Electron Microscopy

The electron microscope is a type of

microscope that uses a beam of electrons to
create an image of the specimen. It is capable
of much higher magnifications and has a
greater resolving power than a light
microscope, allowing it to see much smaller
objects in finer detail. They are large,
expensive pieces of equipment, generally
standing alone in a small, specially designed
room and requiring trained personnel to
operate them

Types of Electron Microscopes

All electron microscopes use electromagnetic
and/or electrostatic lenses to control the path of
electrons. Glass lenses, used in light
microscopes, have no effect on the electron
beam. The basic design of an electromagnetic
lens is a solenoid (a coil of wire around the
outside of a tube) through which one can pass a
current, thereby inducing an electromagnetic
field. The electron beam passes through the
centre of such solenoids on its way down the
column of the electron microscope towards the
sample. Electrons are very sensitive to magnetic
fields and can therefore be controlled by
changing the current through the lens

 The faster the electrons travel, the shorter their

wavelength. The resolving power of a
microscope is directly related to the wavelength
of the irradiation used to form an
image. Reducing wavelength increases
resolution. Therefore, the resolution of the
microscope is increased if the accelerating
voltage of the electron beam is increased. The
accelerating voltage of the beam is quoted in
kilovolts (kV)

Transmission Electron Microscope

(TEM)
The original form of electron microscopy, Transmission
electron microscopy (TEM) involves a high voltage electron
beam emitted by a cathode and formed by magnetic
lenses. The electron beam that has been partially
transmitted through the very thin (and so semitransparent
for electrons) specimen carries information about the
structure of the specimen. The spatial variation in this
information (the "image") is then magnified by a series of
magnetic lenses until it is recorded by hitting a fluorescent
screen, photographic plate, or light sensitive sensor such as
a CCD (charge-coupled device) camera. The image detected
by the CCD may be displayed in real time on a monitor or
computer.
Transmission electron microscopes produce twodimensional, black and white images.

Scanning Electron Microscope

Unlike the TEM, where the electrons in the
primary beam are transmitted through the
sample, the Scanning Electron Microscope
(SEM) produces images by detecting
secondary electrons which are emitted from
the surface due to excitation by the primary
electron beam. In the SEM, the electron beam
is scanned across the surface of the sample in
a raster pattern, with detectors building up an
image by mapping the detected signals with
beam position.

 high-resolution instruments capable of about

2 nm resolution on biological
samples. Because the SEM image relies on
electron interactions at the surface rather
than transmission it is able to image bulk
samples and has a much greater depth of
view, and so can produce images that are a
good representation of the 3D structure of the
sample. SEM images are therefore considered
to provide us with 3D, topographical
information about the sample surface but will
still always be only in black and white.

 In the SEM, we use much lower accelerating

voltages to prevent beam penetration into the
sample since what we require is generation of
the secondary electrons from the true surface
structure of a sample. Therefore, it is
common to use low KV, in the range 1-5kV for
biological samples, even though our SEMs are
capable of up to 30 kV.

Sample Preparation
Materials to be viewed in an electron
microscope generally require processing to
produce a suitable sample. This is mainly
because the whole of the inside of an electron
microscope is under high vacuum in order to
enable the electron beam to travel in straight
lines. The technique required varies
depending on the specimen, the analysis
required and the type of microscope:

Cryofixation - freezing a specimen rapidly, typically to

liquid nitrogen temperatures or below, that the water
forms ice. This preserves the specimen in a snapshot of
its solution state with the minimal of artefacts. An entire
field called cryo-electron microscopy has branched from
this technique. With the development of cryo-electron
microscopy, it is now possible to observe virtually any
biological specimen close to its native state.
Fixation - a general term used to describe the process of
preserving a sample at a moment in time and to prevent
further deterioration so that it appears as close as
possible to what it would be like in the living state,
although it is now dead. In chemical fixation for electron
microscopy, glutaraldehyde is often used to crosslink
protein molecules and osmium tetroxide to preserve
lipids.

 Dehydration - removing water from the

samples. The water is generally replaced with
organic solvents such as ethanol or acetone as
a stepping stone towards total drying for SEM
specimens or infiltration with resin and
subsequent embedding for TEM specimens.
Embedding - infiltration of the tissue with wax
(for light microscopy) or a resin (for electron
microscopy) such as araldite or LR White,
which can then be polymerised into a
hardened block for subsequent sectioning.

