Exploring Uncharted Realms with

Electron
Microscopy
You do not need to take a trip into
outer space to discover an unknown

Table
world. An uncharted realm exists
right here on our planet; it is an
empire full of strange creatures, exotic

of contents
landscapes and unusual structures.

The dimension of micrometers and

nanometers is a mysterious and
almost magical domain that has
An introduction to electron microscopy
captured the human imagination for
Magnifying ten million times 03
decades. For nearly 60 years,
Electron microscopy in the life sciences 04
humankind has been exploring the
Electron microscopy for nanomaterials and devices 04
micro and nano realms with the help New developments in electron microscopy 05
of electron microscopes.
What is microscopy?
The world of microscopes 06
Comparing the scale of different microscopes 08

Key components of electron microscopes

Electron sources 09
The electron microscope column 10
Electromagnetic lenses 12
What happens during electron bombardment? 13

Transmission electron microscopy (TEM)

An overview of TEM 15
3D Imaging techniques 24
Scanning transmission electron microscopy (STEM) 25
X-ray microanalysis 26

Scanning electron microcopy (SEM)

An overview of SEM 28
Application and specimen preparation 33

DualBeam – combining SEM with FIB

An overview of DualBeam technology 35

Glossary of common electron microscopy 39

language
3 | Exploring Uncharted Realms with Electron Microscopy

CHAPTER 1

An introduction
to electron microscopy
Magnifying ten million times
Electron microscopes reveal hidden wonders that are smaller
than the human eye can see. They fire electrons and create
images, magnifying micrometer and nanometer structures
by up to ten million times, providing a spectacular level
of detail that allows us to view single atoms. Observing
Beautiful ornamentation of a spore hidden in
the world through electron microscopes can make the
the hymenophorous tissue of a Russula emetica
invisible visible, expand our horizons, transform our mushroom.
perceptions and open our minds to new possibilities.
Courtesy of Mrs. Anna Siudzinska , PORT Polish
Center for Technology Development
Electron microscopy has the power to answer long-standing Original data collected at a horizontal field width
questions, like “Why is spider silk so strong?” What’s more, of 11 μm.

it will answer questions you never even thought to ask,

such as “What does the mouth of a caterpillar look like?”

Thousands of scientific discoveries have been fueled

by electron microscopy, creating millions of unique images
that not only offer great scientific value, but are art forms
in their own right. When you first look at images from
an electronmicroscope, you may not believe your eyes; Folds of graphite.
the scenes can easily resemble illustrations of fantasy
Original data collected at a horizontal field width
or science fiction.
of 59.5 μm.

The Nanometer An intermediate unit is the micrometer

As distances become shorter, the number of (abbreviated to μm), which is a millionth (10-6) of
zeros after the decimal point becomes larger, so a meter or 1,000 nm. Some literature refers to
microscopists use the nanometer (abbreviated the Ångström unit (Å), which is 0.1 nm and uses
to nm) as a convenient unit of length. One micron for micrometer. A picometer is a trillionth
nanometer is a billionth (10-9) of a meter. (10-12)
4 | Exploring Uncharted Realms with Electron Microscopy

Electron microscopy in the life sciences—life at

microscale
We are surrounded by things that are too small for us
to see. The world at microscale is full of tiny, alien-like
creatures, from the mites that crawl along our eyelashes at
night to tardigrades, who have been proclaimed the most
resilient creatures on earth, despite being only 0.5 mm in size.
What’s more, there may be millions of tiny critters out there
who are yet to be discovered.

From the bacteria trapped underneath our nails to the hidden

world inside our own bodies, electron microscopy has revealed
epic battles between our immune system and diseases. It
has lain bare the tangled mass of fibers that constitutes blood Spider skin, with a hair root and adhered pollen
clots, how diseased cells and tissues wreak havoc on our grains.

health, and how our bodies are vibrant ecosystems in their Courtesy of Maria Carbajo, Universidad de
own right, playing host to millions of bacteria. Extremadura.
Original data collected at a horizontal field width
of 24.9 μm.

Electron microscopy for nanomaterials

and devices
When we zoom in from microscale to nanoscale, we can
observe the building blocks of the materials that comprise
our world. Electron microscopy is essential for
the development of nanotechnology and nanodevices,
transforming a material’s atoms from an abstract concept
to objects we can see with our own eyes, enabling us
to engineer materials atom by atom.

By showing us the structure of materials at the nanoscale,

electron microscopy provides a way to understand the link Hematite nanoparticle with individual atoms
between material composition, structure and performance. visible.
This has led to technological advances, including smaller,
Courtesy of Enrique Díaz Barriga Castro.
faster computers, chemical sensors, targeted drug delivery, Original data collected at a horizontal field width
high-performance materials, water filters and many more. of 14.385 nm.
5 | Exploring Uncharted Realms with Electron Microscopy

New developments in electron Determining the structures of biomolecules

microscopy in their native environment can allow us to create
drugs that are better at targeting specific proteins
In 1959, physicist Richard Feynman proclaimed
or receptors.
“There’s plenty of room at the bottom,” and
called for electron microscopes to be improved
Electron microscopy has initiated a new era of
and increased in power by 100 times to resolve
scientific discoveries that extend far beyond
features as small as one nanometer. This would
simply observing small things, but there are still
allow scientists to truly explore the features
many more discoveries out there, just waiting
of the nano world.
for someone to seek them out. We are on the
threshold of extraordinary advances, driven by our
Since then, we have met Feynman’s demand,
exploration of a reality that is just too small for us
and the possibilities for electron microscopy
to see.
have increased exponentially. In 2017, Jacques
Dubochet, Joachim Frank and Richard Henderson
received the Nobel Prize in Chemistry for their
contributions to the development of the latest
evolution in electron microscopy: cryo-electron
microscopy, which enables us to resolve the
structures of biomolecules frozen in solution.
Each new development in electron microscopy
has opened new doors and allowed scientists to
explore new features of the world at nanoscale.

Once we understand this hidden realm through

exploration, we can use that knowledge to change
our own world.

Knowing how the anatomy of tardigrades makes

them so durable would allow us to create stronger,
more resilient materials. Understanding how
our immune system tackles disease in exacting
detail would let us know what to do when things
go wrong. Examining the nanoscale structures
of materials, sensors and devices can help us
Contact side of an infrared detector array.
understand structure/performance relationships
and ultimately design new or improved materials Courtesy of Sedat Canli.
and technologies. Original data collected at a horizontal field width of 200 μm.
6 | Exploring Uncharted Realms with Electron Microscopy

CHAPTER 2

What is microscopy?
The world of microscopes electrostatic or electromagnetic lenses to focus
the particles. They can observe features as small
Most microscopes can be classified as one
as 0.1 nm (one ten-billionth of a meter), such as
of three basic types: optical, scanning probe
individual atoms. Scanning probe microscopes
or charged particle (electron and ion).
use a physical probe (a very small, very sharp
needle) that scans over the sample in contact
Optical microscopes are the ones that are most
or near-contact with the surface. They map
familiar to everyone and are used in countless
different forces and interactions that occur
places, from the doctor’s office to the high school
between the probe and the sample to create
science lab. They use visible light and transparent
an image. These instruments are also capable
lenses in order to see objects as small as one
of atomic-scale resolution.
micrometer (one-millionth of a meter), such as a
red blood cell (7 μm) or a human hair (100 μm).
A modern light microscope (often abbreviated
to LM) has a maximum magnification of about
Electron and ion microscopes use a beam
1,000× and allows the eye to resolve objects
of charged particles instead of light and use
separated by 200 nm. As inventors and

The first microscopes

Nobody knows for certain who invented
the microscope. The light microscope probably
developed from the Galilean telescope during the
17th century. One of the earliest instruments for
seeing very small objects was made by
the Dutchman Antony van Leeuwenhoek (1632–
1723) and consisted of a powerful convex lens
and an adjustable holder for the object
being studied.

With this remarkably simple microscope, Van

Leeuwenhoek may well have been able to magnify
Phytoplankton sample from Atlantic Ocean
objects up to 400×. With it, he discovered
protozoa, spermatozoa and bacteria, and he was Courtesy of Mr. Daniel Mathys, Universität Basel.
able to classify red blood cells by shape. Original data collected at a horizontal field width of 170 μm.
7 | Exploring Uncharted Realms with Electron Microscopy

scientists worked to attain better resolution, Ernst Ruska at the University of Berlin combined
they soon realized that the resolving power of these characteristics and built the first transmission
the microscope was not only limited by the electron microscope (TEM) in 1931. For this and
number and quality of the lenses, but also by the following work on the subject, he was awarded
wavelength of the light used for illumination. With the Nobel Prize for Physics in 1986. The first
visible light, it was impossible to resolve points in electron microscope used two magnetic lenses,
the object that were closer together than a few and three years later, Ruska added a third lens and
hundred nanometers. demonstrated a resolution of 100 nm, twice as
good as a typical light microscope. Today, electron
microscopes have reached resolutions greater
than 0.05 nm, more than 4,000 times better than
a typical light microscope and 4,000,000 times
better than the unaided eye.

Resolution of the human eye

Given sufficient light, the unaided human
eye can distinguish two points 0.2 mm
Shale sample. apart. If the points are closer together, they
will appear as a single point. This distance
Original data collected at a horizontal field width of 25.4 μm.
is called the resolving power, or resolution
of the eye.
Using light with a shorter wavelength (ultraviolet
or blue) gave a small improvement. Immersing
A lens or an assembly of lenses (as in
the specimen and the front of the objective lens
a microscope) can be used to magnify
in a medium with a high refractive index (such as
this distance and enable the eye to see
oil) offered another small improvement, but these
points even closer together than 0.2 mm.
measures together only brought the resolving
For example, try looking at a newspaper
power of the microscope to just under 100 nm.
picture, or one in a magazine, through
a magnifying glass. You will see that
In the 1920s, it was discovered that accelerated
the image is actually made up of dots
electrons act in a vacuum much like light. They
too small and too close together to be
pass in straight lines and have wavelike properties,
separately resolved by your eye alone.
with a wavelength that is about 100,000 times
shorter than that of visible light. Additionally, it was
The same phenomenon will be observed
found that electric and magnetic fields could be
on an LCD computer display or flat screen
used for shaping the paths followed by electrons,
TV when magnified to reveal the individual
similar to the way glass lenses are used to bend
“pixels” that make up the image.
and focus visible light.
 8 | Exploring Uncharted Realms with Electron Microscopy

Comparing the scale of different microscopes

1m 10-1 m 10-2 m 10-3 m 10-4 m 10-5 m 10-6 m 10-7 m 10-8 m 10-9 m 10-10 m 10-11 m

Human Eye Electron Microscope

Light Microscope

Human Apple Wasp Ant Hair Cell Bacteria Virus DNA Small Atom Electron
Molecule Orbital

Resolution and wavelength permitting better resolution. The wavelength

of an electron in a TEM may be only a few
When a wave passes through an opening in
picometers (1 pm = 10-12 m), more than
a barrier, such as an aperture in a lens, it is
100,000 times shorter than the wavelength
diffracted by the edges of the aperture. Even a
of visible light (400–700 nm). Unfortunately,
perfectly shaped lens will be limited in its resolving
the magnetic lenses used in electron microscopes
power by diffraction. This is why a high-quality
do not approach diffraction-limited performance,
optical lens may be referred to as a diffraction-
so electron microscopes have been unable
limited lens—it is as good as it can be, and any
to take full advantage of the shorter wavelength
further effort to improve the quality of the lens
of the electron.
surface will not improve its resolution.

