AN INTRODUCTION TO ELECTRON MICROSCOPY AND X-RAY

MICROANALYSIS

DR. L.R. TIEDT

W.E. PRETORIUS
2002

LABORATORY FOR ELECTRON MICROSCOPY

CRB
NORTH-WEST UNIVERSITY
POTCHEFSTROOM CAMPUS
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CONTENTS
1. RELATIVE SCALES. 5
2. RESOLVING POWER AND WAVELENGTHS 5
3. HISTORY OF ELECTRON MICROSCOPES 5
3.1 Electron microscope development. 5
3.2 Electron probe microanalysis. 6
4. MICROSCOPES TYPES. 6
4.1 Transmission electron microscope. 7
4.2 Scanning electron microscope. 7
4.3 Environmental scanning electron microscope 10
5. ELECTRON GUN. 10
6. ELECTROMAGNETIC LENSES. 11
7. WORKING DISTANCE. 11
8. MAGNIFICATION. 11
9. DEPTH OF FIELD. 12
10. TYPES OF SIGNAL. 12
10.1 Auger electrons. 13
10.2 Secondary electrons. 13
10.3 Backscattered electrons. 14
10.4 Characteristic X-rays. 14
11. SAMPLE CHARGING. 14
11.1 Charge build up. 15
11.2 Image distortion. 15
11.3 Methods of charge reduction. 15
12. DETECTORS. 16
12.1 Secondary electron detector. 16
12.2 Backscatter detector. 17
13. SCAN COILS AND RASTERING. 17
14. MICROANALYSIS. 17
14.1 X-ray generation. 17
14.2 Qualitative analysis. 19
14.3 Quantitave analysis. 19
14.4 Spectrum processing 20
14.5 Standardless analysis. 21
14.6 Standards analysis. 21
14.7 X-ray mapping. 21
15. ACCELERATING VOLTAGE FOR ANALYSIS AND OVERVOLTAGE. 22
16. SURFACE TOPOGRAPHY. 22
16.1 Effects of topography on matrix corrections. 22
16.2 Topography. 23
16.3 Comparison of EDX and WDX. 23
17. SAMPLE REPARATION FOR MICROANALYSIS (SEM). 23
17.1 Polishing and mounting techniques. 23
18. COATING TECHNIQUES. 25
18.1 Coating. 25
18.2 Sputtering. 26
20. ORGANIC SAMPLE PREPARATION FOR ELECTRON MICROSCOPY. 27
20.1 Narcosis of samples. 27
20.2 Fixation. 27
20.3 Buffers. 28

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20.4 Dehydration. 28
20.5 Critical-point drying (CPD) for SEM. 29
20.6 Embedding for TEM. 29
20.7 Sectioning for TEM. 29
20.8 Staining with heavy metals for TEM. 29
20.9 Negative staining for TEM. 31
21. PROCEDURES FOR SAMPLE PREPARATION IN THE LEM. 31
21.1 Biological sample preparation for sem. 32
21.2 Carbon coating 32
21.3 Coating with gold/palladium 33
21.4 Critical-point drying 33
22. PROSEDURES VAN MATERIAALVOORBEREIDING IN DIE LEM. 33
22.1 Koolstofbedamping. 33
22.2 Metaalbedamping met Au/Pd. 34
22.3 Kritiese droging van materiaal vir SEM. 34
22.4 Osmiumdampfiksering van mikroorganismes 35
22.5 VOORBEREIDING VAN BIOLOGIESE MATERIAAL VIR SEM. 35
23. REFERENCES. 37

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1. RELATIVE SCALES.

In the world around us we are used to only seeing objects with the naked eye. Measurements
are made usually using metres and millimetres and for longer distances, kilometres. These
units form part of the SI system of units. Each unit is related to the other units by 1000 - i.e. 1
kilometre is 1000 meters.

The high magnifications achievable with electron microscopes necessitate using units suitable
for smaller measurements. Units used are micro- and nanometer.

A micron is one thousandth of a millimeter, and similarly a nanometer is one thousandth of a

micron. It is usual to use the exponent notation when relating these dimensions to meters.

The relationship of the various units and their abbreviations are:

1 millimeter (mm) = 1 x 10 -3 meter

1 micron (µm) = 1 x 10 -6 meter
1 nanometer (nm) = 1 x 10 -9 meter
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Historically the Angstrom Unit (Å) was used. 1Å is the same as 1 x 10 meter, and
nanometer is 10 Å.

2. RESOLVING POWER AND WAVELENGTHS.

The resolving power of a microscope governs the point at which two near features are
resolved by the viewer. The eye can usually resolve two points separated by a distance that
can be seen by the human eye.

Given a fixed aperture it can be seen that the limit of resolution is proportional to the
wavelength of the image forming radiation. For an optical microscope ultraviolet
illumination can be used to improve the resolution. Electrons, which have a much shorter
wavelength than light, are the extreme example of the use of short wavelengths to improve
resolution.

3. HISTORY OF ELECTRON MICROSCOPES.

3.1. Electron microscope development.

In 1897 [Link] published the results of his theoretical and experimental studies in
quantitative aspects of cathode rays, demonstrating that they were composed of streams of
negatively charged particles which he named "electrons".

During the following quarter of a century further research into electrons included a number of
developments which were to lead to the construction of the first electron beam machines.
Key events included Wehnelt's 1903 demonstration that an electron beam could be
concentrated by means of an electrostatic field, which established the working principle of the
electron gun. De Broglie's demonstrated in 1924, that an electron could be regarded as a
wave, which initiated research into the electron's waveform possibilities and Busch’s paper in
1926 on the theory of electromagnetic lenses.

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By 1930 a group of German scientists had constructed the first working transmission electron
microscope.

The development of the machines during the pre-war period was slowed by a number of
technical difficulties which remained to be solved. Obtaining the stable power supplies
needed for accurate control of the accelerating voltage and the lens currents was virtually
impossible prior to the wartime development of power stabilisers. Avoidance and control of
lens aberrations was problematic, as was the production and maintenance of high vacuum
inside the microscope column. By the end of the 1940's these problems had been researched
and solved, and an important new development was underway.

3.2. Electron probe microanalysis.

In 1948 Raymond Castaing embarked on a Ph.D project at the University of Paris, the aim of
which was to develop a practical method of electron probe microanalysis by combining an
electron microscope and an X-ray spectrometer. The principle of using characteristic X-rays
for chemical analysis had been promulgated by Moseley in 1913 and put into practice with
the design and construction of X-ray spectrometers during the inter-war years.

In 1947 Hillier had patented the principle of combining spectroscopy and electron
microscopes: in 1950, Castaing arrived at a practical method of achieving this and
demonstrated that the method was capable of discerning compositional differences in
specimens.

The Cameca Company manufactured the first commercially produced microscope/

spectrometer combination in France in 1958.
At the same time, research into the possibility of obtaining larger specimen areas for
microanalysis led to the building of the first scanning electron microscope in 1960.
Further research, carried out in the engineering laboratories at Cambridge University, resulted
in the production of the first commercial SEMs by the Cambridge Instrument Company on
1964.

4. MICROSCOPE TYPES.

In our quest to see the unseen, we have built not only better but different types of
microscopes. Essentially these fall into one of two major categories:

Transmitting - Energy passed through the specimen differentially refracted and absorbed.
Scanning - Probe forming, energy scanned over the surface. Image built up point by point.

