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Kota Miura
Nataša Sladoje
Editors

Bioimage Data
Analysis Workflows
Editors
Kota Miura Nataša Sladoje
Im Neuenheimer Feld 267 Department of Information Technology
Nikon Imaging Center Bioquant BQ 0004 Centre for Image Analysis,
Heidelberg, Germany Uppsala University
Uppsala, Sweden

ISSN 2509-6125     ISSN 2509-6133 (electronic)

Learning Materials in Biosciences
ISBN 978-3-030-22385-4    ISBN 978-3-030-22386-1 (eBook)
https://doi.org/10.1007/978-3-030-22386-1

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 V

Preface

The often-posed question among life science researchers, “Which software tool is the best
for bioimage analysis,” indicates misunderstanding which calls for explanations. It
appears that this question cannot be answered easily, maybe even not at all. Biological
research problems are not general, and each of them questions specific events among
various phenomena seen in biological systems. Therefore, the answer to the “Which is
the best?” question to a high extent depends not only on the biological problem that is to
be addressed but also on the specific goals and criteria to be met. Moreover, the misun-
derstanding seems to be based on an assumption that at some point in the future, there
will be an almighty software tool for bioimage analysis that solves most of the problems
just by clicking on a button. This, most likely, is simply a dream that may never come true.

Software tools are developed with their central value towards having generic applicabil-
ity of offered functionality to be as wide as possible. In a sense, this is the agenda towards
“almighty.” On the other hand, the biological question asked by each researcher is unique
and specific. “Novel findings,” which biologists are seeking, come as answers to specific
and original questions that others have not thought about or by using a novel method
that others have not used to approach the mystery of biological systems. There is a clear
gap between how bioimage analysis tools are developed and how biological questions are
valued. The former is towards generality, and the latter is towards specificity.

The gap can be filled by designing unique combinations of general tools. More precisely,
image analysis software tools should be used by a researcher in a one-and-only, specific
way, by designing a customized workflow combining various suitable implementations
of algorithms, to address a specific biological problem. Such novel designs help the
researchers to see and quantify the biological system in a way that no one has done
before. The highly desired optimal combination of the generality of the available soft-
ware tools and the specificity of biological problem is thus achieved. The outcome can
lead to outstanding scientific results. However, when this gap between generality and
specificity is overlooked, bioimage analysis becomes simply a pain: life science research-
ers do not know how to approach it and benefit from it. Question such as “A great soft-
ware tool is available in my computer but why can’t I solve my problem?” can be rather
frustrating.

As digital image data have become one of the fundamental infrastructures of biological
research activities, students and researchers in the biomedical and life sciences more and
more want to learn how to use the available tools. They want to know how to use various
resources for image analysis and combine them to set up an appropriate workflow for
addressing their own biological question. Getting used to bioimage analysis tools means
learning about the various components that are available as a part of the software and
becoming proficient in combining them for quantifying the biological systems.

The Network of European Bioimage Analysts (NEUBIAS) was established in 2016, with
the aim to promote and share information about rich image analysis resources that have
become widely available nowadays and to encourage, through education, their uses.
VI Preface

Nowadays, we can access many resources. These are good news, but at the same time,
this variety of options may be overwhelming, leading to difficult choices regarding tools
and resources most suitable for a particular problem and their most effective combina-
tions for any specific purpose.

The aim of this textbook is to offer guidance in learning to make such choices. It provides
“guided tours” through the five selected bioimage analysis workflows relevant in real
biological studies, which combine different software packages and tools. Realistically,
these workflows are not general and cannot be directly applied to other problems. How-
ever, the best (if not the only) way to learn to design own specialized workflows is to
study the craft (approaches and solutions) of others. Bioimage Data Analysis (Wiley
2016) was published with the same motivation; this textbook is a sequel, contributing to
the same goal. We hope to continue by including more bioimage analysis workflows and,
by that, inspiring new creative solutions of life science problems.

One prominent contribution of the NEUBIAS team to the life science community is the
conceptual apparatus required for swimming in the sea of rich image analysis resources:
definitions of components, collections, and workflows. These notions are introduced and
explained in 7 Chap. 1 and then utilized in the subsequent ones.

7 Chapter 2 focuses on a workflow for measuring the fluorescence intensity localized to

the nuclear envelope. Automatic segmentation of the nuclear rim, based on thresholding
and mathematical morphology, is iterated through multiple image frames to measure the
changes in fluorescence intensity over time. ImageJ macro commands are recorded by
the command recorder and converted to a stand-alone ImageJ macro.