Sectioning - the production of thin slices of the specimen. For

light microscopy, the sections can be a few micrometres thick but
for electron microscopy they must be very thin so that they are
semitransparent to electrons, typically around 90nm. These ultrathin sections for electron microscopy are cut on an ultramicrotome
with a glass or diamond knife. Glass knives can easily be made in
the laboratory and are much cheaper than diamond, but they
blunt very quickly and therefore need replacing frequently.
Staining - uses heavy metals such as lead and uranium to scatter
imaging electrons and thus give contrast between different
structures, since many (especially biological) materials are nearly
"transparent" to the electron beam. By staining the samples with
heavy metals, we add electron density to it which results in there
being more interactions between the electrons in the primary
beam and those of the sample, which in turn provides us with
contrast in the resultant image. In biology, specimens can be
stained "en bloc" before embedding and/or later, directly after
sectioning, by brief exposure of the sections to solutions of the
heavy metal stains.

 Freeze-fracture and freeze-etch - a preparation method particularly

useful for examining lipid membranes and their incorporated
proteins in "face on" view. The fresh tissue or cell suspension is
frozen rapidly (cryofixed), then fractured by simply breaking or by
using a microtome while maintained at liquid nitrogen temperature.
The cold, fractured surface is generally "etched" by increasing the
temperature to about -95C for a few minutes to let some surface
ice sublime to reveal microscopic details. For the SEM, the sample is
now ready for imaging. For the TEM, it can then be rotaryshadowed with evaporated platinum at low angle (typically about
6) in a high vacuum evaporator. A second coat of carbon,
evaporated perpendicular to the average surface plane is generally
performed to improve stability of the replica coating. The specimen
is returned to room temperature and pressure, and then the
extremely fragile "shadowed" metal replica of the fracture surface
is released from the underlying biological material by careful
chemical digestion with acids, hypochlorite solution or SDS
detergent. The floating replica is thoroughly washed from residual
chemicals, carefully picked up on an EM grid, dried then viewed in
the TEM.

 Sputter Coating - an ultra-thin coating of

electrically-conducting material, deposited by low
vacuum coating of the sample. This is done to
prevent charging of the specimen which would
occur because of the accumulation of static
electric fields due to the electron irradiation
required during imaging. It also increases the
amount of secondary electrons that can be
detected from the surface of the sample in the
SEM and therefore increases the signal to noise
ratio. Such coatings include gold, gold/palladium,
platinum, chromium etc.

Disadvantages of Electron Microscopy

Electron microscopes are very expensive to buy and maintain. They
are dynamic rather than static in their operation: requiring extremely
stable high voltage supplies, extremely stable currents to each
electromagnetic coil/lens, continuously-pumped high/ultra-high
vacuum systems and a cooling water supply circulation through the
lenses and pumps. As they are very sensitive to vibration and
external magnetic fields, microscopes aimed at achieving high
resolutions must be housed in buildings with special services.
A significant amount of training is required in order to operate an
electron microscope successfully and electron microscopy is
considered a specialised skill.
The samples have to be viewed in a vacuum, as the molecules that
make up air would scatter the electrons. This means that the samples
need to be specially prepared by sometimes lengthy and difficult
techniques to withstand the environment inside an electron
microscope. Recent advances have allowed some hydrated samples
to be imaged using an environmental scanning electron microscope,
but the applications for this type of imaging are still limited.

 Transmission Electron Microscopy (TEM)

TEM
The transmission electron microscope is a very powerful tool for material
science. A high energy beam of electrons is shone through a very thin
sample, and the interactions between the electrons and the atoms can be
used to observe features such as the crystal structure and features in the
structure like dislocations and grain boundaries. Chemical analysis can also
be performed. TEM can be used to study the growth of layers, their
composition and defects in semiconductors. High resolution can be used
to analyze the quality, shape, size and density of quantum wells, wires and
dots.
The TEM operates on the same basic principles as the light microscope but
uses electrons instead of light. Because the wavelength of electrons is
much smaller than that of light, the optimal resolution attainable for TEM
images is many orders of magnitude better than that from a light
microscope. Thus, TEMs can reveal the finest details of internal structure in some cases as small as individual atoms.