Transmitting:

1.) Transmitting Light Microscope (TLM) - Visible spectrum or selected wavelengths thereof
passed through the specimen and gathered. Best resolution = 200 nanometers.
2.) Transmission Electron Microscope (TEM) - Electron beam passed through the specimen
and gathered. Differs from TLM principally in its source of illumination. Best resolution = 0.5
nanometers.

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Scanning: Although microscopes, these do not use a lens system to produce the final
magnified image.

Scanning Electron Microscope (SEM) - Electron beam passed over the surface of the
specimen and causes energy changes in the surface layer. These changes are detected and
analysed to give an image of the specimen. Yields information only from the surface or near-
surface of the specimen. Has an advantage over TEM by having a huge depth of field and
gives very pleasing picture. Appears as three dimensional but true 3-D can only be attained by
using two pictures taken at different angles. Best resolution = 10 nanometers.

Light and electron microscopes operate on similar principles. They both magnify objects
such that the human eye can see detail that would otherwise be invisible. Both types of
microscope have a source of illumination, a system of lenses to focus and direct the
illumination and a method of viewing the image.

Within the light microscope the sample is illuminated using visible light, and the light is
focussed and directed using a system of glass lenses. The final image is viewed through an
eyepiece. Light microscopes may be referred to as simple or compound types. A simple type
uses a single lens to magnify the object, whereas a compound microscope uses more lenses to
produce a more highly magnified image in stages.

FIG.1a). Light microscope, b). TEM, c). SEM.

An electron microscope uses an electron gun to provide the illumination, and the electron
beam is focussed and directed using electromagnetic lenses. The sample is viewed either on a
TV type screen in the case of the scanning electron microscope, or on a phosphor screen in
the transmission electron microscope.

4.1 Transmission electron microscope.

The transmission electron microscope allows the investigation of the internal microstructure
of samples, provided that they are thin enough to transmit electrons.

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The source of illumination in a TEM is contained in the electron gun, situated at the top of the
column. Electrons travel at high speed down the column, and are focussed onto the specimen
using a combination of magnetic lenses. The microscope can be operated in a number of
modes, both image and diffraction, and can be fitted with a variety of detectors, making it a
versatile and powerful analytical tool for microstructural investigation at high spatial
resolution.

In image mode, the objective lens produces an image of the internal structure of the specimen,
which is then projected and magnified, using a combination of projector and intermediate
lens, onto the fluorescent screen at the base of the column.

Operation of the TEM in diffraction mode allows crystallographic information, from the
sample, to be obtained.

FIG. 2. Ray paths of light in a LM and electrons in a TEM.

4.2 Scanning electron microscope.

The scanning electron microscope (SEM) is commonly used to examine the microstructure of
bulk specimens.
It is an electron-optical instrument, which uses a source of electrons to illuminate the
specimen. With the new generation of SEM’s, the ESEM or Environmental Scanning Electron
Microscope, wet, dirty, oily, outgassing and samples as hot as 1 500°C can be viewed.

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FIG. 3. The functioning of a SEM

These electrons are accelerated down the column and pass through a combination of
electromagnetic lenses and apertures to form a fine probe at the surface of the specimen in the
chamber area. Both the column and the chamber are held under vacuum to avoid high voltage
discharge and scattering of the electrons along their path by residual gas atoms. The beam
may be scanned in a rectangular raster across the surface of the specimen by means of a series
of scan coils situated above the objective lens. A variety of signals are produced as a result of
the interaction of the beam with the specimen, which may be collected by appropriate
detectors.

This information can be accessed and displayed in a variety of forms, one of which is an
image. The output of the various detectors such as backscattered and secondary electron
detectors are used to modulate the brightness of a cathode ray tube (CRT). The raster of the
electron beam on the specimen is synchronous with that of the CRT so that information from
the sample is built up as a two dimensional image. The SEM is a valuable imaging tool
which allows very fine detail to be resolved and, unlike an optical microscope, offers a large
depth of field. In addition, it may be combined with appropriate detectors, to serve as a
powerful analytical tool. Wqa2a
There are various ways in which to operate the scanning electron microscope, depending on
the information required. If high depth of field images are required then a small convergence a@ßw
angle should be used, so that different heights on an irregular surface are all in focus. This
can be achieved by using a small objective aperture or a long working distance. However, if 2
X-ray microanalysis is to be performed, probe currents of at least 10 -10 amps should be used
for EDS and at least 10-8 amps for WDS.

A higher current will reduce noise in the image. However, the probe diameter increases when
the lenses are adjusted to give higher current, so spatial resolution in the electron image is
compromised. Thus, there is a trade off between good count rate and low noise images and
the ability to see very fine specimen detail. If high-resolution secondary electron images are
required, then a small probe size and short working distance should be used. This can be
achieved by using both a strong condenser and objective lens setting. This will, however,
limit the current in the probe, so the images may appear noisy.

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4.3 Environmental scanning electron microscope ESEM.

As mentioned above, samples for conventional SEM generally have to be clean, dry, vacuum
Tttt5r
compatible and electrically conductive. In recent years the Environmental Scanning Electron
Microscope (ESEM) has been developed to provide a unique solution for problematic
samples. Examples of specimens which pose problems are wool or cotton tissue, cosmetics,
fats and emulsions
(e.g. margarine). Attempts to view a specimen containing volatile components
by placing it in an environmental chamber (see box O) isolated from
the main column by one or more differential pumping apertures used to be hampered by the
lack of a suitable electron detector which can work in the atmosphere of the chamber. The
Gaseous Secondary Electron Detector makes use of cascade amplification not only to enhance
the secondary electron signal but also to produce positive ions which are attracted by negative
charge on the insulated specimen surface and effectively suppress charging artefacts.

Environmental Chamber
The pressure-temperature phase diagram for H2O indicates that true "wet"
conditions only exist at pressures of at least 600 Pa at 0°C (environmental microscopists
usually refer to 4.6 Torr = 4.6 mm of mercury ). In the range 650 to 1300 Pa (5 –10 Torr)
therefore the specimen may be observed whilst at
equilibrium with water. Ice cream in the Cryo-SEM: the amount of emulsion in relation to air
and water (ice) is an important parameter to study.

5. ELECTRON GUN.

The purpose of the electron gun is to provide a stable source of electrons. There are several
types of electron gun routinely used in EMs which vary in their design and emission
characteristics. The most commonly used type of electron gun is the conventional triode
electron gun, which is made up of three components which are kept under vacuum in the gun
chamber.

FIG. 4. The high voltages used in the electron gun

The filament or cathode is the source of electrons and is held at negative potential relative to
earth potential. A typical tungsten filament is made from a bent piece of wire typically
100µm in diameter. A current is applied to the filament to heat the wire to typically 2700 K,
at which point, electrons are emitted from the filament, by a process called thermionic
emission. In order for the electrons in the filament to escape from the material, they require
sufficient energy to overcome the work-function energy of the material. This energy is

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provided by the heat supplied by the filament heating current. The lifetime of the filament
will depend on the temperature to which the filament rises. As the temperature increases, so
the lifetime may decrease. A good vacuum in the gun area is essential to prevent erosion of
the filament by ion bombardment from the gases present in surrounding area.