Ultimately, the resolving power of an electron

The amount of diffraction is a function of the size
microscope is determined by a combination of
of the aperture and the wavelength of the light,
beam voltage, aperture size and lens aberrations.
with larger apertures and/or shorter wavelengths
9 | Exploring Uncharted Realms with Electron Microscopy

CHAPTER 3

Key components of
electron microscopes
Before discussing the different types of electron microscopes and the resulting images that they can
produce, it makes sense to understand the different components of an electron microscope.

In general, an electron microscope has four

key components:
Electron source Electronics The electron
Column Control software An atom is made up of three kinds
of particles: protons, neutrons and
electrons. The positively charged protons
Electron sources and neutral neutrons are held tightly
Three key types of electron sources are employed together in a central nucleus. Negatively
in electron microscopes: tungsten guns, charged electrons surround the nucleus.
lanthanum hexaboride guns (LaB6 - frequently Normally, the number of protons equals
known as “lab six”), and field emission guns the number of electrons so that the atom
(FEG). Each represents a different combination as a whole is neutral. When an atom
of benefits and costs. The choice of source type deviates from this normal configuration
is a significant part of the instrument selection by losing or gaining electrons, it acquires
process. a net positive or negative charge and is
referred to as an ion.
Perhaps the single most important characteristic
of the source is brightness, which characterizes The electrons, which are about 1,800
the electron current density of the beam and times lighter than the nuclear particles,
the angle into which the current is emitted occupy distinct orbits, each of which can
(current density per steradian solid angle); this accommodate a fixed maximum number
eventually determines the resolution, contrast of electrons. When electrons are liberated
and signal-to-noise capabilities of the imaging from the atom, however, they behave
system. FEG sources provide brightness up to in a manner analogous to light. It is this
1,000 times greater than tungsten emitters, but behavior that is used in the electron
they are also much more expensive. In some microscope.
high-current applications, tungsten or LaB6 may
in fact function better than FEG.
10 | Exploring Uncharted Realms with Electron Microscopy

A tungsten gun is comprised of a filament, an to electromagnetic electron generation (rather

anode and a Wehnelt cylinder. These three than thermionic), which results in a higher beam
together compose a triode gun, which is an current despite having a smaller emitting area
extremely stable source of electrons. The tungsten than tungsten and LaB6 sources.
filament is hairpin-shaped and heated to about
2,700 K. By applying a high positive potential
The electron microscope column
difference between the filament and the anode,
electrons are extracted near the filament and then The electron column is comprised of elements
accelerated towards the anode. analogous to those of a light microscope.
The light source of the light microscope is
The anode has a hole in it so that an electron replaced by an electron gun. The glass lenses
beam, in which the electrons could travel faster are replaced by electromagnetic or electrostatic
than 2,000 km/s, emerges and is directed down lenses. Unlike glass lenses, the power (focal
the column. Tungsten sources are the least length) of magnetic lenses can be changed by
expensive, but have limited lifetimes and offer modifying the current through the lens coil.
lower brightness. The brightness of a tungsten
source can be increased, but only at the cost
of a shorter lifetime, as brightness ≈ current/
diameter2.

Like tungsten, LaB6 guns and CeB6 guns rely on

thermionic emission of electrons from a heated
source, a lanthanum (or cerium) hexaboride
crystal. LaB6 sources can offer up to 10 times
more brightness than tungsten and have
considerably longer lifetimes, but need higher
vacuum levels, which increases the cost of
the microscope. The emitting area of LaB6 is
smaller than tungsten, increasing brightness but
decreasing total beam current capability.

Field emission guns, in which the electrons

are extracted from a sharply pointed tungsten
tip by an extremely high electric field, are the
most expensive type of source, but usually
provide the highest imaging and analytical
Mouse brain sample 3D.
performance. This performance increase is due
11 | Exploring Uncharted Realms with Electron Microscopy

(In the light microscope, deviation in magnification light microscope

is attained by changing the lens or by mechanically
moving the lens). The ocular, or eyepiece, is
replaced by a fluorescent screen and/or a digital
camera.

The electron beam is generated by the electron

gun (generally at the top of the column) and is
condensed into a nearly parallel beam at
the specimen by the condenser lenses.
For TEM, the specimen must be adequately thin objective lens

such that it can transmit the electrons, usually

light beam
0.5 μm or less; however, for SEM this is not specimen
important. In TEM, higher energy electrons (that
is, higher accelerating voltages) can penetrate light source
thicker samples.

SEM TEM

electron source
anode electron source
gun align coils
lens 1 

lens 2
first condenser lens
electron beam
second condenser lens
scan and stig coils condenser
aperture objective condenser lens
lens 3
objective minicondenser lens
collector system aperture
specimen (thin)
selected
area objective imaging lens
aperture diffraction lens
secondary electrons intermediate lens
electron beam first projector lens
impact area
specimen (thick) second projector lens
vacuum
projection chamber

turbo/diff pump fluorescent screen

roughing line

Comparison of the light microscope with TEM, SEM and FIB microscopes.
12 | Exploring Uncharted Realms with Electron Microscopy

Electromagnetic lenses
The figure on the right presents a cross-section of
an electromagnetic lens. When an electric current
How do electrons interact
is passed via the coils (C), an electromagnetic
with matter?
field is developed between the pole pieces (P), In the modern view of matter, an atom
which produces a gap in the magnetic circuit. consists of a heavy charged nucleus
By changing the current through the coils, the surrounded by a number of orbiting
strength of the field, and thus the power of the electrons. The number of electrons is
lens, can be varied. equal to the number of protons in the
nucleus and is known as the atomic
This is the essential difference between the number of the atom.The incoming beam
magnetic lens and the glass lens. Otherwise, electron can interact with the nucleus
they act similarly and have the same types of and be backscattered with virtually
aberration: spherical aberration (Cs – the power undiminished energy (just as a space
in the center of the lens differs from that at the probe is deviated by the gravity of a
edges), chromatic aberration (Cc – the power of planet during a fly-by). Or it can interact
the lens varies with the energy of the electrons with the orbiting electrons of sample
in the beam), and astigmatism (a circle in the atoms in a variety of ways, giving up
specimen becomes an ellipse in the image). some of its energy in the process.

electron beam Each type of interaction potentially

constitutes a signal that carries
information about the sample.
C C

For instance, the most frequent

interaction is the ejection of an electron
from the atom with relatively low
P P energy, a few eV. If this occurs near
the sample surface, the liberated
P P electron may escape and be detected
as a secondary electron. Other
signals include characteristic X-rays,
cathodoluminescence, absorbed current
C C and more, each carrying a specific type of
information.

Cross-section of an electromagnetic lens.

C is an electrical coil and P is the soft iron pole piece.
The electron trajectory is from top to bottom.
13 | Exploring Uncharted Realms with Electron Microscopy

What happens during electron bombardment?

Contrary to what could be expected, most specimens are not adversely affected by the electron
bombardment as long as beam conditions are carefully controlled. When electrons impinge on the specimen,
they can cause any of the following:

In crystalline specimens, the electrons are The impinging electrons can knock electrons
scattered in extremely distinct directions that from sample atoms that then escape as
are a function of the crystal structure. low-energy secondary electrons.
Other electrons are scattered over small The impinging electrons could cause specimen
angles, based on the composition and atoms to emit X-rays whose energy and
structure of the specimen. wavelength are related to the specimen’s
Some of the electrons are absorbed as elemental composition; these are called
a function of the thickness and composition characteristic X-rays.
of the specimen. The impinging electrons cause the specimen
Some of the impinging electrons are to emit photons (or light); this is known as
deflected through large angles or reflected cathodoluminescence.
(backscattered) by sample nuclei. Transmitted electrons lose energy as they pass
through the sample.

Colored electrons Since energy can be equated to wavelength,

color EFTEM images, usually made by
We see a world full of color. The color we see
combining multiple images acquired at
comes from our eyes’ ability to distinguish
different energy loss settings, are perhaps the
among various wavelengths of light. However,
closest we can come to color electron images.
most electron detectors see in black and white,
But even EFTEM images are false color
or more accurately, shades of gray. What then
images in the sense that the correspondence
of the beautiful color images that we see in this
between energy loss and color is an arbitrary
publication and elsewhere attributed to electron
assignment made by the creator of the image.
microscopes? In most cases, color has been
added post-imaging for purely aesthetic reasons.
Color is also used to enhance X-ray maps,
where a particular color may be assigned to
There are exceptions. Energy-filtered TEM
a particular element to show its distribution in
(EFTEM) creates images from electrons that have
the specimen.
been selected for a specific level of energy loss
during their passage through the sample.
14 | Exploring Uncharted Realms with Electron Microscopy

Electron velocity
The higher the accelerating voltage, the faster the
electrons. 80 kV electrons have a velocity of 150,000
km/second (1.5 × 108 m/s), which is half the speed
of light. This rises to 230,000 km/second for 300 kV
electrons (2.3 × 108 m/s, more than three-quarters the
speed of light). The wave particle duality concept of
quantum physics asserts that all matter exhibits both
wave-like and particle-like properties. The wavelength
λ of an electron is given by

Image of Trypanosoma brucei gambiense

parasite (blue) intertwined with red blood cells
(red), in a blood capillary (green).