Imaging
The beam of electrons from the electron gun is focused into a small, thin,
coherent beam by the use of the condenser lens. This beam is restricted by
the condenser aperture, which excludes high angle electrons. The beam
then strikes the specimen and parts of it are transmitted depending upon
the thickness and electron transparency of the specimen. This transmitted
portion is focused by the objective lens into an image on phosphor screen
or charge coupled device (CCD) camera. Optional objective apertures can
be used to enhance the contrast by blocking out high-angle diffracted
electrons. The image then passed down the column through the
intermediate and projector lenses, is enlarged all the way.
The image strikes the phosphor screen and light is generated, allowing the
user to see the image. The darker areas of the image represent those areas
of the sample that fewer electrons are transmitted through while the
lighter areas of the image represent those areas of the sample that more
electrons were transmitted through.
Diffraction
The path of a beam of electrons in a TEM from just above the specimen
and down the column to the phosphor screen. As the electrons pass
through the sample, they are scattered by the electromagnetic lens

The Transmission Electron Microscope

The transmission electron microscope (TEM) operates
on the same basic principles as the light microscope
but uses electrons instead of light. What you can see
with a light microscope is limited by the wavelength of
light. TEMs use electrons as "light source" and their
much lower wavelength makes it possible to get a
resolution a thousand times better than with a light
microscope.
You can see objects to the order of a few angstrom (1010 m). For example, you can study small details in the
cell or different materials down to near atomic levels.
The possibility for high magnifications has made the
TEM a valuable tool in both medical, biological and
materials research.

Magnetic Lenses Guide the Electrons

A "light source" at the top of the microscope emits the

electrons that travel through vacuum in the column of the
microscope. Instead of glass lenses focusing the light in the
light microscope, the TEM uses electromagnetic lenses to
focus the electrons into a very thin beam. The electron
beam then travels through the specimen you want to study.
Depending on the density of the material present, some of
the electrons are scattered and disappear from the beam.
At the bottom of the microscope the unscattered electrons
hit a fluorescent screen, which gives rise to a "shadow
image" of the specimen with its different parts displayed in
varied darkness according to their density. The image can
be studied directly by the operator or photographed with a
camera.

 Transmission electron microscope (TEM), type of

electron microscope that has three essential systems:
(1) an electron gun, which produces the electron beam,
and the condenser system, which focuses the beam
onto the object, (2) the image-producing system,
consisting of the objective lens, movable specimen
stage, and intermediate and projector lenses, which
focus the electrons passing through the specimen to
form a real, highly magnified image, and (3) the imagerecording system, which converts the electron image
into some form perceptible to the human eye. The
image-recording system usually consists of a
fluorescent screen for viewing and focusing the image
and a digital camera for permanent records. In
addition, a vacuum system, consisting of pumps and
their associated gauges and valves, and power supplies
are required

 The electron gun and condenser system

The source of electrons, the cathode, is a heated V-shaped tungsten
filament or, in high-performance instruments, a sharply pointed rod of a
material such as lanthanum hexaboride. The filament is surrounded by a
control grid, sometimes called a Wehnelt cylinder, with a central aperture
arranged on the axis of the column; the apex of the cathode is arranged to
lie at or just above or below this aperture. The cathode and control grid
are at a negative potential equal to the desired accelerating voltage and
are insulated from the rest of the instrument. The final electrode of the
electron gun is the anode, which takes the form of a disk with an axial
hole. Electrons leave the cathode and shield, accelerate toward the anode,
and, if the stabilization of the high voltage is adequate, pass through the
central aperture at a constant energy. The control and alignment of the
electron gun are critical in ensuring satisfactory operation.

 The intensity and angular aperture of the beam are

controlled by the condenser lens system between the
gun and the specimen. A single lens may be used to
converge the beam onto the object, but, more
commonly, a double condenser is employed. In this the
first lens is strong and produces a reduced image of the
source, which is then imaged by the second lens onto
the object. Such an arrangement is economical of
space between the electron gun and the object stage
and is more flexible, because the reduction in size of
the image of the source (and hence the final size of
illuminated area on the specimen) may be varied
widely by controlling the first lens. The use of a small
spot size minimizes disturbances in the specimen due
to heating and irradiation

The image-producing system

The specimen grid is carried in a small holder in a
movable specimen stage. The objective lens is usually
of short focal length (15 mm [0.040.2 inch]) and
produces a real intermediate image that is further
magnified by the projector lens or lenses. A single
projector lens may provide a range of magnification of
5:1, and by the use of interchangeable pole pieces in
the projector a wider range of magnifications may be
obtained. Modern instruments employ two projector
lenses (one called the intermediate lens) to permit a
greater range of magnification and to provide a greater
overall magnification without a commensurate
increase in the physical length of the column of the
microscope.