The "Wehnelt" or grid which is held at a few hundred volts relative to the cathode. The
strength of this voltage affects both the form of electrostatic field between the grid and the
filament for a given filament current applied. The form of the electrostatic field focuses the
electrons between the grid cap and the anode.

The anode is positioned at the base of the gun chamber and held at earth potential, therefore,
the electrons are accelerated from the high negative potential of the filament towards the
anode. The hole in the anode allows a proportion of the electrons to shoot down the column
through a combination of lenses and apertures to the specimen.

6. ELECTROMAGNETIC LENSES.

In optical microscopes, the ability to focus light is achieved by using glass lenses. Electron
microscopes use electrons as the source of illumination and the ability to focus electrons in
the microscope is achieved using electron lenses. These may be either electrostatic or
electromagnetic. Electron lenses are all subject to aberrations but less so in the case of
electromagnetic than in electrostatic lenses. The main role of electromagnetic lenses in
electron optical columns is to de-magnify the source of electrons to form a much smaller
diameter probe incident on the sample.

The lens strength can be varied by adjusting the amount of current, which flows through the
windings around the iron core of the magnet. There are two main lenses used in a SEM: the
condenser and the objective. The condenser affects the number of electrons in the beam for a
given objective aperture size, and the objective lens focuses electrons on to the specimen at
the working distance.

7. WORKING DISTANCE.

The working distance is defined as the distance between the lower pole piece of the objective
lens and the position of the specimen at which the electrons are focussed onto the specimen.
The strength of the objective lens essentially changes the distance between the pole piece and
the plane onto which the electrons are focussed. The probe can, therefore, be focussed at
different working distances to cope with different specimen heights. The specimen height can
be adjusted, but for X-ray microanalysis, there is a recommended working distance for
optimum X-ray acquisition, which is specific to the geometry of the detector mount on the
SEM chamber.

8. MAGNIFICATION.

The magnification of an electron image is defined as the ratio of the length of one line of the
electron beam on the monitor to the width of the area scanned on the specimen.
An increase in magnification can be achieved by reducing the width of the area scanned on
the specimen, since the width of the CRT is fixed. In this way magnifications of up to
300,000 can be achieved, provided all the conditions necessary for the best spatial resolution
have been optimised. Above these magnifications, no greater detail is observed and greater
magnification is often referred to as "empty magnification".

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Theoretically, the spatial resolution of the SEM in secondary electron image mode is,
ultimately, determined by the size of the scanning spot. This is influenced by electron optical
parameters such as working distance, lens settings and electron gun brightness and also by
electronic noise in the X and Y deflection currents.

In practice there are a number of other factors that will influence the spatial resolution
practically attainable from the SEM in imaging mode. These include accelerating voltage, the
state of the specimen surface, detector position and set up and external mechanical vibrations.
Spatial resolutions of a nanometer may be obtained with all these conditions optimised.

9. DEPTH OF FIELD.

Depth of field characterises the extent to which image resolution degrades with distance
above or below the plain of best focus. With greater depth of field a microscope can better
image three-dimensional samples. In the SEM, then depth of field is much greater than for
optical microscopes.

FIG. 5. Depth of field in the SEM.

10. TYPES OF SIGNAL.

11. The electron beam interacts with the near surface region of a sample to a depth of
approximately a few microns, depending on both the accelerating voltage and the
density of the material. Secondary electrons have the smallest volume, followed by
backscattered electrons and X-rays. This interaction volume ultimately determines
both the spatial resolution and depth from which analysis can be achieved.

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FIG. 6. Each type of signal originates within a specific volume of interaction

Numerous signals are produced as a result of this interaction, which may be detected, by
appropriate detectors, to provide information about the specimen.
These signals include low energy secondary emission, Auger electron generation,
characteristic X-ray emission, bremstrahlung, backscattered electron emission and
cathodoluminescence.

FIG. 7. The interactions of the beam electrons and the sample atoms generate a variety of
signals.

10.1 Auger electrons.

The bombardment of the specimen by energetic electrons results in ionised atoms to a depth,
depending on accelerating voltage and density of the material, but typically of the order of

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1µm. An ionised atom can emit a characteristic X-ray or the energy released as electron fills
the initial vacancy may eject another electron from the atom in a "radiationless" transition
called the Auger effect.

If an inner K shell electron is ejected and an electron from the L shell fills this vacancy,
releases energy and ejects an Auger electron from the L shell, the Auger transition is termed a
KLL transition. Measurement of the energy of the characteristic Auger electrons forms the
basis of Auger spectroscopy. The energies of the Auger electron peaks allow all elements,
with the exception of hydrogen and helium, to be identified, since a minimum of three
electrons are required for the emission process.

Auger spectroscopy is a surface sensitive technique, since Auger electrons generated deeper
than the top surface layers, will lose their energy signature as they travel out of the specimen.
Therefore, the detected signal comprises of electrons generated from only the first few
monolayers of the sample - those that have sufficient energy to escape.

10.2 Secondary electrons.

Inelastic scattering of an energetic electron with outer valence electrons leads to the emission
of secondary electrons that are characterised by having a kinetic energy less than 50eV. These
electrons may be subject to additional scattering events through which energy is lost and
therefore only electrons which have sufficient energy to overcome the surface barrier energy
and escape the material and contribute to the detected signal; these are electrons at the
specimen surface.
Secondary electron emission is one of the most common signals used for imaging in the SEM,
since most of the signal is confined to a region close to the incident beam, and gives a high-
resolution image. Secondaries are also emitted when backscattered electrons exit the sample,
often some distance away from the beam spot.

10.3 Backscattered electrons.

A significant number of the incident electrons which strike a bulk specimen are re-emitted
through the surface of the material. These electrons are known as backscattered electrons,
which have undergone large angle elastic scattering events in the material, enabling then to
approach the surface with sufficient energy to escape. The scattering intensity is related to
the atomic number of the atom, the higher the atomic number of the material involved, the
higher the backscattering coefficient, and the higher the yield.

This dependence of the backscatter yield with atomic number, forms the basis for
differentiating between different phases, thus providing an ideal starting point to guide further
microanalysis.

Backscattered electrons give useful information about the composition and surface
topography of the specimen.

10.4 Characteristic X-rays.

The interaction of a high-energy electron with an atom may result in the ejection of an
electron from an inner atomic shell. This leaves the atom in an ionised or excited state with a
vacancy in this shell. De-excitation can occur by an electron from an outer shell filling the

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vacancy. The change in energy is determined by the electronic structure of the atom that is
unique to the element.

11. SAMPLE CHARGING.

11.1 Charge build up.

As a result of the interaction of the electron beam with the specimen, a number of interactions
take place, both elastic and inelastic. This injection of charge produces a number of
secondary electrons, and some of the original charge can be re-emitted in the form of
backscattered electrons. However, not all the incident charge is re-emitted via these two
routes.

11.2 Image distortion.

Charging can distort secondary electron images and affect analytical information from the
specimen. The collection of secondary electrons by the detector is influenced by the strength
of the field associated with the Faraday cage on the EHT detector, and also the local specimen
field.

In some cases, the degree of charging is such that the beam can be deflected from its original
position, causing streaks in the image to appear, and in other cases, images can become
distorted. The degree of charging and, hence, the effect this has on the image, depends
critically on the rate at which the beam is scanned across the specimen.