7 Chapter 3 offers a step-by-step guide through a procedure to build a macro for a 3D

object-based colocalization, showing also how to extend and adjust the developed work-
flow to include intensity-based colocalization methods.

7 Chapter 4 aims at teaching the principles and pitfalls of single particle tracking (SPT).
Tracking is, in general, very important for dynamic studies; focus is on propagating
object identities over time and subsequently computing relevant quantities from the
identified tracks. The developed workflow combines tools available in ImageJ/Fiji (for
generating the tracks) and in MATLAB (for analyzing them).

7 Chapter 5 introduces some of the powerful and flexible image analysis methods native
to MATLAB, also providing a crash course in programming for those with no, or limited,
experience. The tools are used to simulate a time series of Brownian motion or diffusion
process, to analyze time-series data, and to plot and export the results as figures ready for
publication. The workflow presented in this chapter is quite powerful in analyzing track-
ing data such as those presented in 7 Chap. 4.

7 Chapter 6 presents the computational approach of registering images from different

modalities based on manual selection of matching pairs of landmarks. The identification
of sites of clathrin-mediated endocytosis by correlative light electron microscopy
(CLEM) is used as an example on how to apply an image registration workflow based on
MATLAB’s image processing toolbox.
 VII
Preface

This textbook is the first bioimage analysis textbook published as an output of the com-
mon efforts of NEUBIAS, the Network of European Bioimage Analysts, funded under
COST Action CA15124. We would like to thank the leaders of workgroups (WGs) in
NEUBIAS: Sebastian Munck, Arne Seitz and Florian Levet (WG1 “Strategy”), Paula
Sampaio and Irene Fondón (WG2 “Outreach”), Perrine Paul-Gilloteaux and Chong
Zhang (WG4 “Webtool biii.eu”), Sébastien Tosi and Graeme Ball (WG5 “Benchmarking
and Sample Datasets”), Julia Fernandez-Rodriguez and Clara Prats Gavalda (WG7
“Short-Term Scientific Missions and Career Path”), and Julien Colombelli (NEUBIAS
Chair). Their efforts to create a synergistic effect of the diverse workgroup activities
towards the establishment of “Bioimage Analysts” are the strong backbone that has led to
the successful realization of this book. We are very much grateful to the reviewers of each
chapter: Anna Klemm, Jan Eglinger, Marion Louveaux, Christian Tischer, and Ulrike
Schulze. Their critical comments largely improved the presented workflows. We are par-
ticularly grateful to the authors of each workflow chapters: Fabrice P. Cordeliéres, Chong
Zhang, Perrine Paul-Gilloteaux, Martin Schorb, Simon F. Nørrelykke, Jean-Yves Tinevez,
and Sébastien Herbert. They have traveled together with selfless commitment to achieve
the demanding publication format we chose, which is to offer both the normal printed
textbook and the “continuously updated” online electronic version. The publication of
this book was enabled by the financial support from the COST Association (funded
through EU framework Horizon2020), through the granted project “A New Network of
European Bioimage Analysts (NEUBIAS, COST Action CA15124).” Finally, we thank all
the members of NEUBIAS who, with their enthusiasm and commitment to the network’s
activities, have contributed to keep the momentum of the initiative constantly high, a
vital element to enable it to reach its objectives, including the publication of this book.

Nataša Sladoje
Uppsala, Sweden

Kota Miura
Heidelberg, Germany
Acknowledgements

This textbook is based upon the work from COST Action CA15124, supported by COST
(European Cooperation in Science and Technology).

COST (European Cooperation in Science and Technology) is a funding agency for

research and innovation networks. Our actions help connect research initiatives across
Europe and enable scientists to grow their ideas by sharing them with their peers. This
boosts their research, career, and innovation.