 For practical reasons of image stability and

brightness, the microscope is often operated to give
a final magnification of 1,000250,000 on the
screen. If a higher final magnification is required, it
may be obtained by photographic or digital
enlargement. The quality of the final image in the
electron microscope depends largely upon the
accuracy of the various mechanical and electrical
adjustments with which the various lenses are
aligned to one another and to the illuminating
system. The lenses require power supplies of a high
degree of stability; for the highest standard of
resolution, electronic stabilization to better than
one part in a million is necessary. The control of a
modern electron microscope is carried out by a
computer, and dedicated software is readily availab

Image recording
The electron image is monochromatic and must be
made visible to the eye either by allowing the electrons
to fall on a fluorescent screen fitted at the base of the
microscope column or by capturing the image digitally
for display on a computer monitor. Computerized
images are stored in a format such as TIFF or JPEG and
can be analyzed or image-processed prior to
publication. The identification of specific areas of an
image, or pixels with specified characteristics, allows
spurious colours to be added to a monochrome image.
This can be an aid to visual interpretation and teaching
and can create a visually attractive picture from the raw
image.

Sample preparation for

Transmission electron microscopy
(TEM)
TEM is a microscopy technique whereby a beam of
electrons is transmitted through an
ultrathin specimen, interacting with the specimen as
it passes through it. An image is
formed from the electrons transmitted through the
specimen, magnified and focused by
an objective lens and appears on an imaging screen,
a fluorescent screen in most TEMs,
plus a monitor, or on a layer of imaging plate, or to
be detected by a sensor such as a CCD
camera.

Biological materials contain large quantities of water. To be able to view it in the

electron microscopy, the first stage in preparing is the fixation, one of the most
important and most critical stages. We need to fixed it in a way that the ultrastructure of
the cells or tissues remain as close to the living material as possible. The sample is then
dehydrated through an acetone or ethanol series, passed through a transition solvent
such as propylene oxide and then infiltrated and embedded in a liquid resin such as
epoxy and LR White resin. After embedding the resin block is then thin sectioned by a
process known as ultramicrotomy, sections of 50 70 nm thickness are collected on
metal mesh 'grids' and stained with electron dense stains before observation in the
TEM. Sectioning the sample allows us to look at crosssections through samples to view
internal (ultra)structure. Many modifications to the basic protocol can be applied to
achieve many different goals, immunogold labeling for example; the in situ localization
of specific tissue constituents using antibody and colloidal gold marker system

 Support film on TEM grids

Formvar film is useful for the support of ultrathin
sections on the finer mesh grids. Using
of support film are ideal for those applications
requiring large viewing areas without
grid bar interference. These films must be strong,
clean and remain attached to the
specimen grids during specimen preparation.
A Formvar film covered with a "light" layer of
carbon will help to stabilize the film when
the film is exposed to the electron beam.

Sectioning with ultramicrotome

Materials for TEM must be specially prepared to
thicknesses which allow electrons to
transmit through the sample, much like light is
transmitted through materials in
conventional optical microscopy. Because the
wavelength of electrons is much smaller
than that of light, the optimal resolution attainable
for TEM images is many orders of
magnitude better than that from a light microscope.
The block is cut into semithin sections (1 m) with a
glass knife, using an
ultramicrotome. The sections are then stained with
Toluidine Blue for LM for
orientation, and for selecting of a small area for
ultrathin sectioning. Ultrathin sections
are made at 5070 nm using a diamond knife and

Positive staining
Details in light microscope samples can be enhanced by stains that absorb light;
similarly TEM samples of biological tissues can utilize high atomic number stains
to enhance contrast. The stain absorbs electrons or scatters part of the electron
beamwhich otherwise is projected onto the imaging system. Uses heavy metals
such as lead and uranium to scatter imaging electrons and thus give contrast
between different structures, since many (especially biological) materials are
nearly "transparent" to electrons (weak phase objects).Heavy metal salts attach
to various organelles or macromolecules within the sections toincrease their
electron density and they appear dark against a lighter background.Uranyl ions
react strongly with phosphate and amino groups so that nucleic acids andcertain
proteins are highly stained. Lead ions bind to negatively charged
componentsand osmiumreacted areas (membranes).Grids are stained with
heavy metals, such as uranyl acetate and lead citrate. The grids,with the
specimen side down, remain in 4% uranyl acetate for 25 minutes and are then
rinsed in a series of four beakers of pure water. After rinsing, the grids are then
stained with 1% lead citrate for 5 minutes, rinsed again in pure water, and stored
in a grid box.