11.3 Methods of charge reduction.

11.3.1 Coating.

If charging is severe, which is the case for most everyday materials, such as minerals,
ceramics, fibres, glasses and biological materials, the specimen generally needs to be coated
with a conducting layer, which creates a path along which the excess charge, caused by the
beam, can flow to ground.

In addition to insulating materials, many metals and semiconductors are also prone to the
build up of insulating oxide layers on the surface. Coating is generally sufficient to prevent
the images of such materials being affected.

11.3.2 Specimen tilt.

The yield of secondary electrons is also a function of the specimen tilt.

As the specimen is tilted away from normal incidence, the secondary electron yield increases
and so reduces charging of the sample.

11.3.3 Probe current.

Lowering the probe current at the specimen can reduce the effect of charging. This allows the
injected charge into the specimen to leak away. The effectiveness of this charge leakage is
material dependant.

11.3.4 Scan rate

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Charging of the specimen can be reduced by scanning the beam as quickly as possible over
the specimen, and building up the image frame by frame, rather than acquiring the image with
a long dwell time scan.

11.3.5 Accelerating voltage.

The total electron emission depends on the primary electron energy. Adjustments of this
energy will dictate whether the specimen surface will charge negative or positive.
The charging can be reduced by lowering the accelerating voltage, since the secondary
electron coefficient generally rises as the beam energy is reduced, whilst the backscattered
yield is not a strong function of beam energy.

11.3.6 Choice of detector.

Secondary electron images are extremely sensitive to charging, due to the strength of the bias
on the secondary electron detector, and the typically low energies of secondary electrons
(<50eV). Whilst secondary electron images are sensitive to low levels of charging, the
backscattered component is less so, because of the greater energy of these electrons.
A surface potential of a few volts will not have a substantial effect on the trajectory of a
backscattered electron, whose energy could be as great as the incident beam energy.

12. DETECTORS.

Two types of detector are commonly used to detect these electrons: a scintillator type detector
(SE), or a solid state type (BSE).

12.1 Secondary electron detector.

The standard detector used in the scanning electron microscope is a combined secondary and
backscattered detector, known as the Everhart-Thornley detector.
The detector consists of a Faraday cage, a scintillator, light pipe and photomultiplier tube.
Electrons, incident on the scintillator, produce photons, which are then "guided" down the
light pipe, via total internal reflection, to the photomultiplier. At the photomultiplier, the
signal falls
on the first photocathode, where it is turned back into an electron current. The electrons are
accelerated onto successive electrodes, finally producing a cascade of electrons.
The detector is usually operated in two modes. The most common of these is with a positive
bias of around 500V on the Faraday cage. The effect of this is to deflect the trajectories of the
secondary electrons, emitted from the sample, into the detector.

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FIG. 8. The Everhart-Thornley detector.

The bias will also accelerate the electrons onto the scintillator. Electrons can be collected
from the sample, even if there is not a direct line sight from the specimen to the detector. The
collection efficiency of the detector approaches 100% on a flat surface. Backscattered
electrons, which have a direct line of sight to the detector, will also be detected and contribute
to the signal observed. The positive bias will have little to no effect on these electrons.

With the bias turned off, only those secondary electrons with a direct line of sight to the
detector, will be detected, along with the backscattered electrons mentioned above.

12.2 Backscatter detector.

The solid state backscattered detector is mounted close to the specimen, under the final lens
and is usually split into four quadrants. The detector will have a hole in the middle for the
beam to pass through. The detector is slim enough to be usually left in place, and also to
permit simultaneous x-ray microanalysis.

13. SCAN COILS AND RASTERING.

The deflection coils or scan coils are situated above the objective lens in the column and may
be used to raster the electron beam over a rectangular area on the surface of the specimen.
This raster mode may be used to generate information from more than one location of the
beam, in order to build up an image using the emitted signals from the sample

Other such deflection coils in the SEM are the gun shift and tilt coils and the image shifting
coils, which are used to align the gun and move the image respectively. The scanning action
is achieved by applying current to each of the horizontal and vertical scan coils, so that the
probe is deflected to cover a rectangular area on the specimen.
As a result of the interaction of the beam with the specimen, a variety of signals are produced,
which may be collected by appropriate detectors.

Such signals are detected end amplified and used to modulate the brightness of the electron
beam in the cathode ray tube (CRT). The raster of the electron beam on the specimen is
synchronous with that of the electron beam in the CRT.
In this the way, variations in the signal emitted over the scanned area are displayed as
variations in brightness on the CRT, and hence, the appropriate information can be displayed
in image form. Since the area scanned on the CRT is much bigger than the area scanned on
the sample, magnification is achieved.

14. MICROANALYSIS.

As the name suggests, this refers to the analysis of a sample on a microscopic scale, resulting
in structural, compositional and chemical information about the sample.
There exists a whole host of analytical detectors that exploit the many signals that may be
generated within the sample. X-ray microanalysis specifically gives information about the
elemental composition of the specimen, in terms of both quantity and distribution.

14.1 X-ray generation.

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As a result of the inelastic scattering of the incident electrons with matter, two main processes
can produce X-rays: characteristic X-ray emission and Continuum X-ray emission or
bremsstrahlung.

The interaction of a high-energy electron with an atom may result in the ejection of an
electron from an inner atomic shell. This leaves the atom in an ionised or excited state, with a
vacancy in this shell. De-excitation can occur by an electron from an outer shell filling the
vacancy. The change in energy is determined by the electronic structure of the atom that is
unique to the element.

This "characteristic" energy can be released from the atom in two ways. One is the emission
of an X-ray photon with a characteristic energy specific to that transition and hence, to the
element. Detection of such X-rays gives information about the elemental composition of the
specimen, in terms of both quantity and distribution. The second way is releasing so called
Auger electrons.

The continuum X-ray spectrum or bremsstrahlung (literally translated as braking radiation) is

generated, in addition to characteristic X-ray emission, when electrons interact with matter.
The emission of these X-ray photons is associated with the deceleration of the atom core.
Since the energy lost by the electron can range anywhere between zero up to the value of the
incident electron energy, a continuous spectrum of X-ray energies is produced.

FIG. 9. . X-ray generation.

An X-ray spectrometer collects the characteristic X- rays. The spectrometer counts and sorts
the X- rays, usually on the basis of energy (Energy Dispersive Spectrometry – EDS). The
resulting spectrum plots number of X-rays, on the vertical axis, versus energy, on the
horizontal axis. Peaks on the spectrum correspond to the elements present in the sample.

The EDS Detector

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How the EDS Detector Works

The EDS detector converts the energy of each individual X-ray into a voltage signal of proportional
size. This is achieved through a three stage process. Firstly the X-ray is converted into a charge by the
ionization of atoms in the semiconductor crystal. Secondly this charge is converted into the voltage
signal by the FET preamplifier. Finally the voltage signal is input into the pulse processor for
measurement. The output from the preamplifier is a voltage ‘ramp’ where each X-ray appears as a
voltage step on the ramp.

EDS detectors are designed to convert the X-ray energy into the voltage signal as accurately as
possible. At the same time electronic noise must be minimized to allow detection of the lowest X-ray
energies.