Courtesy of David Pérez-Morga, Université Libre

where h is Planck’s constant and is the relativistic de Bruxelles.
momentum of the electron. Knowing the rest mass of Original data collected at a horizontal field width
an electron , and its charge , we can calculate the of 45.76 µm.

velocity imparted by an electric potential as

and wavelength at that velocity as

Finally, since the velocities attained are a significant

fraction of the speed of light , we add a relativistic
correction to get

SEM image of alginic acid crystals.

Courtesy of Dr. Maria Carbajo, Universidad de Extremadura.

The wavelength of the electrons in a 10 kV SEM is Original data collected at a horizontal field width of 37μm.
then 12.3 × 10-12 m (12.3 pm), while in a 200 kV TEM
the wavelength is 2.5 pm.
15 | Exploring Uncharted Realms with Electron Microscopy

CHAPTER 4

Transmission electron
microscopy (TEM)
An overview of TEM
It is possible to compare a transmission electron microscope
with a slide projector. In a slide projector, light from a light source
is made into a parallel beam by the condenser lens; this travels
through the slide (object) and is then focused as an enlarged
image onto the screen by the objective lens.

In the electron microscope, the glass lenses are replaced by

magnetic lenses, the light source is replaced by an electron
source, and the projection screen is replaced by a fluorescent
screen, or, more often in modern instruments, an electronic
imaging device such as a charge-coupled device (CCD) camera.

The whole trajectory from source to screen is under vacuum,

and the specimen (object) has to be extremely thin to allow the
electrons to pass through it. All specimens cannot be made thin
enough for the TEM. If individuals want to look at the surface of
the specimen, rather than a projection through it, they use
A modern transmission electron microscope -
a scanning electron or ion microscope. the Thermo Scientific™ Themis™ ETEM.

The electron gun

High-resolution TEM, based on phase contrast, Field emission sources are available in two types,
requires the high spatial coherence of a field cold field emission and Schottky (thermally
emission source, i.e., the field emission source assisted) field emission. Cold field emission offers
should create waves of regular phase and extremely high brightness but varying beam
shape. The greater current density and higher currents. It also needs frequent flashing in order
brightness provided by these sources produce to clean contaminants from the tip. Schottky field
smaller beams with higher currents for better emission offers high brightness and high, stable
spatial resolution and faster, more precise current with no flashing. The newest generation
X-ray analysis. of Schottky field emitters (XFEG) retains its
present stability while attaining brightness levels
close to cold field emission.
16 | Exploring Uncharted Realms with Electron Microscopy

As a rule of thumb, if the application demands Finally, transmitted beam electrons can be
imaging at magnifications up to 40,000–50,000× counted and then sorted by an energy loss
in TEM mode, a tungsten source is usually spectrometer according to the amount of energy
not only adequate but the best source for the they have lost in interactions with the specimen.
application. When the TEM imaging magnification The energy loss carries information about
is between 50,000–100,000×, the brightest the chemical, elemental and electronic states of
image on the screen will be produced with the the sample atoms. In a standard TEM, the mass
help of a LaB6 source. If magnifications greater thickness is the key contrast mechanism for non-
than 100,000× are needed, a field emission crystalline (biological) specimens, while phase
source provides the better signal. In the case contrast and diffraction contrast are the most
of small probe experiments such as scanning vital factors in image formation for crystalline
or analytical techniques, a field emission gun is specimens (most non-biological materials).
always preferred.

The electromagnetic lenses

In a standard TEM, spherical aberration, which
is primarily determined by the lens design and
Electron penetration manufacture, is the main limitation to enhanced
Electrons are easily stopped or deflected image resolution. Chromatic aberration can be
by matter. (An electron is nearly 2,000× decreased by keeping the accelerating voltage as
smaller and lighter than the smallest atom.) stable as possible and employing extremely thin
That is why the microscope has to be specimens. Astigmatism can be corrected by
evacuated and why specimens—for using variable electromagnetic compensation coils.
the transmission microscope—have to be
very thin. Typically, for electron microscopy The condenser lens system focuses the electron
studies, a TEM specimen must be no beam onto the specimen under investigation as
thicker than a few hundred nanometers. much as necessary in order to suit the purpose.
Different thicknesses provide different The objective lens generates an image of
types of information. the specimen that is then magnified by the
remaining imaging lenses and projected onto
For present day electron microscopy the viewing device.
studies, thinner is almost always better.
Specimens as thin as a few tenths of If the specimen is crystalline, a diffraction pattern
a nanometer can be created from some will be developed at a point below the objective
materials using modern preparation lens called the back focal plane. By varying
techniques. While thickness is a primary the strengths of the lenses immediately below
consideration, it is equally important that the objective lens, it is possible to enlarge the
the preparation preserves the specimen’s diffraction pattern and project it onto the viewing
bulk properties and not alter its atomic device. The objective lens is then followed by a
structure—not a trivial task. number of projection lenses used to focus, magnify
and project the image or diffraction pattern onto
17 | Exploring Uncharted Realms with Electron Microscopy

the viewing device. To promise high stability This results in a general blurring of the image and
and to attain the highest possible lens strength/ also in a phenomenon known as delocalization,
magnification, the lenses in a modern TEM are in which periodic structures appear to extend
generally water-cooled to prevent the build up of beyond their actual physical boundaries.
heat, which would result in more noise and lower
quality data. In a light microscope, spherical aberration can be
reduced by integrating lens elements that have
On the way from the source to the viewing opposing spherical aberrations. This method
device, the electron beam travels through cannot be applied in electron microscopes, as
a series of apertures with varied diameters. the round magnetic lenses they use display only
These apertures stop those electrons that are positive spherical aberration. Multi-pole correcting
not needed for image formation (for example, elements (with fundamentally negative aberration)
scattered electrons). Using a special holder were described by Otto Scherzer in 1947.
carrying a number of differently sized apertures,
the objective lens, the diameter of the apertures However, their effective commercial
in the condenser lens, and the diffraction lens can implementation needed solutions to a range
be changed as needed. of practical issues. Some are comparatively
simple to achieve; for instance, the diameter
of the electron column can be increased to
provide the mechanical stability needed to see
the advantage of enhanced optical performance.
Others were very multifaceted, such as designing
adequately stable power supplies and developing
techniques and software controls that are
advanced enough to reliably measure and then
rectify the aberrations by autonomously exciting
the multi-pole elements.
An atomic resolution image of an advanced logic semiconductor
device is depicted. Some of the layers critical to chip
The ability to correct spherical aberration
performance are only a few atoms in width.
leaves chromatic aberration effects as
Original data collected at a horizontal field width of 60 nm. the next key challenge in refining TEM
performance. Chromatic aberration correctors
have been effectively added into modern S/
Aberration-corrected TEM
TEM instrumentation, but their design and
The latest development of a dedicated
operation are considerably more complex than
commercial aberration-corrected TEM has
spherical aberration correctors. At the same
allowed major advances in both STEM (Scanning
time, substantial progress has been made in
Transmission Electron Microscope) and TEM
decreasing the energy spread of electrons
capability. Without correction, TEM resolution is
passing through the lenses. (The energy
limited primarily by spherical aberration, which
spread establishes the magnitude of chromatic
causes information from a point on the object to
aberration’s deleterious effects.)
be spread over an area in the image.
18 | Exploring Uncharted Realms with Electron Microscopy

Variations in electron energy may originate as

the beam is created in the electron gun, or they
may be introduced in transmitted electrons by
interactions with sample atoms. The first of these,
beam energy spread, has been addressed by
designing extremely stable high-voltage and
lens current power supplies, by using specially
improved field emission electron sources and by
directing the beam through a monochromator,
which passes only a very narrow band of
energies.
This image displays the formation of planes that are visible when
The energy spread among electrons conveyed nanoparticles of titanium dioxide are used as a catalyst in the
through the specimen can be reduced by process of photocatalysis.

minimizing sample thickness with modern sample Courtesy of Dr. Maria Carbajo, Universidad de Extremadura.
preparation methods. Original data collected at a horizontal field width of 63 nm.

Image resolution and information limit Moiré-fringe image extracted

from the original TEM image
Prior to the development of spherical aberration taken on a spherical-aberra-
correctors, scientists knew that a TEM was tion corrected S/TEM.
capable of providing higher spatial resolution than
Courtesy of Craig Johnson,
what could be observed directly in the image.
CEMES-CNRS.
Original data collected at
This directly observable resolution, known a horizontal field width of
as point resolution, was limited by spherical 34 nm.
aberration of the lenses. However, by
appropriately combining data from multiple
images in a “throughfocus series” (acquired over
a range of defocus values), researchers could
reconstruct a model image exhibiting higher
resolution information.

The highest resolution information the instrument

is capable of transferring is known as its
Comparison of HR-TEMs with (left) and without (right) Cs-
information limit. With spherical aberration correction on the same Si (110) grain boundary at 300 kV.
correctors, the point resolution is extended to
the information limit, and the distinction
disappears for most practical purposes.
19 | Exploring Uncharted Realms with Electron Microscopy

Observing and recording the image

Originally, TEMs used a fluorescent screen, which emitted
light when influenced by the transmitted electrons, for real-
time imaging and alterations. A film camera was used to
record permanent, high-resolution images (electrons have
the same impact on the photographic material as light).
The screen was under vacuum in the projection chamber
but could be perceived through a window using a binocular
magnifier, if required.

The fluorescent screen typically hinged up to allow the image

to be projected on the film below. Advanced instruments rely
mainly on solid-state imaging devices, such as a charge-
coupled device (CCD) camera, for image capture. They may
still include a fluorescent screen, but it can be observed by a
video camera. In this article, unless particular aspects of an
imaging system are being discussed, the instrument is implied
to have a solid-state imaging device.

The subsequent introduction of a direct electron detector

promised great improvements in image resolution and
contrast, mainly in signal-limited applications. A conventional
CCD camera uses a scintillator material over the image
detector elements to convert incident electrons to light, which
then produces charge in the underlying CCD element.

The scintillator introduces some loss of resolution, and

the conversion process reduces the efficiency with which
Dynamic random-access memory (DRAM) electrons add to image contrast. This can be an issue in
capacitors, a type of random-access memory
applications that are sensitive to damage by the electron
that stores each bit of data in a separate
capacitor within an integrated circuit. beam, such as cryogenically prepared samples of delicate
biological materials, where it is vital to extract the highest
Courtesy of Dr. Neerushana Jehanathan,
amount of information from a faint, noisy signal before the
Chipworks
Original data collected at a horizontal field width sample is damaged.
of 1.5 µm.