7 www.­cost.­eu
 IX

Contents

1 Workflows and Components of Bioimage Analysis . . . . . . . . . . . . . . . . . . . . . . . 1

Kota Miura, Perrine Paul-Gilloteaux, Sébastien Tosi, and Julien Colombelli

2 Measurements of Intensity Dynamics at the

Periphery of the Nucleus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Kota Miura

3 3D Quantitative Colocalisation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Fabrice P. Cordelières and Chong Zhang

4 The NEMO Dots Assembly: Single-Particle Tracking and Analysis . . . . . . . 67

Jean-Yves Tinevez and Sébastien Herbert

5 Introduction to MATLAB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  97

Simon F. Nørrelykke

6 Resolving the Process of Clathrin Mediated Endocytosis

Using Correlative Light and Electron Microscopy (CLEM) . . . . . . . . . . . . . . . . 143
Martin Schorb and Perrine Paul-Gilloteaux

Supplementary Information
Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Contributors

Julien Colombelli Perrine Paul-Gilloteaux

Advanced Digital Microscopy Core Facility, SFR-Santé MicroPICell Facility, UNIV Nantes,
Institute for Research in Biomedicine, IRB INSERM, CNRS, CHU Nantes
Barcelona, Spain Nantes, France

Barcelona Institute of Science and INSB France BioImaging

Technology, BIST Nantes, France
Barcelona, Spain [email protected]
[email protected]
Martin Schorb
Fabrice P. Cordelières Electron Microscopy Core Facility,
Bordeaux Imaging Center, UMS 3420 EMBL Heidelberg
CNRS – Université de Bordeaux – US4 INSERM Heidelberg, Germany
Bordeaux, France [email protected]

Pôle d’imagerie photonique, Centre Broca

Jean-Yves Tinevez
Nouvelle-Aquitaine
Image Analysis Hub – C2RT – Institut Pasteur
Bordeaux, France
Paris, France
[email protected]
[email protected]

Sébastien Herbert
Sébastien Tosi
Image Analysis Hub – C2RT – Institut Pasteur
Advanced Digital Microscopy Core Facility,
Paris, France
Institute for Research in Biomedicine, IRB
[email protected]
Barcelona, Spain

Kota Miura Barcelona Institute of Science and

Im Neuenheimer Feld 267 Technology, BIST
Nikon Imaging Center Bioquant BQ 0004 Barcelona, Spain
Heidelberg, Germany [email protected]
[email protected]
Chong Zhang
Simon F. Nørrelykke SimBioSys Group, Pompeu Farba University
Image and Data Analysis Group, Barcelona, Spain
Scientific Center for Optical and Electron [email protected]
Microscopy, ETH
Zurich, Zurich, Switzerland
[email protected]
 1 1

Workflows and Components

of Bioimage Analysis
Kota Miura, Perrine Paul-Gilloteaux, Sébastien Tosi,
and Julien Colombelli

1.1 Introduction – 2

1.2 Types of Bioimage Analysis Software – 2

Bibliography – 6

© The Author(s) 2020

K. Miura, N. Sladoje (eds.), Bioimage Data Analysis Workflows, Learning Materials in Biosciences,
https://doi.org/10.1007/978-3-030-22386-1_1
 2 K. Miura et al.

What You Learn from This Chapter

1 Definitions of three types of bioimage analysis software—Component, Collection, and
Workflow—are introduced in this chapter. The aim is to promote the structured designing
of bioimage analysis methods, and to improve related learning and teaching.

1.1 Introduction

Software tools used for bioimage analysis tend to be seen as utilities that solve problems
off-the-shelf. The extreme version of such is like: “If I know where to click, I can get good
results!”. In case of gaming software, as the user gets more used to the software, the user
can achieve the final stage faster. To some extent, this might be true also with bioimage
analysis software, but there is a big difference. As bioimage analysis is a part of scientific
research, the goal to achieve is not to clear the common final stage that everyone heads
toward, but something original that others have not found out. The difficulty of the usage
of bioimage analysis software does not only reside in the hidden commands, but also in
the fact that the user needs to come up with more-or-less original analysis. Then, how can
we do something original using tools that are provided in public?
In this short chapter, we define several terms describing the world of bioimage analysis
software, which are “workflows”, “components”, and “collections”, and explain their rela-
tionships. We believe that clarifying the definition of these terms can contribute largely to
those who want to learn bioimage analysis, as well as to those who need to design the
teaching of bioimage analysis. The reason is that these terms link the generality of software
packages provided in public, with the specificity and the originality of the analysis that one
needs to achieve.