How the crystal converts X-ray energy into charge

When an incident X-ray strikes the detector crystal its energy is absorbed by a series of ionizations
within the semiconductor to create a number of electron-hole pairs. The electrons are raised into the
conduction band of the semiconductor and are free to move within the crystal lattice. When an
electron is raised into the conduction band it leaves behind a ‘hole’, which behaves like a free positive
charge within the crystal. A high bias voltage, applied between electrical contacts on the front face and
back of the crystal, then sweeps the electrons and holes to these opposite electrodes, producing a
charge signal, the size of which is directly proportional to the energy of the incident X-ray.

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FIG.10. An EDS spectrum displays peaks at energies characteristic of elements present in the
sample.

14.2 Qualitative analysis.

Qualitative analysis identifies the elements present in the analysed volume of a specimen, i.e.
it answers the question: "what is there?"
An X-ray spectrum is recorded, over a range of energy, within which, relevant lines may be
present. The lines, and therefore, the elements, are identified by reference to tables or
databases.

14.3 Quantitave analysis.

Quantitave analysis determines how much of a particular element is present in the analysed
volume of a specimen, i.e. it answers the question: "How much is there?" or "What is the
composition?"
Intensities of X-ray lines from the specimen, are compared with those from standards of
known composition. Corrections are made for background and instrumental effects. The
composition of the analysed volume is then calculated by applying "matrix corrections",
which take into account various factors governing the relationship between the measured
intensity and composition. It is important that the volume being analysed is homogeneous,
and is representative of the specimen. The sample must be flat and perfectly polished.
Polished lines, steps formed by strong chemical etching or electric polishing lead to intensity

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variations caused by differences in X-ray absorption due to the topography rather than to the
chemical nature of the specimen.
After polishing the sample must be cleaned by washing in water or alcohol and drying in hot
air. The use of an ultrasonic cleaner is recommended.

14.4 Spectrum processing

Spectrum processing :
Processing option : All elements analyzed (Normalised)
Number of iterations = 4

Standard :
C CaCO3 1-Jun-1999 12:00 AM
O SiO2 1-Jun-1999 12:00 AM
S FeS2 1-Jun-1999 12:00 AM

Element Weight% Weight% Atomic%

Sigma
CK 44.79 0.30 55.82
OK 39.30 0.29 36.76
SK 15.91 0.18 7.43

Totals 100.00

Processing option : Oxygen by stoichiometry (Normalised)

Number of ions calculation based on 8.00 anions per formula
Number of iterations = 4

Element Weight% Weight% Atomic% Compd% Formula Number

Sigma of ions
CK 21.01 0.18 27.34 76.96 CO2 3.21
SK 9.23 0.10 4.50 23.04 SO3 0.53
O 69.77 0.20 68.17 8.00
Totals 100.00
Cation sum 3.74

FIG. 11. Quantification results with Wt% (weight %), At% (atomic %),

14.4.5 Spectral artefacts.

Escape peaks- These peaks are caused by the fluorescence of X-rays in the detector. The
effects of escape peaks is usually small.
Sum peaks- When two X-rays enter the detector nearly simultaneously in less than the
resolving time, their energy will be summed. This occurs at high count rates ( ± 10000 CPS).

14.5 Standardless analysis.

The same basic approach can be applied without measuring standards. The standards are pure
elements, but we can calculate the relative pure element intensities from theory easily with a
small computer. So the measured pure element intensities can be replaced with calculated
relative pure element intensities. Standardless analysis is as accurate as the ability to calculate
pure intensities and as the assumption that the sum of all the concentrations is known.

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14.6 Standards analysis.

Standards are materials which are used to relate the intensity of a peak in a spectrum to the
concentration of that element in the specimen. They are materials in which the concentration
of all the elements are accurately known.
Standards may be pure elements or compounds which contain a known concentration of the
element. In order to make a direct comparison between intensity and concentration, a
standard specimen is referred to.
Once this is known, this ratio can be used to determine the concentration of that element in an
unknown specimen. All the intensities are assumed to have been corrected for background.
These corrections account for differences in X-ray absorption, atomic number and the degree
fluorescence in the specimen matrix.
The ratio between the apparent concentration and the concentration is a measure of the true
concentration is a measure of the ZAF correction, which needs to be included in the
calculation. The need for the correction is minimised if the composition of the standard is as
similar as possible to the composition of the unknown specimen. Since these matrix
corrections can be calculated with only a certain degree of accuracy, the choice of standard
material is very important if the quantification is to be as accurate as possible.

For all these methods the set up is done through a dialog box in the software. If a standard has
been measured, ie, a spectrum has been obtained, then the concentrations are entered into the
box and RZAF is used to calculate pure element intensities.

When standards are used, the SAME kV and beam current must be used for both the standard
and the sample. This is also applicable when comparing different samples in a project.

14.7 X-ray mapping

The distribution of elements over a particular area of the sample can be viewed by the
acquisition of element specific maps. X-ray mapping utilises the X-ray signal from a
specified energy range in the X-ray spectrum in order to show this elemental distribution.
There are several modes of mapping, qualitative digital mapping and quantitative digital
mapping.

Qualitative mapping gives information on the X-ray intensity distribution over the required
field of view. The grey scale value for a given pixel in any digital map corresponds simply to
the number of X-rays which enter the X-ray detector within the energy range and, therefore,
gives an idea of the elements. Quantitative mapping involves full spectrum processing,
including escape peak removal, background subtraction, followed by matrix corrections for
each pixel within the image.

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14.8 Point and I D Mapping + Cameo +

By using point and ID, multiple spot or area analysis can be done on one sample and presnent
the results in an Excel format.
15. ACCELERATING VOLTAGE FOR ANALYSIS AND OVERVOLTAGE.

Two physical phenomena come into play when thinking of the choice of voltage. First, the
incoming electron must be higher in energy than the critical excitation energy of the element
to be excited. If not the atom will not be excited. As the energy of the electron exceeds KeV,
more efficient excitation of the element occurs, and we say we have more “overvoltage”. As
the “overvoltage” increases, the X-rays are produced deeper in the sample. At some point the

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depth become so great that the absorption of the X-rays leaving the sample dominates over
the increases in the number of X-rays produced.
The "overvoltage" is define as:
U= kV/KeV
It will be different for each element in the sample. The rule of thumb for the selection of
accelerating voltage is that U should be >2, but <20 for general work. For quantitative work,
U should be >2 but < 10.

16. SURFACE TOPOGRAPHY.

16.1. Effects of topography on matrix corrections.

If the surface of the specimen is irregular, it is clear that X-rays produced, within the X-ray
production volume, will have to travel through different distances as they exit the specimen,
thus suffering differing degrees of absorption. The dimensions of the roughness, in
conjunction with the mean atomic number of the material and the accelerating voltage, will
ultimately determine the errors in successfully correcting for matrix effects.

If the specimen is so rough, such that the line of sight to the detector is obstructed, then X-
rays may be absorbed in the shadowing material and fail to reach the detector. Furthermore,
remote excitation of X-rays by electrons scattered from the original beam position on a rough
surface can occur and these remote X-rays may still reach the detector.
Even for a flat specimen, matrix corrections are affected if the surface is not normal to the
incident beam. The volume from which X-rays are produced is closer to the surface of the
specimen than for an untitled specimen.