Eliminating the scintillator with a direct electron detector

enhances image resolution and increases detector efficiency
by up to three times.
20 | Exploring Uncharted Realms with Electron Microscopy

The vacuum The size of the sample chamber in a TEM is

Electrons act like light only when they are very constrained by the requirements of lens
controlled in a vacuum. As has already been design—the sample is essentially located inside
mentioned, the entire column from source to the objective lens. The development of aberration
the fluorescent screen (including the camera) is correctors promises to lessen some of these
evacuated. Various levels of vacuum are essential; constraints, creating extra flexibility for larger,
the maximum vacuum is around the specimen and more complex experimental apparatuses in ETEM.
in the source, while a lower vacuum is found in the
projection and camera chambers.

Different vacuum pumps are used to acquire

and maintain these levels. The vacuum in a field
emission electron gun may be as high as (i.e.,
“pressure as low as”) 10-8 Pa. To avoid the need
to evacuate the entire column every time
a specimen, photographic material or filament is
exchanged, many airlocks and separation valves
are incorporated. In advanced TEMs,
the vacuum system is totally automated, and
Bright Field TEM of a 3D transistor used in microprocessors.
the vacuum level is continuously scrutinized and The gate of the transistor is wrapped around the source drain to
fully protected against defective operation. increase performance.

Original data collected at a horizontal field width of 190 nm

Environmental TEM
Environmental TEM (ETEM) uses a particularly The electronics
designed vacuum system to allow researchers
To acquire the very high resolution that advanced
to detect specimens in a variety of conditions
TEMs are capable of, the accelerating voltage
approaching more “natural” environments, with
and the current through the lenses must be
gas pressures in the sample chamber as high as
very stable. The power supply cabinet contains
a few percent of atmospheric pressure. This can
several power supplies whose output voltage
be vital for observing interactions between the
or current does not deviate by more than
environment and the sample, as, for example,
1/10,000,000th of the value selected for
the action of a solid catalyst particle in a
a specific purpose. Such stabilities necessitate
gaseous reaction environment. ETEM depends
very advanced electronic circuits.
on pressure-limiting apertures and differential
vacuum pumping to allow less restrictive vacuum
Enhanced electron optical design has made
conditions in the vicinity of the sample while
a variety of progressively complicated electron-
maintaining a high vacuum in the rest of the
optical methods possible. This, in turn, has
electron column.
brought on the need to streamline instrument
operation, allowing more users with less
specialized training to produce data efficiently
21 | Exploring Uncharted Realms with Electron Microscopy

and effectively. Digital electronic methods,

in general, and microprocessor-based methods,
in particular, play a key role in this respect.

Advanced electron microscopes employ a

computer to control, monitor and record the
operating conditions of the microscope. This
results in a significant reduction in the number
of control knobs, compared with previous
models, and a microscope that is easier to use,
particularly when numerous accessories require
simultaneous optimization.

Additionally, it allows special methods and

experiments to be embedded in the instrument
so that the operator can perform them using Cross-section view of the myelin sheath of an axon. Myelin is a
the same controls. The computer can be fatty substance that electrically insulates the axon of a nerve cell,
speeding up the transmission of impulses between neurons.
linked to a network to allow automatic backups
and data sharing. Courtesy of Professor Juan Carlos Leon-Contreras, INCMNSZ.
Original data collected at a horizontal field width of 900 nm.

Specimen orientation and manipulation

The goniometer itself offers motorized X, Y and Z
The TEM specimen stage must provide a range
movement and tilt along one axis.The specimen
of movements to control and orient the sample.
is mounted near the tip of a rod-shaped holder,
Tilt and X, Y and Z translation are used to move
which in turn is added into the goniometer
the correct region of the sample into the field
through an airlock. It is the specimen holder rod
of view of the microscope. Tilt along a second
that provides the additional tilt axis, rotation,
axis is needed to allow precise orientation of
heating, cooling or straining, with a special holder
crystalline samples with respect to the beam for
required for each purpose.
diffraction studies and analysis along a specific
crystallographic orientation or grain boundary.
Specimen preparation
Specialized stages may also provide heating, A TEM can be used in any branch of science
cooling and straining of the specimen for and technology where it is informative to examine
experiments in the microscope. The elementary the internal structure of specimens down to
movements are provided by a goniometer the atomic level. The sample must be stable
mounted very close to the objective lens. and small enough (about 3 millimeters in diameter)
The specimen is usually located in the objective to allow its introduction into the evacuated
lens field between the pole pieces because it is microscope column and thin enough to allow
there that the lens aberrations are smallest and the transmission of electrons.
the resolution is highest.
22 | Exploring Uncharted Realms with Electron Microscopy

Different thicknesses are required for various Cryo-TEM allows biological molecules to be
applications. For critical high-resolution materials tested in their natural context, in connection
studies, the sample cannot be thicker than with other molecules that are frequently vital
20 nm or so; for bio-research, the film can to their form and function. Moreover, vitrified
be 300–500 nm thick. samples are, quite plainly, frozen in time, allowing
researchers to examine time-based phenomena
Every branch of research has its own specific such as the structural dynamics of flexible
sample preparation techniques for electron proteins or the aggregation and dissociation
microscopy. In biology, for instance, there may of protein complexes.
first be a chemical treatment that removes water
and preserves the tissue (as much as possible)
in its original state, followed by embedding in
a hardening resin. After the resin has hardened,
What is cryo-electron
slices (sections) with an average thickness
microscopy?
of 0.5 μm are cut with an instrument called
an ultramicrotome, which contains a glass Cryo-electron microscopy is a form
or diamond knife. The minute sections created of transmission electron microscopy
this way are placed on a specimen carrier, that uses cryogenic temperatures to
typically a 3 mm diameter copper specimen enable proteins, viruses and sub-cellular
grid that has been coated with a 0.1 μm thick structures to be observed in aqueous
structureless carbon film. environments. Recent technological
advancements, including improved
Cryogenic freezing and vitrification detectors and computer algorithms, have
allowed scientists to use cryo-electron
Cryo (freezing) methods avoid the sample
microscopy to determine 3D structures
damage inevitably caused by conventional
of biological macromolecules with atomic
drying, fixing and sectioning preparations.
resolution, taking structural biology and
However, traditional freezing methods, while
biochemistry into a new era.
avoiding the introduction of foreign materials,
can still damage the sample, as the formation
of expanding ice crystals may disrupt delicate
biological structures.

Vitrification is a fast freezing process that occurs

so rapidly that water molecules do not have
time to crystallize, instead forming a vitreous
(amorphous) solid that does little or no damage
to the sample structure.The low temperature of HIV spike protein
the vitrified sample also decreases the damage structure solved by
single particle analysis,
caused by beam electrons during observations, visualized by Chimera.
allowing more (or longer) exposures at higher PDB ID 6MEO, Shaik et
beam currents, producing better quality images. al, Nature 565, 318-323
(2019)
23 | Exploring Uncharted Realms with Electron Microscopy

By measuring the variability within a set of images, each

capturing the shape of a molecule at an instant in time,
researchers can calculate the range of motion and
the intramolecular forces operating in flexible proteins. Similarly,
a collection of images might offer a freeze frame sequence
of the assembly of a protein complex or conformational
changes during antigen binding. Automated vitrification
tools allow precise control of the process, ensuring reliable,
repeatable results.

Focused ion beam milling

In metallurgy, a 3 mm diameter sample disc (~0.3 mm thick)
is chemically treated in such a way that the material in the
center of the disc is completely etched away. Around this
hole, there will typically be areas that are appropriately thin
(approximately 0.1 μm) to allow electrons to pass through.
For studies in aberration corrected systems, this thickness
cannot be over a few dozen nanometers.The use of a focused
ion beam to mill and thin a sample from a bulk specimen is
more and more important, mainly in semiconductor and other
Automated systems, like the Thermo Scientific™
nanoscience applications where the sample site must be Vitrobot™ System, provide fast, easy and
precisely situated. reproducible sample preparation.

Challenges of determining the 3D It is not possible to use traditional electron

structure of macromolecules microscopy techniques to image proteins and
Macromolecules, like proteins, are too small to see macromolecules, as interactions between the
with visible light. For decades, researchers have sample and the electrons can cause extensive
been using X-ray diffraction (XRD) to determine damage to the sample. Lower electron doses
the structures of biological macromolecules. can be used to protect the sample and reduce
However, to conduct XRD, samples must damage, but this often results in poor signal-to-
be crystallized, which is often challenging for noise ratios.
proteins and macromolecules. Nuclear magnetic
resonance (NMR) is also widely used to study Furthermore, electron microscopes operate in
the structure of biological molecules. However, a vacuum, which damages biological samples
NMR works best for small biomolecules and is and does not allow for an aqueous environment.
less effective for characterizing larger biological As biomolecules exist largely in water, the
structures such as proteins and viruses. true structure of these samples can only be
characterized in their native, aqueous states.
24 | Exploring Uncharted Realms with Electron Microscopy

3D imaging techniques It is similar to the way someone would turn

Comprehending the organization of matter an object about in their hand while looking at it
in three dimensions has become increasingly to appreciate its 3D shape. Electron tomography
important. Semiconductor manufacturers has been limited by the technique’s inability to
regularly create nanometer-scale structures acquire information from perspectives that lie
that they must observe and measure in order to close to the plane of the thin sample. Here,
regulate their manufacturing processes. Perhaps the trajectory of the beam through the sample
the most vital application of 3D microscopy is in becomes extremely long, resulting in a region
the sciences, where investigators are separating known as the missing wedge.
the multifaceted molecular interactions that are
the basis of life. Most of these rely directly on the The development of dual-axis tomography, in
intricate 3D shapes of the interacting molecules. which the sample is also rotated about a second
axis perpendicular to the first, has enhanced
results—decreasing the missing wedge to
a missing pyramid. Tomography looks at a single
instance of the subject structure, which allows it
to examine differences within a population
of such structures, but also restricts the analysis
to the data that can be attained from that single
sample (frequently a biological entity quite
vulnerable to beam damage). Presently, the best
spatial resolution available from the tomographic
analysis is a few nanometers.

Single particle analysis (SPA, a slightly misleading

name) obtains images of a large number of
arbitrarily oriented, nominally identical particles
and uses a computer to categorize them into
groups of similar orientation, creates composite
projected images representative of each
In situ cryo-electron tomogram of the native Chlamydomonas
orientation and integrates the composited images
Golgi. Image courtesy of Ben Engel. into a 3D model.