Immunogold labelling
This technique uses antibodies to detect the intracellular
location of structures of particular proteins by electron
microscopy. Ultrathin sections are labelled with antibodies
against the required antigen and then labelled with gold
particles. Gold particles of different diameters enable two or
more proteins to be studied EMLab can offer postembedding
immunogold labelling of samples in resin (Epoxy, LR White and
Lowicryl) and on frozen hydrated ultrathin sections
(Tokuyasumethod).The investigator must supply the primary
and secondary antibodies. The investigator should do
immunolabelling at the fluorescent light microscopy level
before attempting it at the EM level

Cryo techniques/Low temperature

Cryoultramicrotomy is the ultrathin sectioning of unfixed/fixed, cryoprotected and/or
rapidly frozen samples at very low temperatures. The Leica EM FC6 cryochamber is
designed for low temperature sectioning of samples at temperatures from 15 to 160 C.
Freeze substitution is a process where the water molecules within the samples are
exchanged with a solvent (usually methanol or acetone), then, the solvent with a resin
(Lowicryl, LR White or Epoxy resins). This method, working at temperatures below 0C,
reduces the loss of components from the sample and minimizes the denaturization of
the proteins. In the end, the sample is fully infiltrated with pure resin. Polymerization of
the resin is performed outside (Epoxy resin) or inside the machine when using Lowicryl
resin. This latter resin is polymerized under a UV lamp, starting at 45C, then gradually
moving up to room temperature (Lowicryl HM20). At the end of the process hard plastic
blocks are generated ready to be cut by ultramicrotomy. The Leica EM AFS is capable of
freeze substitution, progressive lowering of temperature techniques, and low
temperature embedding and polymerization of resins using UV light instead of heat for
improved preservation of ultrastructure and antigenicity.

 TEM services include:

Conventional specimen fixation, dehydration and embedding.
Specimen sectioning:
Semithin sectioning (1 m) with Toluidine blue stain
Ultrathin sectioning (50 70 nm) of resin embedded material
(Epoxy,
LR White, Lowicryl etc)
Cryoultrathin sectioning for Tokuyasumethod (sucroseinfiltrated)
Cryoultrathin sectioning for Xray microanalysis (unfixed)
Freeze substitution followed by resin embedding (Epoxy, LR White,
Lowicryl etc)
Immuno labelling of sections (resin and frozen)
Positive staining
Preparation of samples for Xray microanalysis
Coating of grids (formvar)
Image processing (software iTEM and TIA)

Scanning Electron Microscopy

(SEM)
A typical SEM instrument, showing the electron column,
sample chamber, EDS detector, electronics console, and
visual display monitors.
The scanning electron microscope (SEM) uses a focused
beam of high-energy electrons to generate a variety of
signals at the surface of solid specimens. The signals that
derive from electron-sample interactions reveal
information about the sample including external
morphology (texture), chemical composition, and
crystalline structure and orientation of materials making
up the sample. In most applications, data are collected
over a selected area of the surface of the sample, and a
2-dimensional image is generated that displays spatial
variations in these properties.

Fundamental Principles of Scanning

Electron Microscopy (SEM)
Accelerated electrons in an SEM carry significant amounts
of kinetic energy, and this energy is dissipated as a variety
of signals produced by electron-sample interactions when
the incident electrons are decelerated in the solid sample.
These signals include secondary electrons (that produce
SEM images), backscattered electrons (BSE), diffracted
backscattered electrons (EBSD that are used to determine
crystal structures and orientations of minerals), photons
(characteristic X-rays that are used for elemental analysis
and continuum X-rays), visible light (cathodoluminescence
CL), and heat. Secondary electrons and backscattered
electrons are commonly used for imaging samples:
secondary electrons are most valuable for showing
morphology and topography on samples and backscattered
electrons are most valuable for illustrating contrasts in
composition in multiphase samples (i.e. for rapid phase
discrimination

X-ray generation is produced by inelastic

collisions of the incident electrons with electrons
in discrete ortitals (shells) of atoms in the sample.
As the excited electrons return to lower energy
states, they yield X-rays that are of a fixed
wavelength (that is related to the difference in
energy levels of electrons in different shells for a
given element). Thus, characteristic X-rays are
produced for each element in a mineral that is
"excited" by the electron beam. SEM analysis is
considered to be "non-destructive"; that is, x-rays
generated by electron interactions do not lead to
volume loss of the sample, so it is possible to
analyze the same materials repeatedly.