When analysing particles, the position of the electron beam and detector relative to the curved
surface of the particle may clearly have a profound effect on the degree of absorption.
Furthermore, since electrons may escape from the sides and beneath the particle, the
backscatter correction may be considerably different to that for a bulk specimen.

16.2 Topography.

Whilst a polished specimen makes it easy to calculate matrix corrections, it may not always
be feasible to polish the specimen surface, and in doing so, the required detail, such as
impurities exposed at facets of a fracture surface, may be destroyed. The consequence of
analysing a specimen, with an undulating surface, is often an incorrect estimate of
composition. In such cases, different approaches can be tried, which include normalising the
results, applying analytical solutions for specific geometry’s, or using a peak to background
method

16.3 Comparison of EDX and WDX.

Wavelength dispersive spectrometry and energy dispersive spectrometry is complementary

techniques.

Historically, the WD spectrometer was the first type of elemental X-ray detector used on an
electron microscope. As semiconductor technology improved in the 1960's and 70's, the
faster ED spectrometer gained popularity making it today the first choice of a general purpose
X-ray detector on electron optic columns.

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However, despite its popularity, ED suffers from limitations, notably limit of detection and
resolution, in this case its ability to separate small differences in energy. These weaknesses
are compensated by the advantages of the WD spectrometer

17. SAMPLE PREPARATION FOR MICROANALYSIS (SEM).

17.1 Polishing and mounting techniques.

It is important that specimens for microanalysis are mounted correctly. This means that there
must be a good electrical connection between the specimen end the microscope stage, to
prevent charging. The type of mounting employed will depend on the specimen. It is also
sometimes necessary that the sample is flat, if accurate information is to be obtained from the
sample. Various polishing methods can be used to ensure a flat surface, depending on the
type of sample.

Whatever sample mounting and polishing techniques are used, it is important that the
information present in the sample is preserved, and not distorted.

17.1.1 Polishing.

Polishing techniques are varied, but similar in principle. The rough surface of the specimen is
abraded through a series of polishing papers, starting with rough grit papers and progressively
working through to finer papers. The polishing process will lubricate with water. Once the
finest paper has been used, fine diamond polishing wheels may be used. These will be pads,
with small amounts of diamond paste, with appropriate lubricants. The final finish will be to,
usually, 1 or 0.5µm finish. The specimen must be thoroughly cleaned and dried after the final
polish.

When polishing it is important to consider if the process will damage the sample. For
example, will the water used to lubricate the wet and dry papers used in the first stages,
damage the sample? Sometimes care must be taken when polishing specimens with small
precipitates. Removing too much material at a time may dislodge the precipitates of interest.
Different hardness materials will be polished at different rates, and will cause relief on the
surface of the specimen. Similarly, specimens such as sections of coated components, e.g.
tool bits, turbine blades, may need to be coated with another harder metal, to prevent the
coating of interest from spalling away.

The mechanical action of polishing will mechanically strain the surface region, and if, for
example, an imaging technique is being used, the relies on a contrast forming mechanism that
takes place near the surface, then a more gentle polishing technique must be used. These will
include final polishing with alumina rather than diamond or chemical and electro polishing.
These latter two techniques will require additional equipment, but will result in a
mechanically undamaged surface.

17.1.2 Mounting.

Specimens can be either mounted directly onto the sample stub, or are sometimes mounted in
another medium, prior to, say, polishing. The specimens should also be cleaned, if possible,

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to remove any hydrocarbon or other contaminants using mild solvents. If a low vacuum
microscope is being used, these requirements are less stringent.

If the specimen is being mounted directly onto the stub, it needs to be fixed down, with either
conductive tape or cement. Carbon, aluminium or copper double-sided conductive tape is
widely available. Stick some tape to the stub, and then simply mount the specimen onto the
tape. Sometimes it may also be necessary to coat the specimen with a conductive layer
(carbon or gold/palladium). If the specimen is irregular, make sure that the cement is in
contact with any conductive coating.

Metallographic specimens can also be mounted to make micro sections. A small amount of
the material will be embedded in a resin mount. Conductive mounting resins are available.
They are to be preferred if microanalysis is needed. Most mounting resins are hot press resins
carried out under pressure using a mounting press. The resultant mount can then be polished
and examined in the electron or optical microscope. If a non-conductive resin is used, a
conductive path must be provided from the specimen to the microscope stage. This will
usually be achieved with a thin evaporated or sputtered coating, or by a small trail of
conductive paint, containing carbon or silver.

Geological type samples will be cut into thin slices, using diamond type saws, and then fixed
onto glass slides using a strong adhesive. The mount will then be polished, and subsequently
coated with a conductive layer.

Powder specimens can be sprinkled directly onto conductive tape, or almost dry paint. Take
care to remove any excess unfixed particles in case they become loose inside the microscope
chamber. Again a conductive coating may be necessary, depending on the materials
involved.

Biological specimens need to be dried before they are fixed onto the stub. Alternatively, they
can be examined using cryo-preparation techniques, or with a low vacuum microscope.
These latter two techniques will preserve more information within the specimen.

18. COATING TECHNIQUES.

18.1 Coating.

In order to obtain a good image of most non-conductive specimens in the SEM the sample
must first be covered with a thin coating. A coating serves a number of purposes including:
a) increased conductivity,
b) reduction of thermal damage,
c) increased secondary and backscattered electron emission, and
d) increased mechanical stability.

Conductivity is the single most important reason for coating a specimen. As the primary beam
impinges on the specimen the increased electrical potential must be dissipated in some way.
For a conductive specimen such as most metals this is not a problem and the charge is
conducted through the specimen and eventually is grounded by contact with the specimen
stage. On the other hand non-conductive specimens or “resistors” can not dissipate this excess
negative charge and so localised build up charges cause a dielectric breakdown and gives rise
to an artefact known as charging. Charging results in the deflection of the beam, deflection of
some secondary electrons, periodic bursts of secondary electrons, and increased emission of

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secondary electrons from crevices. All of these serve to degrade the image. In addition to
coating the sample, the specimen should be mounted on the stub in such a way that a good
electrical path is established. This is usually accomplished through the use of a conductive
double-sided adhesive carbon tape or conductive adhesive silver or colloidal carbon paint.
A conductive coating can also be useful in dissipating the heating that can occur when the
specimen is bombarded with electrons. By rapidly transferring the electrons of the beam away
from the region being scanned, one avoids the build up of excessive heat.

Because secondary electrons are more readily produced by elements of a high atomic number
than by those of a low atomic number a thin coating of specimen can result in a greatly
improved image over what could be produced by the uncoated specimen. In cases where
backscattered electrons or characteristic X- rays are of primary interest a coating of heavy
metal such a gold or gold/palladium could obscure differences in atomic number that we
might be trying to resolve. In this case a thin coating of a low atomic number element (eg.
carbon) serves the purpose of increasing conductivity without sacrificing compositional
information.
The fourth and final purpose of using conductive coatings is to increase mechanical stability.
Although this is somewhat related to thermal protection, very delicate or beam sensitive
specimens can benefit greatly from a thin layer of coating material that actually serves to hold
the sample together. Fine particulates are a prime example of a case where a coating of carbon
or heavy metal can add physical stability to the specimen.