By combining numerous images, SPA builds

Electron tomography is in some ways similar
contrast and enhances the signal-to-noise
to larger scale medical imaging technologies
ratio of the resulting model. In theory, it could
such as CAT scans or MRIs. Tomography
continue refining by merely increasing the number
obtains a series of projected images from
of images, though the diminishing returns from
different perspectives as the sample is rotated
incremental increases impose a practical limit.
incrementally about an axis perpendicular to the
SPA results have been reported with a spatial
viewing direction. A computer then combines
resolution of a few tenths of a nanometer.
these images into a 3D model of the sample.
25 | Exploring Uncharted Realms with Electron Microscopy

Automation plays a key role in both approaches scattered beam electrons, secondary electrons,
to 3D imaging. In the tomographic analysis, characteristic X-rays and electron energy loss.
the entire series acquisition can be automated.
Automation is virtually indispensable in SPA, While the technique can be used in both
which may require the analysis of tens of an SEM and TEM, the higher accelerating
thousands of particles. In both cases, automation voltages available in a TEM allow the use of
can also help to decrease sample damage thicker samples, and the additional lenses
by guaranteeing consistent use of low-dose below the sample greatly expand the number
methodologies. (Low-dose imaging refers to of possibilities for gathering information. TEM-
methods that reduce the exposure of the sample based STEM, using a condenser lens aberration
to damaging radiation from the electron beam.) corrector, has attained a resolution of 0.05 nm.
It is vital in 3D analysis (mainly of biological
materials) to guarantee that the highest amount Similar to SEM, the STEM technique is capable of
of information is acquired before the sample is scanning a very finely focused beam of electrons
damaged or destroyed. across the sample in a raster pattern. Interactions
between the beam electrons and sample atoms
produce a serial signal stream, which is correlated
Scanning transmission electron
with beam position. This builds a virtual image
microscopy (STEM)
in which the signal level at any location in the
TEM can also be combined with SEM to give sample is represented by the gray level at the
scanning transmission electron microscopy corresponding location in the image.
(STEM). The first commercial instrument in which
the transmission and scanning techniques were Its main advantage over conventional SEM
incorporated was a Philips EM200 equipped with imaging is the enhancement in spatial resolution,
a STEM unit, produced in 1969 by Ong Sing which results from eradication of the electron
Poen of Philips Electronic Instruments in the U.S. scattering that occurs in bulk specimens as the
It had a resolving power of 25 nm. Modern beam electrons penetrate into the sample.
TEM systems equipped with STEM capability
can attain resolutions down to 0.05 nm in Secondary electrons (SE) are electrons from
STEM mode. sample atoms that have been scattered by beam
electrons. They have extremely low energies and
Scanning transmission electron microscopy are capable of escaping from the sample only if
incorporates the principles of TEM and SEM and they originate extremely close to the surface. SE
can be carried out on either type of instrument. is the primary imaging signal in SEM, where they
Like TEM, STEM needs extremely thin samples offer good spatial resolution and high topographic
and looks primarily at beam electrons transmitted sensitivity. SE are not often used in STEM mode
by the sample. One of its principle benefits over but are mentioned here for completeness.
TEM is in enabling the use of other signals that
cannot be spatially correlated in TEM, including
26 | Exploring Uncharted Realms with Electron Microscopy

X-ray microanalysis The primary restrictions on the speed and

Electrons bombarding the specimen lead it to precision of X-ray analysis are the fraction of
emit X-rays whose energy is characteristic outgoing X-rays that can be gathered, the energy
of the elemental composition of the sample. resolution of the detector and the speed with
X-ray microanalysis uses an energy dispersive which X-rays can be detected and measured.
X-ray (EDX) spectrometer to count and then sort That rate, at which a single detector can examine
characteristic X-rays according to their energy. X-rays, has been increased considerably by
the development of silicon drift detectors.
The resulting energy spectrum displays distinctive
peaks for the elements present, with the peak
heights indicating the elements’ concentrations.
Analysis of the spectrum can help determine X-ray analysis
accurate elemental concentration with a spatial
The impinging electrons in the primary
resolution down to the 100 nm scale in bulk
beam may eject an electron from a sample
SEM specimens and the 10–20 nm scale in thin
atom. If the ejected electron originates
specimens for SEM-based STEM.
from one of the inner orbitals, the resulting
vacancy may be filled by an electron from
Sub-angstrom (10-10 m) spatial resolution has
an outer orbital of the same atom with
been reported for X-ray microanalysis in the TEM-
the concurrent emission of an X-ray. The
based STEM. Due to the extremely small volume
energy of the emitted X-ray is equal to the
analyzed at any given instant, X-ray microanalysis
energy difference between the orbitals and
can detect extremely small quantities of elements
is thus “characteristic”of the elemental
(down to one-thousandth of a picogram (10-15 g)
identity of the emitting atom.
or less). It is mainly useful for detecting locally
concentrated occurrences of elements that are
An X-ray spectrometer counts and
present at extremely low bulk concentrations,
measures the energy of emitted X-rays.
such as grains of precious metal ores.
The relative intensity of the X-ray signal
at each energy (the energy spectrum)
can be used to calculate the quantitative
elemental composition of the sample
within the volume of interaction—the
region within the sample from which
the X-ray signal originates as the beam
electrons penetrate and scatter.

Energy-dispersive X-ray map of a superalloy.

Original data captured at 3.6 µm.

27 | Exploring Uncharted Realms with Electron Microscopy

Custom-designed systems, optimized for rapid elemental

analysis in applications such as SEM-based automated
mineralogy, may also employ multiple detectors in order to
increase the total area of the detectors, and thus the number
of X-rays they intercept.

Adding detectors is a vital design problem since the detectors

must be arranged close to the specimen without interfering
with other functions of the microscope. TEMs specifically
optimized for X-ray analysis have achieved collection of solid
angles approaching 1 steradian, significantly enhancing
minimum detectable mass performance.

Overall, this allows users to extract more information from the

sample within a short time frame, a key factor when looking at
samples that may be damaged or changed under the electron
beam. This also decreases the time needed to develop
elemental maps from samples.
3D EDS tomogram of Ag-Pt core-shell
nanoparticles, where most of the Ag cores (red)
are covered by Pt shells (green). Pores in the Pt Wavelength dispersive X-ray (WDX) spectrometry measures
partially expose the cores. and then counts X-rays by their wavelength (a correlate
of energy). A wavelength spectrometer uses a crystal or
Sample courtesy of Professors Yi Ding and
Jun Luo of the Center for Electron Microscopy, grating with known spacing in order to diffract characteristic
Tianjin University of Technology, China. X-rays. The angle of diffraction is a function of the X-ray
Original data collected at a horizontal field width
wavelength, and the crystal is mechanically scanned over
of 550 nm.
a range of angles while a detector measures varying intensity.

WDX is usually much slower than EDX but offers greater

spectral (energy) resolution (which helps to prevent inference
among closely spaced spectral peaks) and better sensitivity to
light elements. Several WDX spectrometers are needed with
different crystals in order to cover the complete range
of elements. Their size usually limits their application to SEM
or dedicated electron probe instruments. Electron energy loss
spectrometry (EELS) examines transmitted electrons to
define the amount of energy they have lost in interactions
with the sample. It supplies information about the interacting
atoms, including chemical bonding, elemental identity, valence
and conduction band electronic properties, surface properties
and element-specific pair distance distribution functions.
EELS is mainly used with the TEM-based STEM.
28 | Exploring Uncharted Realms with Electron Microscopy

CHAPTER 5

Scanning electron
microscopy (SEM)
An overview of SEM
A scanning electron microscope (SEM), like a TEM,
uses a vacuum system, an electron optical column,
electronics, detectors and software to capture
a nanoscale image of a sample.

SEM uses a short column because the only lenses needed

are those above the specimen (used to focus the electrons
into a fine spot on the specimen surface). The specimen
chamber, on the other hand, is large because the SEM
method does not impose any limits on specimen size (unlike
TEM) other than that set by the size of the chamber itself.

The fine spot produced by the beam can be as small as 1 nm

in diameter on the specimen surface. This beam is scanned
in a rectangular raster over the sample and the intensities
of various signals formed by interactions between the beam
electrons and the specimen are measured and recorded. A modern scanning electron microscope - the
Thermo Scientific™ Apreo SEM.

Scanning microscopy A scanning electron microscope uses an electron

Imagine yourself alone in an unknown darkened beam instead of a flashlight, an electron detector

room with only a narrowly focused flashlight. instead of your eyes, and computer memory

You might start exploring the room by scanning instead of your brain to build an image of a

the flashlight systematically from side to side, sample’s surface.

gradually moving down (a raster pattern) so that

you could build up a picture of the objects in
the room in your memory.
29 | Exploring Uncharted Realms with Electron Microscopy

The stored values are then mapped as If the sample is thin, the SEM may be worked
differences in brightness on the image display. in scanning electron transmission microscope
The secondary electron (SE) signal is the (STEM) mode with a detector located below
most commonly used signal. It differs with the the sample to collect transmitted electrons.
topography of the sample surface much like an
aerial photograph: edges are bright, recesses are
dark. The ratio of the size of the exhibited image
to the size of the area scanned on the specimen
gives the magnification. The magnification can
be increased by decreasing the size of the area
scanned on the specimen.

The most significant differences between TEM

and SEM are:
Instead of the wide static beam used in TEM,
the SEM beam is concentrated to a fine point
and scanned line by line over the sample SEM image of orchid flower, courtesy of Nurshaiba Md. Nasir.

surface in a rectangular raster pattern.

Original data collected at a horizontal field width of 330 μm.
The accelerating voltages in SEM are a lot
lower than in TEM because it is no longer
essential to penetrate the specimen; in an
SEM they range from 50 to 30,000 volts.
SEM magnification
The SEM specimen need not be thin,
significantly simplifying specimen preparation. SEM magnification is simply the length of
one line scanned in the image (usually the
The interactions between the beam electrons width of the image) divided by the length
and sample atoms are similar to those defined for of the line scanned on the sample surface
TEM: (usually the width of the raster pattern). A
The specimen itself produces secondary high-resolution computer display might be
electrons (SE). half a meter wide and display 2,000 pixels
over that distance (pixel width = .25 mm).
Some of the key electrons are reflected
backscattered electrons (BSE). These
If each pixel represents one square
backscattered electrons can also cause
nanometer on the sample surface, then
the emission of secondary electrons as they
an image that fills the display represents
move through the sample and exit
a scanned area 2,000 nm (2 µm) wide,
the sample surface.
and the magnification of the image on
Electrons are absorbed by the specimen. the display is 250,000×.
The specimen releases X-rays.
The specimen occasionally emits visible light
(cathodoluminescence).
30 | Exploring Uncharted Realms with Electron Microscopy

All these phenomena are interconnected, and all of them rely to a certain extent on the topography,
the atomic number, structure and the chemical state of the specimen. The most commonly imaged signals in
SEM are SE and BSE. Secondary electrons, because of their very low energies, can escape the sample to be
detected only if they originate very close to the sample surface. This gives SEM images high spatial resolution
and robust topographic contrast. The BSE signal is used predominantly for its strong atomic number
contrast. Characteristic X-rays are also extensively used in SEM for elemental microanalysis.

