 53 generated pattern 122
Atlas Tab 81 Define one or more Acquisition Areas 142
Auto Functions Tab 64 Define the Acquisition Area Pattern and Filter
Auto-Functions (TEM) tasks 65 Settings for a Manual Selection Session 118
Automated Acquisition task 152 Define the Acquisition Area Pattern and Filter
Settings for an Automated Selection Session 112
Automatically create a Manual Selection in all
selected Grid Squares 120, 135 Define the Atlas Preset 50
Automatically generate a pattern of Acquisition Areas Define the Autofocus Area 146
142 Define the Autofocus Preset 40
Define the Data Acquisition Preset 28
C
Define the Defocus List for a Lacey Carbon specimen
Calibrate I0 task 58 125
Calibrate Image Shifts task 55 Define the Defocus List for one or more Acquisition
Calibration tasks 75 Areas 144
Calibrations 183 Define the Drift Measurement Area 147
Camera Settings for all camera types 18 Define the Drift Measurement Preset 42
Change the order of the EPU Multigrid Sessions in Define the Foil Hole dimensions and Filter Settings
the Queue 164 for a Manual Selection Session 132
Define the Foil Hole dimensions and Filter Settings
for an Automated Selection Session 126
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Chapter | Index

Define the GridSquare Preset 48 Guidelines for the Drift Measurement Function and
Define the Hole/EucentricHeight Preset 36 the Delay After Stage Shift Time 149

Define the Thon Ring Preset 44 Guidelines for the Hole/EucentricHeight Preset when
using Phase Plates 39
Define the Zero Loss Preset 47
Guidelines for the Optics Settings when using Phase
Deflectors 175 Plates 27
Description of the Camera Settings 18 Gun and Condenser 172
Description of the Optics Settings 25
Description of the Session Setup options 98 H
Detailed Preconditions for Successful EPU Usage Handpick the Acquisition Areas for a Manual
170 Selection Session 115
Determine the Phase Plate Activation Time 62 Handpick the Foil Holes for a Manual Selection
Direct Alignments and Astigmatism 182 Session 130
Disable Beam Settings > Intensity Zoom 171 Histogram side panel 11, 11
Dose Rate for Thermo Scientific cameras 22 Hole Selection task for Quantifoil specimens 126
Home Tab 16
E How to improve the Autofocus Calibration result 77
EPU Tab 94 How to plan for high microscope utilization 152
Edit an EPU Multigrid Session in the Queue 163
Enable the Smart Plugins in the Template Definition I
149 Image Information side panel 12
Eucentric Correction Calibration (Optional) 183 Image and plot display area 13
Exchange the specimen on a Tundra system 90 Import and export a Foil Hole Template 149
Export an image to file 15 Import and export the Acquisition and Optics Presets
Exposure Settings for Thermo Scientific Falcon 3EC 52
and Falcon 4(i) cameras 19 Inspect a Foil Hole to assess suitability 139
Exposure Settings for the Thermo Scientific Ceta-F Inspect an Acquisition Area to assess suitability
camera 21 123
Inspect the Acquired Images 168
F Introduction 4
FEI1 Extended Header specification 198
FEI2 Version 2 Extension to the Extended Header L
specification 204 Limitations to loading or acquiring an Atlas after
Focus Calibration 183 Session Setup 106
Load a specimen on the stage 87
G Load an existing Atlas Session 82
Getting Started 6 Log in on Thermo Scientific Athena 7
Guidelines for the Autofocus Preset when using
Phase Plates 41 M
Guidelines for the Data Acquisition Preset when MRC image pixel format for Gatan K2 and K3
using Phase Plates on a microscope with a C3 lens cameras 100
31
Magnification Calibrations 183
Guidelines for the Data Acquisition Preset when
using Phase Plates on a microscope without a C3 Manually define one or more Acquisition Areas
lens 34 143
Messages side panel 10