Scanning Electron Microscopy

(SEM) Instrumentation

Electron Source ("Gun")

Electron Lenses
Sample Stage
Detectors for all signals of interest
Display / Data output devices
Infrastructure Requirements:
Power Supply
Vacuum System
Cooling system
Vibration-free floor
Room free of ambient magnetic and electric fields

SEMs always have at least one detector (usually a

secondary electron detector), and most have
additional detectors. The specific capabilities of a
particular instrument are critically dependent on
which detectors it accommodates.

Applications
The SEM is routinely used to generate high-resolution
images of shapes of objects (SEI) and to show spatial
variations in chemical compositions: 1) acquiring
elemental maps or spot chemical analyses using EDS,
2)discrimination of phases based on mean atomic number
(commonly related to relative density) using BSE, and 3)
compositional maps based on differences in trace
element "activitors" (typically transition metal and Rare
Earth elements) using CL. The SEM is also widely used to
identify phases based on qualitative chemical analysis
and/or crystalline structure. Precise measurement of very
small features and objects down to 50 nm in size is also
accomplished using the SEM. Backescattered electron
images (BSE) can be used for rapid discrimination of
phases in multiphase samples. SEMs equipped with
diffracted backscattered electron detectors (EBSD) can be
used to examine microfabric and crystallographic
orientation in many materials.

Strengths and Limitations of

Scanning Electron Microscopy
There is arguably no other instrument with the
breadth of applications in the study of solid
materials that compares with the SEM. The SEM is
critical in all fields that require characterization of
solid materials. Most SEM's are comparatively easy
to operate, with user-friendly "intuitive" interfaces.
Many applications require minimal sample
preparation. For many applications, data acquisition
is rapid (less than 5 minutes/image for SEI, BSE, spot
EDS analyses.) Modern SEMs generate data in digital
formats, which are highly portable.

Limitations
Samples must be solid and they must fit into the microscope chamber.
Maximum size in horizontal dimensions is usually on the order of 10 cm,
vertical dimensions are generally much more limited and rarely exceed 40
mm. For most instruments samples must be stable in a vacuum on the
order of 10-5 - 10-6 torr. Samples likely to outgas at low pressures (rocks
saturated with hydrocarbons, "wet" samples such as coal, organic
materials or swelling clays, and samples likely to decrepitate at low
pressure) are unsuitable for examination in conventional SEM's. However,
"low vacuum" and "environmental" SEMs also exist, and many of these
types of samples can be successfully examined in these specialized
instruments. EDS detectors on SEM's cannot detect very light elements
(H, He, and Li), and many instruments cannot detect elements with
atomic numbers less than 11 (Na). Most SEMs use a solid state x-ray
detector (EDS), and while these detectors are very fast and easy to utilize,
they have relatively poor energy resolution and sensitivity to elements
present in low abundances when compared to wavelength dispersive xray detectors (WDS) on most electron probe microanalyzers (EPMA). An
electrically conductive coating must be applied to electrically insulating
samples for study in conventional SEM's, unless the instrument is capable
of operation in a low vacuum mode.

 Sample preparation can be minimal or elaborate for

SEM analysis, depending on the nature of the samples
and the data required. Minimal preparation includes
acquisition of a sample that will fit into the SEM
chamber and some accommodation to prevent charge
build-up on electrically insulating samples. Most
electrically insulating samples are coated with a thin
layer of conducting material, commonly carbon, gold,
or some other metal or alloy. The choice of material for
conductive coatings depends on the data to be
acquired: carbon is most desirable if elemental analysis
is a priority, while metal coatings are most effective for
high resolution electron imaging applications.
Alternatively, an electrically insulating sample can be
examined without a conductive coating in an
instrument capable of "low vacuum" operation.