Many of the negative effects of imaging an uncoated specimen can be reduced by using a
lower energy primary beam (Kv) to scan the sample. Whereas this will tend to reduce such
things as localised charge build up, thermal stress, and mechanical instability it has the
distinct disadvantage of reducing overall signal. By carefully adjusting factors such as
accelerating voltage and spot size, many of these same effects can be reduced but a fine
coating of the specimen is still usually required.

Non-conductive specimens are coated with a conductive material prior to observation in the
SEM, to prevent charge up, and sometimes to increase the emission of secondary electrons.
Coatings are usually applied by using a vacuum evaporator, or a sputtering device. For
microanalysis, carbon is the preferred coating material, which does not generally interfere
with elements of interest in the specimen. Gold, usually used when a good secondary electron
image is required, is not recommended for microanalysis, because of the large number of lines
in the gold X-ray spectrum.

18.2 Sputtering.

Various types of sputtering machines are available. This technique is good for coating
specimens that are not smooth, and coating will be obtained even where there is not a direct
line of sight to the target. The target atoms suffer many collisions before they reach the
specimen, and are travelling in all different directions. Complete coating can be achieved
without rotating or tilting the specimen. Materials such as gold, platinum or gold/palladium
are used for sputtering. These materials have a high secondary electron emission resulting in
good imaging performance. Carbon cannot be deposited effectively with a sputter coater.

20. ORGANIC SAMPLE PREPARATION FOR ELECTRON MICROSCOPY.

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Organic samples, such as biological materials and maybe foodstuffs, need special handling if
they are to be observed in the electron microscope. These materials contain large amounts of
water, and this will cause problems when the specimen is put under vacuum. The samples
may be susceptible to beam damage.

The procedure of sample preparation depends on different factors, ea. the type and
characteristics of the sample, the environment from which it comes the effect of chemicals on
the sample and the purpose of the investigation.
There is a basic procedure which all biological material has to pass through: killing, fixing,
dehydration, (for TEM embedding, sectioning and staining) and drying and coating for SEM.
To keep damage to samples to a minimum, small sample holders are used. This also makes
the handling of small samples easier.

20.1 Narcosis of samples.

To relax small organisms, the following can be used:

 In water, gradually heated till the organism is dead.
 Putting it in 10% methanol or ethanol, ether vapour, osmium tetroxide or osmium tetroxide
vapour.

20.2. Fixation.

The fixation of organisms is to keep post-mortem changes or disintegration to a minimum and

to have the condition as close as possible to that of the living organism.

The value of fixing is:

 To keep the condition of the organism as close as possible to the natural state.
 To prohibit structural damage.
 To preserve cytological elements.
 To harden the tissue.
 To enhance the penetration of the stain.
 To prohibit shrinkage during the preparation procedure.

It is important to fix the samples as quickly as possible. Fixing is done by 4oC or at room
temperature. If the samples float on or near the surface of the fixative, they must be put under
vacuum to remove the air.

20.2.1. Primary fixatives.

This group includes glutaraldehyde (GA) and formaldehyde (FA).

GA is a di-aldehyde and the most effective aldehyde fixative for the preservation of
ultrastructure and have the ability to cross-link proteins and retain lipids.
FA is a mono-aldehyde and is used in the fixation of compact tissue as its penetration ability
is faster than that of GA.
Usually GA and FA are used in combination. Additives that can be used in low
concentrations are picric acid and calcium chloride. Calcium chloride is used for the
stabilisation of membranes.
Examples of fixative mixtures are Todd's, Bullock and Carnovski.
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20.2.2 Secondary fixatives.

Osmium tetroxide is used as a secondary fixative to harden the tissue, as well as an electron
dense stain. It stains tissue black and thus makes it more visible and easier to handle.
Osmium tetroxide vapour is used as fixative for fungi and bacteria and other samples where
aquatic fixatives can not be used. Over-fixation with osmium tetroxide causes protein
extraction.

20.2.3 Tertiary fixatives.

These fixatives react fast with proteins and stabilise structures before extraction takes place.
ea. Uranyl acetate. It can also be used as a stain for thin sections in concentrations of 2-5%.
20.3 Buffers.

Buffers are used for dissolving fixatives and to carry the fixative through the tissue and for
washing of samples between fixations.

To do this the buffer must have the correct:

 pH, usually between 7,2 - 7,6

 osmolarity to prohibit expansion or shrinkage.
 ion concentrations to prohibit extraction or precipitation of substances during fixation.
Examples of buffers are Phosphate -, Sodium cacodylate and Pipes.

When used in washing, it is usually done 3X for 10 - 15 min. depending on the size of the
samples.

20.4 Dehydration.

Following chemical fixation the fixative must be removed from the tissue by way of rinsing.
This is usually done be removing the fixative solution and replacing it with a pure buffer of
the same concentration and pH. Two to three changes of buffer over a period of 10-20 min. is
usually sufficient for removing most of the fixative. Additionally it is useful to rinse out the
buffer with distilled water. This helps to eliminate the possibility that residual salts might
affect polymerisation. The water is removed by a bringing the sample through a graded series
of either ethanol or acetone. Since the shrinkage problems that often accompany dehydration
are more pronounced with sudden changes in solvent concentration it is preferable to have a
number of short exposures to gradually increasing concentrations of solvent.

Generally one of two procedures are followed prior to infiltration and embeddment with
epoxy resins. The first involves bringing the sample through a graded acetone series from 70-
100% up to 100% followed by another change in 100% acetone. There is some evidence that
acetone causes less specimen shrinkage and lipid extraction than does ethanol. It is also non-
reactive with osmium tetroxide and will not interfere with epoxy resin polymerisation.
Phospholipids are particularly immune to extraction by acetone. Acetone is completely
miscible with most epoxy resins and is not know to radically alter protein antigenicity.

The most significant problems caused by dehydration are those of shrinkage and extraction of
cell constituents. For these reasons dehydration times should be kept to a minimum. As with
fixation this process is aided by having small pieces of material in which the time it takes for

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diffusion to occur is reduced. In some cases the dehydration and infiltration schedule can be
shortened to a few hours however these procedures should not be applied in standard fixation
protocols. Some resins can tolerate a small amount of water and one can thus begin the
infiltration process before the dehydration is complete.
Related to dehydration is the problem of infiltration. Like dehydration infiltration involves the
replacement of the solvent with the unpolymerised resin. Because this is a diffusion
dependent event several factors can influence both dehydration and infiltration.
One of course is size of the sample. Another is the use of a slow rotator to keep the sample
moving and always coming in contact with fresh fluid. Placing the sample under vacuum can
also aid in this process.

20.5 Critical-point drying (CPD) for SEM.

After dehydration with 100% acetone or ethanol, the sample holders with samples are placed
in the CPD-chamber filled with the same fluid as used for dehydration. The lid is screwed on
and the dehydration fluid is replaced with the transitional fluid, liquid carbon dioxide. After
repeating this (4-5X), the holder is heated with water to 45oC for the liquid carbon dioxide to
sublimate to carbon dioxide gas. The CO2 gas is slowly released till the CPD-chamber reaches
atmospheric pressure. The samples are then removed for mounting on SEM-stubs.