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Chapter | Index

Monitor the progress of the Automated Acquisition

R
run 157
Remove an EPU Multigrid Session from the Queue
164
N
Reset the Slot Position status 86
Navigate and pan in a zoomed image 14
Run the 'Auto-eucentric by beam tilt' auto-function
67
P
Run the 'Auto-eucentric by stage tilt' auto-function
Pause and resume the Automated Acquisition run
68
158
Run the Auto Zero-Loss auto-function 74
Perform Automated Acquisition for a single EPU
Multigrid Session from the Queue 164 Run the Autocoma auto-function 70
Perform Automated Acquisition for all EPU Multigrid Run the Autofocus auto-function 65
Sessions in the Queue 166 Run the Autostigmate auto-function 69
Perform the Atlas Optics Alignment 53 Run the Drift Stabilization auto-function 70
Perform the Autofocus Calibration for other Presets Run the Optimize Beam task 64
77
Perform the Autofocus Calibration for the Autofocus S
Preset 75
Screening task 83
Perform the Eucentric Correction Calibration 80
Select Grid Squares for high quality data acquisition
Perform the I0 Calibration for the GridSquare Preset 107
59
Select the bad Foil Holes in the generated pattern,
Perform the I0 Calibration for the Hole/ then invert the selection 137
EucentricHeight Preset 60
Select the bad areas in the generated pattern, then
Perform the Image Shift Calibration 56 invert the selection 122
Perform the Phase Plate Microprobe (uP) Alignment Select the suitable Foil Holes in the generated
31 pattern 137
Perform the Phase Plate Nanoprobe (nP) Alignment Select the suitable areas in the generated pattern
34, 32 121
Perform the Template Definition procedure 141 Session Creation task 95
Phase Plate position logging 160 Session Setup task 98
Pixel sequence in the MRC2014 format 205 Set the Autofocus and Drift Stabilization values
Pre-select the Grid Squares for the Automated 124
Acquisition run 92 Set up a new EPU Session based on Advised
Preconditions for the microscope 170 Preferences 17
Preconditions for the microscope alignments 172 Set up a new EPU Session based on a Saved
Preference 17
Preconditions for the specimen and specimen holder
170 Set up an EPU Session 104
Preparation Tab 18 Setup Session task 81
Prepare for Autofocus Calibration 75 Skip a Grid Square, Foil Hole or Acquisition Area
159
Prepare for Image Shift Calibration 55
Specify the Maximum Image Shift and Delay Timers
Prepare for an EPU session 6
148
Prepare for the Eucentric Correction Calibration 79
Square Selection task 107
Projector 176
Start the Automated Acquisition run 155
Start the EPU software 6
Status side panel 11

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Chapter | Index

System and software compatibility 5 The pixel format for Gatan Dose Fraction images
100
T The recommended order to define the Acquisition
TIFF image pixel format for Gatan K2 and K3 and Optics Presets 27
cameras 101 The red crosshair 15
Target audience for this user manual 5 Troubleshoot an ongoing Automated Acquisition run
Template Definition task for Quantifoil specimens 159
141 Troubleshooting: Atlas 184
Template Execution task for Quantifoil specimens Troubleshooting: Automated Acquisition 194
151 Troubleshooting: GridSquare 186
Test the Condenser Alignment 173 Troubleshooting: Symptoms and Solutions 184
Test the Condenser Focus Alignment 175 Troubleshooting: Template Definition 188
Test the Condenser Zoom on Titan Systems 174
Test the Eucentric Focus 176 U
Test the Gun Alignment 172 Unselect the bad Foil Holes in the generated pattern
Test the Image/Beam Shift Calibration 175 137

Test the Lens Series Magnification Center Alignment Unselect the bad areas in the generated pattern
176 122

Test the Magnification-Dependent Beam and Image Use the Sherpa APM function to maintain the
Shift - TEM mode 178, 179 microscope alignments 172

Test the Magnification-Dependent Focus 177 User Interface 9

Test the Parallel Beam range on Titan Systems User interface panels 9
173
The Acquisition Mode for Quantifoil specimens 98 V
The Athena settings 101 Validate the Foil Hole Template 151
The Autofocus Calibration task 75 Verify that the microscope is ready for high quality
data acquisition 7
The Automated Acquisition task in the EPU Multigrid
workflow 164 Verify that there is sufficient free disk space and
sufficient liquid nitrogen (LN2) 153
The EPU Multigrid Option 161
Verify the on-plane illumination on a microscope with
The EPU Session Preferences file 95 a C3 lens 33
The Eucentric Correction Calibration task 78 Verify the on-plane illumination on a microscope
The Extended Header specification 197 without a C3 lens 35
The MRC image pixel data encoding for Thermo View and post-process MRC images with Thermo
Scientific Ceta cameras 206 Scientific Velox software 169
The MRC2014 Image Format 197 View the JPEG images 168
The Main Header and Extended Headers in an MRC View the MRC, EMI, DMX and other microscope
file 197 images 168
The Screening task for Tundra systems 89 View the images in Windows Explorer and Photo
The Screening task for systems with an Autoloader Viewer 168
83
The automatic Dose Fractions calculation for Falcon W
3EC and Falcon 4(i) cameras 22 When to reset and renew the Image Shift Calibration
The impact of Hole Position Refinement on the 'Move 55
stage to location' function 139

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Chapter | Index

Z
Zoom in on an individual Atlas Tile image 109
Zoom in/out 14

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