1.2 Types of Bioimage Analysis Software

Software packages such as ImageJ (Schneider et al. 2012),1 MATLAB,2 CellProfiler

(Carpenter et al. 2006)3 or ICY (de Chaumont et al. 2012)4 are often used to analyze image
data in life sciences. These software packages are “collections” of implementation of
image processing and analysis algorithms. Libraries such as ImgLib2 (Pietzsch et al.
2012),5 OpenCV (Bradski 2000),6 ITK (Johnson et al. 2015a,b),7 VTK (Schroeder et al.
2006),8 and Scikit-­Image (van der Walt et al. 2014)9 are also packages of image processing
and analysis algorithms, although with a different type of user interface that is not graph-
ical. We invariably refer to them as “collections”. To scientifically analyze and address
an underlying biological problem, one needs to hand-pick some algorithms from these

1 7 https://imagej.org
2 7 https://nl.mathworks.com
3 7 https://cellprofiler.org/
4 7 http://icy.bioimageanalysis.org
5 7 https://imagej.net/ImgLib2
6 7 https://opencv.org
7 7 https://itk.org
8 7 https://vtk.org
9 7 https://scikit-image.org
Workflows and Components of Bioimage Analysis
3 1
collections, carefully adjust their functional parameters to the problem and assemble
them in a meaningful order. Such a sequence of image processing algorithms with a spec-
ified parameter set is what we call a “workflow”. The implementations of the algorithms
that are used in the workflows are the “components” constituting that workflow (or
“workflow components”). From the point of view of the expert who needs to assemble a
workflow, a collection is a package bundling many different components. As an example,
many plugins offered for ImageJ are mostly also collections (e.g. Trackmate (Tinevez
et al. 2016),10 3D Suite (Ollion et al. 2013),11 MosaicSuite12…), as they bundle multiple
components. On the other hand, some plugins, such as Linear Kuwahara filter plugin,13
are a single component implemented as a single plugin.
Each workflow is uniquely associated with a specific biological research project
because the question asked therein as well as the acquired image quality are often unique.
This calls for a unique combination of components and parameter set. Some collections,
especially those designed with GUI, offer workflow templates. These templates are pre-­
assembled sequences of image processing tasks to solve a typical bioimage analysis prob-
lem; all one needs to do is to adjust the parameters of each step. For example, in the case
of Trackmate plugin for ImageJ (Tinevez et al. 2016), a GUI wizard guides the user to
choose an algorithm for each step among several candidates and also to adjust their
parameters to achieve a successful particle tracking workflow (see 7 Chap. 4). When
these algorithms and parameters are set, the workflow is built. CellProfiler also has a help-
ful GUI that assists the user in building a workflow based on workflow templates
(Carpenter et al. 2006). It allows the user to easily swap the algorithms for each step and
test various parameter combinations. . Figure 1.1 summarizes the above explanations.
Though such templates are available for some typical tasks, collections generally do
not provide helpful clues to construct a workflow—choice of components to be used and
approach taken to assemble those components depend on expert knowledge, empirical
knowledge or testing. Since the biological questions are so diverse, the workflow often
needs to be original and might not match any available workflow templates. Building a
workflow from scratch needs some solid knowledge about the components and the ways
to combine them. It also requires an understanding of the biological problem itself. Each
workflow is in essence associated with a specific biological question, and this question
together with the image acquisition setup affect the required precision of the analysis. For
example, image data in general should not be analyzed at a precision higher than the
physical resolution of the imaging system that captures those data.14 In some cases, a
higher precision does not imply more meaningful results just because such precision can
be irrelevant to the biological question. These aspects should be carefully considered dur-
ing the planning of the analysis and the choice of the components, together with the
choice of statistical treatment.
Many biologists feel difficulty in analyzing image data, because of the lack in skills
and knowledge to close the gap between a collection of components and a practical

10 7 https://imagej.net/TrackMate
11 7 http://imagejdocu.tudor.lu/doku.php?id=plugin:stacks:3d_ij_suite:start
12 7 http://mosaic.mpi-cbg.de/?q=downloads/imageJ
13 7 https://imagej.net/Linear_Kuwahara
14 If the model-based approach designed to compute sub-pixel resolution results is used e.g. single
molecule localization microscopy, precision does go beyond the given optical resolution and the
approach is thus validated.
 4 K. Miura et al.