Electron detection Generally, at lower voltages, where the beam

Detectors for backscattered and secondary electrons do not travel far into the sample,
electrons are typically either a scintillation the size of the spot is the main determinant
detector or a solid-state detector. In of image resolution. At higher voltages, the
the scintillator case, electrons strike a fluorescent volume of interaction, from which the signal
screen, which releases light that is amplified originates, may become the main consideration.
and converted into an electrical signal by
a photomultiplier tube or diode. The solid Presently, the best SEMs offer resolution below
state detector works by amplifying the minute 1 nm with voltages below 1 kV up through
signal created by the incoming electrons in the entire range of accelerating voltages, allowing
a semiconductor device. the operator to select a beam energy to suit
the requirements of the analysis; for instance,
The third type of detector monitors the net higher energy to provide a broad energy
current absorbed by the specimen, where net spectrum for X-ray analysis, or lower energy to
current = beam current - (SE + BSE), or improve surface specificity, avoid charging and/or
the current induced in a semiconductor junction beam damage.
by the incoming beam electron. These absorbed
current and electron beam induced current
(EBIC) measurements allow the study of dynamic
electrical phenomena in electronic devices.

Resolution
Resolution in an SEM relies on the degree to
which the signal, at any instant in time, can be
related to the position of the electron beam.
Specifically, for a particular beam location, how
large is the region within the sample from which
the signal originates? This can be influenced by
a number of factors, including the type of signal,
composition of the sample, the size of the spot
Atomic scale HAADF image of potassium tungsten niobate
formed by the beam, the energy of the beam Sample.
and more. Original data collected at a horizontal field width of 23.75 nm.
31 | Exploring Uncharted Realms with Electron Microscopy

Beam deceleration Image treatment

Beam deceleration offers extra flexibility Since the image in an SEM is completely
in the choice of accelerating voltage. With electronically produced, it can be subjected to
beam deceleration, the beam traverses most sophisticated analysis and manipulation with
of the column at high energy to decrease the help of modern digital techniques. This is
the adverse effects of chromatic aberration and comprised of contrast enhancement, inversion
is then decelerated by an opposing electrical (black becomes white, etc.), filtering, mixing
potential. The beam electrons thereby land with of images from various detectors, subtraction
decreased energy. of the image from one detector from that
generated by a different detector, and color
coding. The application of these techniques must
be guided by the key goal of extracting the best
possible information from the specimen.

Resolution and magnification their resolving power. This text will emphasize
resolving power as the primary measure
The resolving power of a microscope
of an instrument’s imaging capability, and refer
determines its maximum useful magnification.
to magnification only to provide a relative sense
For instance, if a microscope has a resolving
of scale among various electron microscopy
power of 200 nm (typical of a light microscope),
techniques. When a more precise usage of
it is only useful to magnify the image by a
magnification is required, it will be cited explicitly.
factor of 1,000 to make all the available
information visible.
Magnification is often quoted for an image
because it gives a quick idea of how much
At that magnification, the smallest details that
the features of the specimen have been enlarged.
the optical system can transfer from the object
However, a magnification that was accurate for
to the image (200 nm) are large enough to be
the original image will be inaccurate when that
seen by the unaided eye (0.2 mm). Further
image is projected on a large screen as part
magnification makes the image larger (and
of a presentation or reproduced at a smaller size
more blurred), but does not reveal additional
in a printed publication.
detail. Magnification in excess of the maximum
useful magnification is sometimes referred
For this reason, most microscopes now routinely
to as “empty resolution.” Notwithstanding
include reference scale markers of known length
the limiting principle of maximum useful
that scale accurately as the image is enlarged or
resolution, it is often convenient, for a variety
reduced for various uses.
of practical or aesthetic reasons, to use higher
magnifications; and commercial instruments
typically offer magnification capability well beyond
the maximum useful magnification implied by
32 | Exploring Uncharted Realms with Electron Microscopy

Vacuum
Generally, a sufficiently good vacuum for an
Resolution and accelerating voltage, SEM is generated by either an oil diffusion
spot size and volume of interaction pump or a turbomolecular pump (the current
The resolution of an SEM is determined by standard for most SEMs), in each case backed
the size of the region from which the signal by a mechanical pre-vacuum pump. These
originates. Certainly, this will not be smaller combinations also offer reasonable exchange
than the extent of the spot illuminated by the times for filament, specimen and aperture (less
beam on the sample surface. In conventional than a few minutes).
SEM, it is easier to form a smaller spot at
higher beam energies because the degrading Vacuum airlocks could also be used for huge
effects of chromatic aberration are relatively chambers and in high-volume applications when
less significant. However, at higher beam fast sample exchange has great value. Modern
energies, the beam electrons penetrate SEM vacuum systems are wholly automatically
deeply and scatter widely within the sample, controlled and protected against operating
contributing signal from locations well failures. Samples for standard SEM usually
outside the spot, thereby degrading image have to be dry, clean, vacuum-compatible
resolution. and, preferably, electrically conductive.

When beam energy is reduced, spot size In recent years, the environmental scanning
increases as the fixed energy spread among electron microscope (ESEM) has expanded
electrons in the beam becomes larger the variety of samples and sample environments
relative to the nominal beam energy, and that can be accommodated in the SEM chamber.
the adverse effects of chromatic aberration Examples of specimens that pose problems
increase. At some point, the benefit of are wool or cotton tissue, cosmetics, fats
reducing penetration is overwhelmed by the and emulsions (for example, margarine).
cost of increasing spot size.
Early attempts to view a specimen containing
A monochromator reduces the energy volatile components (by placing it in an
spread of the beam by eliminating beam environmental chamber isolated from the main
electrons that fall outside a selected range. column vacuum by small, differential pumping
Combined with a field emission electron apertures) were hindered by the inability
gun, monochromator-equipped SEMs have of standard secondary electron detectors to work
demonstrated sub-nanometer resolution in a low-vacuum or non-vacuum environment.
at accelerating voltages below 1 kV.
Monochromator technology avoids The ESEM’s gaseous secondary electron
restrictions on sample type and size that detector uses gas molecules in the sample
have limited the utility of other approaches environment in a cascade amplification in order to
to low-voltage imaging, such as “in-the-lens” detect and amplify the secondary electron signal
configurations and chromatic aberration while, at the same time, generating positive ions.
correctors. This effectively suppresses charging artifacts, as
33 | Exploring Uncharted Realms with Electron Microscopy

they are attracted by any negative charge A number of samples can be brought into
accumulating on insulated specimen surfaces. the chamber without preparation of any kind. If
the specimen contains any volatile components,
Variable pressure and low pressure are terms such as water, these must be removed by
used to describe SEMs that work in an a drying process. In some circumstances,
intermediate vacuum range between high- the sample can be frozen solid prior to being
vacuum SEM and ESEM. These instruments used in a high-vacuum system. Non-conducting
offer some of the sample flexibility of ESEM, specimens will accumulate charge under electron
though they are not usually capable of providing bombardment and may need to be coated with
pressure/temperature conditions that will sustain a conducting layer.
liquid water.
Iridium provides a fine-grained coating and
is effortlessly applied in a sputter coater. It
Application and specimen preparation
offers a good yield of secondary electrons
An SEM can be used whenever information is and, consequently, a good quality image of
needed about the surface or near-surface region the surface. Other metals, such as platinum,
of a specimen. It finds application in almost every chromium and gold, are options as well.
branch of technology, science and industry. Carbon is considered to be an alternative when
The only requirement is that the specimen must the X-ray emissions from iridium might interfere
be capable of withstanding the vacuum of with elemental analysis. The layer itself must be
the chamber and bombardment by the electron thick enough to provide a continuous conductive
beam. Since there is no need for a thin sample, film, but also not so thick that it obscures surface
SEM sample preparation is significantly simpler details of interest. Typical thicknesses are in
than the preparation of specimens for TEM. the range of 1–10 nm based on the sample
and application.

The Vacuum in an SEM Normal atmospheric air pressure at sea level is

represented as 1 atmosphere (atm), which is
A vacuum is a body of space not occupied by
approximately equivalent to 1 bar.
matter. Creating a perfect vacuum is a near-
impossible task, though SEM columns are close.
Normal air pressure = 1 bar = 1,000 mbar =
Gaseous matter exerts a pressure on the vessel
100, 000 Pa = 760 torr = 760 mm of Hg.
inside which it is contained, and therefore
The usual residual pressure of the vacuum in an
pressure is used as a measure of vacuum
electron microscope = 2.5 × 10–5 Pa.
strength, where a perfect vacuum exerts no
pressure whatsoever.
At this pressure, the number of gas molecules
per liter is around 7 × 1012, and the chance of an
Physicists use the Pascal (Pa) as the SI unit of
electron striking a gas molecule while traversing
pressure, but microscopists frequently use torr,
the column is almost zero.
millimeters of mercury (mm Hg) and millibar as well.
34 | Exploring Uncharted Realms with Electron Microscopy

Sometimes, it is extremely important to prevent

any alteration of the sample during preparation,
as is the case for, forensic specimens or silicon
wafers examined during the integrated circuit
(IC) manufacturing process as well as the ICs
themselves, which will have to be studied while
in operation. In such cases, special techniques,
such as low-voltage SEM, are employed in order
to prevent charging without the use of conductive
coatings. Cryo preparations are also used in
SEM, mainly in biological applications or with
organic materials (polymers).