20.6 Embedding for TEM.

After the 100% dehydration step, the samples are transferred to a 1:1 mixture of resin and
100% acetone or ethanol, depending on which one was used during dehydration, and left for 2
- 4 hours. The samples are then transferred to 100% resin, left till they have sunk to the
bottom of the vials, and left for a further 2-3 hours (this is done twice). This is to ensure that -
the exchange of resin and the acetone is complete. During sample preparation the vials must
be rotated continuously.

20.7 Sectioning for TEM.

The embedded sample blocks are trimmed in the laboratory and not on the microtome. First,
semi-thin sections of ±0,5 µm are made on the microtome, placed on a drop of distilled water
on a microscope slide, dried on a hotplate, stained with 0,5% toluidine blue in 1% boracs over
moderate heat, washed, dried and viewed under a light microscope. This is done to determine
the quality of the fixation and the orientation of the sample.
The sample block is the trimmed further with a scalpel under a stereo microscope. A new
knife is placed in the microtome and ultra thin sections of ±100 nm are made. The sections
are stretched with xilol vapour, oriented with an eyebrow-hair stick, picked up with a 200--
mesh grid and dried on moist filter paper to remove the water.

20.8 Staining with heavy metals for TEM.

In order to visualise a specimen in the TEM one must have contrasting regions of electron
transparency and electron opacity. Just as in light microscopy differences in contrast can be
accentuated through the use of a stain. To be of use in a TEM a stain must have the ability to
stop or strongly deflect the electrons of the electron beam so that they do not contribute to the
final image. The most commonly used stains in electron microscopy are made up of heavy
metal salts. These have atoms of high atomic weight which are especially good at deflecting
electrons.

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Electron staining falls into one of two categories:

1) positive staining in which contrast is imparted to the specimen itself and
2) negative staining in which the area surrounding the specimen is given increased electron
opacity while the specimen itself remains more translucent. We will discuss positive
staining here.

20.8.1 Positive Staining:

We have already discussed one type of positive stain, that being Osmium tetroxide. When
OsO4 reacts with biomolecules in the specimen the Osmium atom serves as a bridge between
the reacted sites. With an atomic weight of 190 it is of sufficient size to effectively deflect
electrons. Because it reacts more readily with lipids than it does with proteins osmium
tetroxide has the added of advantage of being somewhat structure specific positive stain.
The two most commonly used post-fixation positive stains are uranyl acetate (MW = 422) and
lead citrate (MW = 1054) the two heavy metals being uranium and lead respectively. Both
UA and lead citrate are heavy metal salt stains and are both categorised as general or non-
specific stains. Because they are heavy metal salts they are quite toxic and should be handled
and disposed of with great care. UA ions are believed to react with phosphate and amino
groups (found in nucleic acids and certain proteins) while lead ions are thought to bind to
negatively charged molecules such as hydroxyl groups. Because of this ability to stain
different cellular components UA and lead citrate are often used in conjunction with one
another though not simultaneously for reasons we will see in a few minutes.
Positive stains may be applied either prior to embedding or after sectioning. When applied to
the specimen before dehydration this type of staining is referred to as en bloc staining
meaning “on the block.” Because it is prone to forming image degrading precipitates lead
citrate is not used as an en bloc stain. UA on the other hand is a very useful en bloc stain and
is believed by some to actually act as a fixative in its ability to retain structural detail. When
used as an en bloc stain UA is applied to the specimen as a 0.5% - 4.0% aqueous solution
after the initial fixatives (glut and osmium) have been thoroughly rinsed from the specimen.
After several hours in the stain the specimen is dehydrated and infiltrated as normally done.
The dehydration step should not be long as UA is soluble in solvents and extended storage in
a dehydrating agent will remove most of the UA. En bloc staining can greatly improve the
contrast of membranous structures such as mitochondria, golgi, ER, as well as DNA and other
fine filaments.

20.8.2 Post-embedding Staining:

Sections that have been picked up and dried can be stained on their grids. Usually this is done
by floating the grids on a drop of 1%-4% UA for 15-30 minutes. The grids are then
thoroughly rinsed, dried, and either stained with lead citrate or stored until they are examined
in the TEM. Although grids can theoretically be stained any time after sectioning it is best to
do so within 24 hours of having cut the sections. Grids that have been exposed to the energy
of the electron beam will not absorb stain. Some resins are particularly difficult to penetrate
and therefore do not stain well. In these cases one can try to either elevate the temperature of
the stain or by staining in a methanolic UA solution. UA can be dissolved in 100% methanol
and the grids placed into it. All of the steps are the same as for aqueous UA staining with the
exception that the grids must be rehydrated through a graded methanol series before being

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rinsed in dH2O and finally dried. If this is not done the sections will wrinkle badly due to the
temporary disassociation of the sections from the support film.
Lead citrate is often used to stain grids after they have been stained in UA. Because Lead
citrate is very sensitive to CO2 (it quickly reacts to form a precipitate that can ruin a section)
every effort must be made to eliminate this gas from the staining procedure. For this reason
very clean glassware, CO2-free water, and other precautions must be followed in preparing
lead citrate for use. Sections are stained by floating the grids on drops of lead citrate for 3-5
minutes at room temperature. After staining the sections are rinsed in a 1M NaOH solution (to
wash off the lead citrate) and then thoroughly rinsed in dH2O (to rinse off the NaOH). Grids
are then blotted dry and stored until needed.

20.9 Negative staining for TEM.

In order to visualise a specimen in the TEM it is necessary to create what is known as image
contrast. By this we mean that there must exist regions of varying electron opacity such that
differences can be detected and therefore information about the structure of the specimen can
be discerned. This is accomplished by the differential scattering, deflecting, and stopping of
illuminating electrons. For an object to have a level of electron opacity there must be enough
nuclear mass present to accomplish this deflection of electrons. A thick biological specimen
meets this requirement by having a large number of relatively low atomic weight atoms
aligned relative to the incident electron beam.

A second way of accomplishing this is to have a relatively thin layer of high atomic weight
elements aligned relative to the incident beam. This same principle is utilised in the positive
staining of biological specimens to selectively give electron contrast to different portions of
the specimen. The primary distinction between positive and negative staining is that in
positive staining the stain forms a complex with the specimen whereas in negative staining
the stain and the specimen do not react with one another. Also as the name implies a positive
stain will impart increased electron opacity to the specimen creating a darker specimen
whereas in negative staining the specimen remains more electron translucent relative to the
surrounding stain. By pooling up around the edges and crevices of the specimen and not as
much on the top portions of the specimen an image of differential contrast of the specimen
can be made. One of the limitations of negative staining is that only information about the
microtopography of the specimen is produced. Little or nothing is learned about the internal
structure.
A drop of the sample, in suspension, is placed on a Formvar coated grid, a drop of 2%
phosphotungsten acid or uranyl acetate is added, the grid is dried on filter paper, left to air dry
and examined under the TEM.

21. REFERENCES.

1. Oxford Instruments Plc, The Oxford guide to X-ray Microanalysis, CD-ROM Tutorial,
(England, 1997).
2. Philips Electron Optics, All you wanted to know about electron microscopy, but didn’t
know what to ask., 1-17, Netherlands.
3. Philips Electron Optics, Environmental Scanning Electron Microscopy., 2nd Ed, (Robert
Johnson Associates, California, 1996).
4. Microanalysis and Scanning Electron Microscopy, Maurice, F., Menly, L. & Tixtier, R.,
(1978), Summer School, St-Martin-d'Hères, France.

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