1 Collection Workflow
Biological Image Data

Component Component Component

Component Component
Component
Component Component

Component Component
Component
Component Component

Component Component Component

Numbers, Plots, Stats,

Visualization

..      Fig. 1.1 Relationship between components, collection and workflow. Components (e.g. Gaussian
blurring filter) are selected from collection (e.g. ImageJ) and assembled into a specific workflow (red
arrow) for analyzing image data in each research project (e.g. scripts associated with journal papers)

workflow. A collection bundles components without workflows, but it is often errone-

ously assumed that installing a collection is enough for solving bioimage analysis prob-
lem. The truth is that expert knowledge is required to choose components, adjust their
parameters and build a workflow (. Fig. 1.1 red arrows). The correct assembly of compo-
nents as an executable script is in general even more difficult, as it requires some pro-
gramming skills. The use of components directly from library-type of collections, which
host many useful components, also requires programming skills to access their
API. Bioimage analysts may fill this gap but even they, who professionally analyze image
data, need to always search for the most suitable components to solve problems, reaching
the required accuracy or coping with huge data in a practical time.
Another important aspect and difficulty is the reproducibility of workflows. We often
want to know how other people have performed image analysis and to learn from others
new bioimage analysis strategies. In such cases, we look for workflows addressing a sim-
ilar biological problem. However, many articles do not document the workflows they
used in sufficient details to enable the reproducibility of the results. As an extreme exam-
ple, we found articles with their image analysis description in Materials and Methods
merely documenting that ImageJ was used for the image analysis. Such a minimalism
should be strictly avoided. On the other hand, some workflows are written as a detailed
text description in Materials and Methods sections in the publications. We go even fur-
ther and recommend to publish workflows as executable scripts, i.e. a computer pro-
gram, with documented parameter sets for clarity and reproducibility of analysis and
results. In our opinion, the best format is a version-tracked script because the version
Workflows and Components of Bioimage Analysis
5 1
used for the published results can be clearly stated and reused by others. A script embed-
ded in a Docker image is even better for avoiding problems associated with a difference
in execution environments.
Towards a more efficient designing of workflows, The Network of European Bioimage
Analysts (NEUBIAS) has been developing a searchable index named Bioimage Informatics
Search Engine (BISE). This service is accessible online at 7 https://biii.­eu and hosts the
manually curated registry of collections, workflows and components.
Two ontologies are used for annotating resources registered to BISE: The BISE ontol-
ogy for properties of resources e.g. programming language; and the EDAM Bioimaging
Ontology (Kalaš et al. 2019)—an extension of the EDAM ontology (Ison et al. 2013) devel-
oped together with ELIXIR15—for applications of these resources, e.g. image processing
step and imaging modality. “Component”, “Workflow” and “Collections” are implemented
as part of the BISE ontology for classifying the type of software, for more distinctive filter-
ing of search results.
While BISE allows researchers to search for bioimage analysis resources at all these
levels, general web search engines, such as Google, typically return hits of collections but
not to the details of their components. In addition, workflows are in many cases hidden
in biological papers and difficult to be discovered. BISE is also designed to feature users
impressions on the usability of components and workflows so that individual experi-
ences can be swiftly shared within the community.

Take Home Message

Within the world of bioimage analysis software, various types of tools, which can be
classified as “collections”, “components”, or “workflows”, coexist and are flatly provided
to the public as “software tools”. Clear definition of these types and recognition of the
role of each is a foundation for learning and teaching bioimage analysis.

kFurther Readings
1. Miura and Tosi (2016) discusses the general challenges of bioimage analysis.
2. Miura and Tosi (2017) provides more details on the structure and designing of
bioimage analysis workflows.
3. Details about NEUBIAS can be found at the following web pages:
55 7 http://neubias.­org
55 7 https://www.­cost.­eu/actions/CA15124: The Memorandum of Understanding
describes the objectives of the network, that includes the motivation to create
the registry 7 http://biii.­eu.

Acknowledgements We are grateful to Nataša Sladoje for critically reading this text. We
thank Matúš Kalaš for checking the text and correcting our mistakes.

15 7 https://www.elixir-europe.org
 6 K. Miura et al.

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Workflows and Components of Bioimage Analysis
7 1
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 9 2

Measurements of Intensity
Dynamics at the Periphery
of the Nucleus
Kota Miura

2.1 Introduction – 10

2.2 Tools – 11

2.3 Dataset – 11

2.4 Workflow – 12
2.4.1 S egmentation of Nucleus Rim – 12
2.4.2 Integration: The Measurement Over Time – 24
2.4.3 Integrating Segmentation and Measurements – 25

2.5 Results and Conclusion – 29

2.6 Exercise Answers – 31

2.6.1 E xercises 2.1–2.4 – 31
2.6.2 Exercise 2.5 – 31

Bibliography – 32

© The Author(s) 2020

K. Miura, N. Sladoje (eds.), Bioimage Data Analysis Workflows, Learning Materials in Biosciences,
https://doi.org/10.1007/978-3-030-22386-1_2
 10 K. Miura