Specimen orientation and manipulation

The quality of the image in an SEM relies on
the orientation and distance of the specimen from
the final lens and the detectors.The specimen Water droplets on the hydrophobic top side a leaf (SEM).
stage allows it to be shifted in a horizontal plane
Courtesy of Dr. Jim Buckman.
(X and Y directions), up and down (Z direction),
Original data collected at a horizontal field width of ~400 μm.
rotated and tilted as needed. These movements
are usually motorized and controlled by a
There are special stages or attachments for
computer using a mouse or joystick.
cooling, heating and straining specimens,
but because of the wide range of possible
The different SEM models in a range differ in
sample sizes, these stages are frequently
the size of their specimen chambers, allowing
produced by specialized firms.
samples of different sizes to be introduced
and manipulated. The larger the specimen
If the specimen in an SEM is thin enough to
chamber, the larger the stage mechanism
transmit electrons, a detector located below
needed to move and manipulate the sample
the specimen may be used for collecting these
and the larger the pumping system needed
electrons, offering STEM capabilities similar
to attain and maintain a good vacuum. The
to those described earlier for TEM. The lower
simplest models accept specimens of a few
accelerating voltages and lack of post-specimen
centimeters in diameter and can move them
lenses limit the ultimate resolution and flexibility
50 mm in the X and Y directions. Bigger models
of SEM-based STEM. However, it can be
can accommodate samples up to 300 mm in
a powerful technique, extending the contrast
diameter. Most models also allow samples to
capabilities and resolution seen in SEM imaging
be tilted to high angles and rotated through 360
of bulk samples. It can also enhance the spatial
degrees.
resolution of X-ray microanalysis by decreasing
the large volume of interaction from which X-rays
can originate in bulk specimens.
35 | Exploring Uncharted Realms with Electron Microscopy

CHAPTER 6

DualBeam – combining
SEM with FIB
An overview of DualBeam technology
Thus far, the focus has been on electron
microscopy and the beneficial information
that can be obtained using an electron beam.
However, electrons are not the only charged
particles that can be fast-tracked and focused SEM
using electric and magnetic fields. Usually, FIB
an atom is neutral since there are an equal
number of electrons and protons. However,
Diagram illustrating the DualBeam system, a combined
an atom that has lost one or more of its application of a focused ion beam (FIB) and SEM on a sample
outermost electrons has a positive charge and (orange).

can be accelerated, deflected and focused in

the same way as a negatively charged particle molecules are known as secondary ions, which
(electron). can be used for imaging and compositional
analysis. Ion bombardment also forms secondary
The most vital difference lies in the mass of the electrons that can be used for imaging, just as
ions. The lightest ion has nearly 2,000 times the they are in an SEM.
mass of an electron, and heavier ions can be
another 250 times as massive. The ion beam directly alters (mills) the surface (via
the sputtering process), and this milling can be
In an SEM, the comparatively low-mass electrons regulated with nanometer precision. By carefully
interact with a sample non-destructively regulating the energy and intensity of the ion
to produce secondary electrons which, when beam, it is possible to perform highly precise
collected, provide superior quality image nano-machining to create minute components or
resolution, down to the sub-nanometer range. to eliminate undesirable material.
A focused ion beam (FIB) instrument is
virtually identical to an SEM but uses a beam Futhermore, ion beam-assisted chemical vapor
of ions instead of electrons. The higher-mass deposition can be used to deposit material with
ions dislodge neutral and charged particles a level of precision similar to FIB milling. A small
(molecules, atoms and multimolecular particles) quantity of a precisely selected precursor gas
from the sample surface in a process known is injected into the beam vicinity, where it is
as sputtering. Ionized specimen atoms and
36 | Exploring Uncharted Realms with Electron Microscopy

decomposed by the beam, depositing the non- In a DualBeam instrument, the electron and ion
volatile products on the specimen surface, beams intersect at a 52° angle at a coincident
while the volatile products are removed by point near the sample surface, allowing
the vacuum system. instant, high-resolution SEM imaging of the
FIB-milled surface. Such systems integrate
Other reactive gases can be used with the ion the benefits of both the SEM and FIB and
beam, which, based on the particular gas and provide complementary imaging and beam
substrate, can enhance the milling rate, increase chemistry capabilities.
the milling selectivity for specific materials, or
subdue the redeposition of milled material. A FIB
Ion column
becomes even more robust when it is joined with
an SEM, as in a Thermo Scientific™ DualBeam™ FIB columns must deliver a beam of energetic
system. ions for use in all three application categories:
imaging, analysis and sample modification. High-
resolution imaging demands small spot sizes with
low currents. Analysis requires higher currents to
produce sufficient signal for precise measurement.

Sample modification requires a variety of beam

currents, from the very lowest for precise spatial
control to the very highest for high material
removal rates. Low-energy final polishing, to
eliminate the amorphous and/or ion-implanted
damage layer left by high-energy milling,
is also a vital capacity. Over the whole variety
of applications, higher beam current to spot size
ratios generally enhance system performance.

Ion source
Most FIBs use a liquid metal ion source (LMIS)
to provide charged ions for the beam. Other
types of sources may be used in special
applications, such as those necessitating very
Cross-section made by FIB milling of a multilayer photovoltaic
high beam currents for rapid milling. The LMIS
panel.
has a sharply pointed tungsten needle coated
Courtesy of Dr. Maria Carbajo, Universidad de Extremadura. with a liquid metal. Gallium provides the ideal
Original data collected at a horizontal field width of 29.8 μm.
combination of large atomic number, low vapor
pressure and ease of use.
37 | Exploring Uncharted Realms with Electron Microscopy

A wire, welded to the needle, holds the needle in FIB

position and heats it to burn off contamination.
A coiled wire below the needle holds a reservoir Ga+ LMI source
suppresser
of gallium to refill the coating. The needle points extractor
in the direction of an aperture in a negatively
lens 1 
biased extraction electrode. The field formed
by the extraction electrodes accelerates ions octopole alignment
from the needle tip through the aperture. blanking plates
blanking aperture
The extraction field is extremely strong at scan and stig octopoles
lens 2 
the sharply pointed needle tip. continuous
dinode detector

In this field, the liquid gallium coating flows into

an even more sharply pointed cone. A balance secondary electrons or ions
between electrostatic and surface tension forces ion beam impact area
establishes the shape of this point, known as specimen (thick)
a Taylor cone. If the apex of the cone were to
become flawlessly sharp, the extraction field
would be extremely strong. turbo/diff pump

roughing line
The ion density is subsequently very high near
the tip, and the ions exert significant Coulomb
forces on each other. As they hurry away from
the tip in the extraction field, they spread out, and Components of a FIB microscope.

their coulombic interactions reduce. This process

eliminates gallium from the tip and decreases its
When the FIB column is improved for image
sharpness. Thus, a balance exists at the tip
resolution (i.e., low beam current, small spot size
of the cone between the elimination of gallium,
and small apertures), the spherical aberrations
through ionization, and the replenishment
of the column lenses are significantly reduced
of gallium, through fluid flow into the tip region.
and system performance is restricted by certain
features of the source, namely, its apparent size
These forces essentially create a protrusion, or
and energy distribution. Though the radius of
jet, at the tip of the cone. The jet is extremely
the ion jet is just a few nanometers, its apparent
small, having a radius of possibly five nanometers.
size (i.e., the radius of the region from which
The ion trajectories out of the jet typically lie
the ions seem to originate when their trajectories
within twenty to thirty degrees of the needle axis.
are plotted backward through the optical system)
Even with a low total emitted current, around
is larger by a factor of ten, about 50 nm.
one microampere, the small source size and
narrow emission angle give the LMIS a brightness
of over a million amperes per square centimeter
per steradian.
38 | Exploring Uncharted Realms with Electron Microscopy

Therefore, energy distribution, apparent source

size and beam shape are all affected unfavorably
by space charge effects in high current density
beams. Anything that increases the current
density near the emitter tip increases the space
charge effects and degrades performance.
Therefore, LMISes are always used at the lowest
possible total emitted current.

Minimization of space charge effects, through

a cautious balance of electrode geometry and
field strength, is the main concern in the design
of advanced high-intensity LMIS. A higher
extraction field and larger extractor-electrode
spacing decrease the time the ions spend in
the high interaction zone of the ion jet while still
keeping a low level of total emission current.

From a practical point of view, the gallium supply

Platinum nano-features fabricated by FIB microscopy and
imaged using SEM. of the LMIS is consumed during use, and so the
source must be changed periodically. Similarly,
Courtesy of Guillaume Audoit.
the various beam-limiting apertures in the column
Original data collected at a horizontal field width of 5 μm.
will be eroded by the ion beam and, therefore,
also need periodic replacement. Source lifetime
This apparent source is the signal that the optical and ease of replacement are key considerations.
system must demagnify onto the sample surface. Lifetime relies on the size of the liquid metal
The enlargement of the apparent source is mostly reservoir; however, larger reservoirs increase
because of perturbations in particle trajectories the total size of the source, making the source
caused by coulombic interactions between ions. harder to integrate into the column design as an
easily replaceable module.
These same interactions cause an increase in
the energy spread of the ions, which results in Current ion sources have lifetimes in excess
increased chromatic aberration in the optical of 1,000 hours and exchange times, including
system. Preferably, the beam should have a system pump-down, of less than four hours.
Gaussian intensity profile. In practice, beam tails Removable source-end structures that simplify
extend many times the full-width-half-maximum source replacement have also been developed.
diameter of the beam. These tails can be credited
to the transverse energy spread resulting from
coulombic interactions.
39 | Exploring Uncharted Realms with Electron Microscopy

CHAPTER 7

Glossary of common
electron microscopy
language
A Amplitude

Aberration The maximum value of a periodically varying

The deviation from perfect imaging in an optical parameter, as in the height of a wave crest above

system, caused by imperfections in the lens or by the mean value.

non-uniformity of the electron beam.