What You Learn from This Chapter

The aim of this chapter is to learn how to construct a workflow for measuring the fluores-
cence intensity localized to the nuclear envelope. For this purpose, the nucleus image is
2 segmented to create a mask along the nuclear rim. The reader will learn a typical technique
for automatically delineating the segmented area by post-processing using the mathemat-
ical morphology algorithm, and how to loop that piece of ImageJ macro and iterate through
multiple image frames to measure changes in fluorescence intensity over time. This chapter
is also a good guide for learning how to convert ImageJ macro commands recorded by the
Command Recorder to a stand-alone ImageJ macro.

2.1 Introduction

In some biological research projects, we encounter problems that should be studied by

measuring fluorescence intensity at the boundary between two different compartments.
Here, we pick up an example analysis of the Lamin B receptor protein density targeting
inner nuclear membrane. The protein changes its location from the cytoplasmic area
(Endoplasmic Reticulum, ER) to the nuclear envelope (Boni et al. 2015).
We analyze a two-channel time-lapse image stack, a sequence of the process of the
protein re-localization that causes increases in the protein density at the nuclear envelope.
The data was acquired by Andreas Boni (Jan Ellenberg lab, EMBL Heidelberg) and have
been used in many training workshops in EMBL as a great example for learning bioimage
analysis. His work, with more advanced bioimage analysis workflows for analyzing the
protein targeting dynamics, is published in The Journal of Cell Biology (Boni et al. 2015).
Those codes and image data used in his study, which might be interesting for you after
going through this chapter, are accessible through the supplementary data section in the
journal website.1
Two images shown in . Fig. 2.1 are from the first and the last time points of a time-
lapse sequence.2 Compare these images carefully. The green signal broadly distributed in
the cytoplasmic area at time point 1 becomes accumulated at the periphery of nuclei (red)
at time point 15—between these image frames, the signal changed its localization from ER
to the nuclear envelope. We construct a workflow that measures this accumulation pro-
cess by writing an ImageJ macro. The workflow involves two steps: First, we segment the
rim of nucleus—nuclear membrane—using the first channel (histone). Second, we use
that segmented nuclear rim as a mask to measure the intensity changes over time in the
second channel.
Segmentation of nucleus using its marker (e.g. DAPI) is a popular image analysis tech-
nique used in many biological research projects, but to measure more specific location—
in our case nuclear envelope—we need to add several more steps to refine the
region-of-interest. When we are successful in determining the area of nuclear envelope,
the measurement of intensity in that region over time is rather trivial. We just need to loop
the same process for each time point. Especially for the analysis of time-lapse sequence,
programming is highly recommended to iterate the measurement for each time point.

1 7 http://jcb.rupress.org/content/209/5/705
2 The images shown in the . Fig. 2.1 are from a 4D hyperstack “NPC1.tif”, which can be downloaded
using ImageJ plugin “CMCI-EMBL”. More details are in “Dataset” section.
Measurements of Intensity Dynamics at the Periphery of the Nucleus
11 2

a b

..      Fig. 2.1 Lamin receptor localization difference at two time points: More Lamin receptor in nucleus
periphery. a Time point 1. b Time point 15

This chapter should be a good guide not only limited to study the intensity changes
occurring at the nuclear envelope, but also in general for segmenting the edge (perimeter)
of biological compartments such as the edge of organelle, plasma membrane and tissue
boundaries. In principle, similar post-processing strategy is also applicable to 3D volumes
by using 3D morphology filters.

2.2 Tools

We use Fiji (Fiji Is Just ImageJ) for image analysis.

55 Fiji
55Download URL: 7 https://imagej.­net/Fiji/Downloads
55Please choose the latest version.

In addition, a plugin is required for loading the sample image data. Using the “Update
sites” function, please add “CMCI-EMBL” to your Fiji installation. Please restart Fiji after
this plugin installation.