Amplitude contrast

Accelerating voltage Image contrast caused by the removal

of electrons (or light) from the beam by
The potential difference in an electron gun
interactions with the specimen.
between cathode and anode over which
electrons are accelerated. The higher the voltage,
Ångström
the faster the electrons (or ions) and the more
Unit of length, 1 Å = 0.1 nm = 10-10 m.
penetrating power they have. Voltages may range
from a few hundred volts up to several hundred
Anode
thousand.
In an electron gun, the negatively charged
Airlock electrons are accelerated towards the anode,

A chamber within the electron microscope that which has a positive charge relative to the

can be isolated from the rest of the instrument to filament (cathode) from which the electrons

allow the specimen to be inserted. The airlock is emerge. In practice (for ease of construction),

then pumped out, and the specimen moved into the filament has a high negative charge and the

the chamber (SEM, FIB) or column (TEM) anode is at ground potential.

vacuum. This reduces the amount of air and

Aperture
other contaminants brought into the column
and speeds sample exchange. A small hole in a metal disc used to stop
those electrons that are not required for image
formation (e.g., scattered electrons).
40 | Exploring Uncharted Realms with Electron Microscopy

Astigmatism Chromatic aberration

A lens aberration in which the power of the See aberration. The power of the lens varies with
lens is greatest in one direction and least in the the wavelength of the electrons in the beam.
perpendicular direction. It causes a round feature
in the object to assume an elliptical shape in the Column
image. The physical structure of an electron microscope
that accommodates the evacuated electron
Atom beam path, the electromagnetic lenses, the
The smallest unit of physical matter that retains aperture mechanisms and the specimen in TEM.
its elemental identity. There are many ways
of looking at the atom. The most useful one Condenser lens
for electron microscopists is to think of it as Part of the illumination system between the gun
consisting of a positively charged nucleus and the specimen designed to form the electron
(containing positively charged protons and beam, usually into a parallel configuration as it
uncharged neutrons) surrounded by negatively transits the sample (TEM) or enters the objective
charged electrons in discrete orbits. lens (SEM). It may also be used to form a finely
focused spot on the specimen (STEM).
Atomic number
The number of protons in the atomic nucleus. Crystal
This number determines the chemical nature
A material in which the atoms are ordered into
of the atom. An atom of iron, for example, has 26
rows and columns (a lattice). This periodicity
protons, an atom of oxygen 8, and so on.
causes electrons, whose wavelength is about
the same size as the spacing between atoms, to
B
undergo diffraction.
Backscattered electrons
Primary (beam) electrons that have been D

deflected by the specimen through an angle Detector

generally greater than 90° so that they exit the A device for detecting particular electrons or
sample with little or no loss of energy. photons in the electron microscope.

Binocular magnifier Diffraction

A light microscope built into a TEM for viewing a Deviation of the direction of light or other wave
fine-grain fluorescent screen for critical focusing motion when the wave front passes the edge of
and astigmatism correction. an obstacle.

C Diffraction contrast
Cathodoluminescence Constructive interference of waves caused by
The emission of light photons by a material under interaction with a periodic structure.
electron bombardment.
41 | Exploring Uncharted Realms with Electron Microscopy

E sample chamber, up to that required to sustain

EDX water in its liquid phase.

Energy dispersive X-ray analysis or spectrometry

ETEM
(sometimes called EDS). An EDX spectrometer
Environmental transmission electron
makes a spectrum of X-rays emitted by the
microscope—a transmission electron microscope
specimen on the basis of their energy.
that can accommodate a wider range of
environmental conditions and apparatuses in the
EELS
sample space to enable in situ examination of
Electron energy loss spectroscopy (or
materials and processes.
spectrometry) analyzes transmitted electrons on
the basis of energy lost to interactions with Excitation
sample atoms. Energy loss provides information
The input of energy to matter, which can lead to
about the sample atoms’ elemental identity,
the emission of radiation.
chemical bonding and electronic states.

Excited atom
Electron
An atom that has a vacancy in one of its inner
Fundamental sub-atomic particle carrying a
electron orbitals (see also, ion) and, therefore, has
negative charge and conventionally described as
higher energy. It returns to its ground state when
orbiting the nucleus of the atom. Free electrons
an electron from an outer orbital drops down
can easily flow in a conductor and can be
to fill the vacancy, emitting the excess energy
extracted into a vacuum by an electric field.
as radiation (typically an X-ray). The energy
difference between orbitals and, thus, the energy
Electron microscope
of the X-ray, is characteristic of the emitting
A microscope in which a beam of electrons is
atom’s elemental identity.
used to form a magnified image of the specimen.

F
Electrostatic lens
FEG
Device used to focus charged particles into
a beam. Although it may also be used with Field emission gun. An electron source in which
electrons, it is most frequently used with ions in electrons are extracted from a sharply pointed
a FIB column. The much greater mass of ions tungsten tip by a very strong electric field.
requires the stronger optical power available from
an electrostatic lens. Lighter electrons can be FIB
effectively focused by a weaker magnetic lens. Focused ion beam. Similar to an SEM but
uses an ion beam instead of an electron beam.
ESEM DualBeam instruments combine FIB and SEM.
Environmental scanning electron microscope—a
scanning electron microscope that can
accommodate a wide range of pressures in the
42 | Exploring Uncharted Realms with Electron Microscopy

Filament Ion getter pump

Metal wire, usually in the form of a hairpin, which, Vacuum pump that uses electric and magnetic
when heated in vacuum, releases free electrons. fields to ionize and trap residual gas molecules by
This provides a source of electrons for an embedding them in the cathode of the pump.
electron microscope.
L
Fluorescent screen
Lattice
Large plate coated with a material (phosphor) that
Regular three-dimensional array of atoms
gives off light (fluoresces) when bombarded by
in a crystal.
electrons. A TEM may project its electron image
onto a fluorescent screen to make it visible in real
Lens
time.
In a light microscope, a piece of transparent
Focal length of a lens material with one or more curved surfaces that is
used to focus light. In an electron microscope, a
The distance (measured from the center of the
similar effect is achieved on a beam of electrons
lens in the direction of the beam) at which
by using a magnetic (or electrostatic) field.
a parallel incident beam is brought to a focus.

LMIS
Focusing
Liquid metal ion source. An ion source in which
The act of making the image as sharp as possible
ions are extracted by a strong electric field from a
by adjusting the power of the objective lens.
layer of liquid metal Ga+ coating a sharply pointed
electrode.
G

Goniometer M
Specimen stage allowing linear movement of the Micrometer
specimen in two or more directions and rotation
Unit of length (distance). One micrometer (μm)
of the specimen in its own plane and tilting about
is a millionth of a meter (10-6 m) or 1,000 nm.
one or more axes that remain fixed with respect
to the beam. Microtome
Instrument for cutting extremely thin sections
Ground state
from a specimen prior to examination in
The lowest energy state of an atom.
the microscope. In electron microscopy, this
is usually referred to as an ultramicrotome.
I

Ion N
An atom or molecule that has lost or gained an Nanometer
electron and, therefore, has a net positive or
Unit of length (distance). One nanometer (nm)
negative electric charge.
is a billionth of a meter (10-9 meter).
43 | Exploring Uncharted Realms with Electron Microscopy

O Primary electrons

Oil diffusion pump Electrons in the beam generated by the

Vacuum pump in which the pumping action microscope.

is produced by the dragging action of a stream

Q
of oil vapor though an orifice.
Quantum
Objective lens A discrete packet of energy, as a photon of light.
In a TEM, this is the first lens after the specimen.
Its function is to focus transmitted electrons into R
an image. In an SEM, it is the last lens before Raster
the specimen, and it produces the extremely fine
The track of the beam in an SEM or STEM.
electron spot with which the specimen
It is analogous to eye movements when reading
is scanned. Its quality largely determines
a book: left to right, word by word, and down
the performance of the microscope.
the page line by line.

P
Refraction
Phase
Changes in direction of a beam of light (or
Relative position in a cyclical or wave motion. It electrons) as the beam passes through regions in
is expressed as an angle, with one cycle or one which its propagation speed changes.
wavelength corresponding to 360°.
Refractive index
Phase contrast
The ratio of the speed of light in a vacuum to that
Image contrast caused by the interference in a given medium such as glass, water or oil.
among transmitted electrons with phase shifts
caused by interaction with the sample. Resolving power
The ability to make points or lines that are closely
Phase diagram
adjacent in an object distinguishable in an image.
Graph of temperature and pressure showing
the range of each under which a given material Resolution
can exist in the solid, liquid or vapor phase.
A measure of resolving power.

Photomultiplier
S
Electronic tube in which light is amplified to
Scanning
produce an electrical signal with very low noise.
Process of investigating a specimen by moving
Photons a finely focused probe (electron beam) in a raster
pattern over the surface.
Discrete packets of electromagnetic radiation.
A light beam is made up of a stream of photons.
44 | Exploring Uncharted Realms with Electron Microscopy

Scintillation detector STEM

Electron detector used in SEM or STEM in which Scanning transmission electron microscope or
electrons are accelerated towards a phosphor, scanning transmission electron microscopy.
which fluoresces to produce light, which is
amplified by means of a photomultiplier to Steradian
produce an electrical signal. Standard unit for solid angles, also called square
radian. On the surface of a sphere with radius r,
Secondary electrons 1 steradian results in a region with area
Electrons scattered from sample atoms by = radius squared.
interactions with beam (primary) electrons.
T
SEM TEM
Scanning electron microscope or scanning Transmission electron microscope or
electron microscopy. transmission electron microscopy.

Semiconductor detector Turbomolecular pump

Electron detector used in SEM or STEM in which Vacuum pump in which the molecules are moved
a high-energy electron is detected by the current against the pressure gradient by collisions with
it generates as it dissipates its energy in a solid rapidly rotating, angled vanes.
state diode.
V
Spectrometer
Vacuum
Instrument for obtaining a spectrum.
A region of reduced (lower than ambient)
Spectrum gas pressure.

A display produced by the separation of W

a complex radiation into its component intensity
Wavelength
as a function of energy or wavelength.
The distance on a periodic wave between two
Spherical aberration successive points at which the phase is the
See aberration. The power of a lens varies with same; for example, two crests.
radial distance from its center.
WDX

Sputter coater Wavelength dispersive X-ray analysis or

spectrometry. An alternative to energy dispersive
Instrument for coating a non-conducting specimen
spectrometry for X-ray analysis. In WDX, X-rays
with a very thin uniform layer of a conducting
are dispersed into a spectrum by diffraction from
element such as gold or iridium to eliminate
a crystal or grating. The crystal is mechanically
artifacts caused by accumulating charge.
scanned through a range of angles while
a detector measures changes in signal intensity.
45 | Exploring Uncharted Realms with Electron Microscopy

Wehnelt cylinder
An electrode between the cathode (filament) and
the anode (ground) in a triode electron gun. Used
to form the beam and control its current.

Working distance
In an SEM, the physical distance between the
external metal parts of the objective lens and
the specimen surface. This is the space
available for placing certain electron, X-ray and
cathodoluminescence detectors. For highest
resolution, the working distance has to be made
as small as possible, which leads to compromises.

X-rays
Electromagnetic radiation with wavelengths much
shorter than visible light, ranging from 10 to 0.01
nm. In the electron microscope, characteristic
X-rays are used to analyze elemental composition
with high spatial resolution.