2.3 Dataset

All ImageJ macro codes can be downloaded from the Github repository.3
The image data we used in this chapter can be downloaded using the plugin
“CMCI-­EMBL”. After installation of this plugin, select the menu item [EMBL > Sample
Images > NPCsingleNucleus.tif] to load the image data. This is a time-lapse

3 7 https://github.com/miura/NucleusRimIntensityMeasurementsV2/
 12 K. Miura

sequence of a cell, extracted from “NPC1.tif ” which can be also downloaded through the
same plugin.
55 Cell Type: Hela Cells
2 55 Scale: 0.165 μm/pixel
55 Frame Rate: 400 Sec/Frame
55 Channels
55Red channel (C1): H2B-mCherry (ex:561nm)
55Green Channel (C2): Lamin B Receptor-GFP (ex:488nm)

2.4 Workflow

To simplify the development, we focus on a single cell/nucleus to construct the workflow.

Load the image stack NPCsingleNucleus.tif. This is a hyperstack sequence. Slide the scroll
bar at the bottom back-and-forth to watch the process of intensity changes. H2B-­mCherry
signal (red), used as a marker for nucleus, is more or less constant with its distribution. On
the other hand, the Lamin receptor signal (green) exhibits strong accumulation to the
nuclear membrane. To study this accumulation process, our aim is to measure the inten-
sity changes of green signal intensity at the rim of the nucleus over time. The outline of the
workflow is shown in the diagram (. Fig. 2.2).
To achieve this aim we first need to identify the region of nucleus rim (“segmenta-
tion”)—in other words, we create a mask of the nucleus rim. Using this mask we measure
the changes in intensity over time.

2.4.1 Segmentation of Nucleus Rim

We first write a macro for the nucleus rim segmentation by taking following steps:
1. Split the original multi-channel image stack and create two image stacks of each
channel for processing them independently (. Fig. 2.3a)
2. Blur the image to attenuate noise (. Fig. 2.3b)
3. Nucleus segmentation: Binarize the image by intensity thresholding (. Fig. 2.3c)
4. Remove other Nuclei: At the right-bottom corner of the image, a small part of
different nucleus is present. This should be removed.
5. Duplicate the image
(a) Erode the original (. Fig. 2.3e)
(b) Dilate the duplicated (. Fig. 2.3d)
6. Subtract the eroded from the dilated (. Fig. 2.3f)

In the following we record these steps as macro commands using the Command Recorder
([Plugins > Macros > Record…]). We recommend you NOT to launch the com-
mand recorder from the beginning. Please first try to reproduce the workflow using mouse
and the graphical user interface (GUI). This is like a rehearsal before recording your actions.
When you become clear with the steps you have to take, record the processing steps. When
you use the command recorder, be sure that “Macro” is selected in the “Record:” drop down
menu at the top-left corner of the recorder.
Measurements of Intensity Dynamics at the Periphery of the Nucleus
13 2

2-Channel Time
Series

Channel
Splitting Block

Ch1
Nucleus

Nucleus Rim Segmentation Block

Pre-processing
Gaussian blur

Intensity
Thresholding (Otsu)

Dilation Erosion

Image
Measurement Block
Subtraction

Ch2
NPC
Define the region /
measurements

..      Fig. 2.2 The outline of the workflow

2.4.1.1 Block 1: Splitting Channels

To split the multichannel image stack from the GUI menu, do [Image > Color >
Split Channels]. In the Recorder you will see the following command.

run("Split Channels");

run function is the most frequently used build-in macro function.

 14 K. Miura

a b c

d e f

..      Fig. 2.3 The strategy of segmentation. a Original nucleus image. b Blurred nucleus image.
c Binarized image, after thresholding. d Dilated binary image. e Eroded binary image. f Subtraction
result, the rim

run(”command”[, ”options”])
Executes an ImageJ menu command. The optional second argument contains values that
are automatically entered into dialog boxes (must be GenericDialog or OpenDialog). Use
the Command Recorder (Plugins>Macros>Record) to generate run() function calls. Use
string concatenation to pass a variable as an argument. With ImageJ 1.43 and later, variables
can be passed without using string concatenation by adding “&” to the variable name.

The run function takes a menu item as the first argument and optional values (values you
fill-in in a dialog window) in the second argument. In case of channel splitting, there is no
such optional value so the second argument is ignored.
We then process the nucleus image. Click the nucleus image window to bring it up to
the top—We call this action as “activating a window”. By this clicking, we activated
Channel 2 (red, nucleus image).
Please confirm that a new command shown below, is added to the recorder after acti-
vating the nucleus image.
selectWindow("C1-NPCsingleNucleus.tif");
…Here is the explanation from the macro function reference.

selectWindow(”name”)
Activates the window with the title ”name”.