Sample Preparations
•
For the examination of images of topographic contrast from metal
and ceramic specimens, the only specimen preparation necessary
is to ensure that the specimen is thoroughly degreased so as to
avoid hydrocarbon contamination and , in the case of insulators, to
provide a conductive coating.
•
The techniques for cleaning surfaces include solvent cleaning and
degreasing in an ultrasonic cleaner, mechanical brushing, replica
stripping, and chemical etching. These techniques should be
applied starting with the least damaging one and employing only
the minimum cleaning necessary. Usually the first step is to use a
solvent wash such as acetone, toluene, or alcohol in an ultrasonic
cleaner.
•
If low-voltage microscopy is to be used, the cleaned surface can be
examined without the need for the conductive coating.
•
If the specimen needs to be kept as received, without any
alteration, an environmental SEM may be used.
Assorted SEM mounts.
Assorted SEM specimens.
Media you can use to attach specimens to mounts
Carbon tape is spongy.
Fresh tape
Tape placed in a vacuum
Small blocks, shims, risers,
brackets and other devices
can be added to a mount to
position a specimen.
Clean specimens is especially important. Plasma cleaning can
destroy surface organics that interfere with imaging and analysis.
Specimen sitting in plasma
glow discharge
Surface contaminants, such as oil
or grease, should be removed with
a solvent (acetone), soap and water,
alcohol, and plasma.
Specimens need to be electrically conductive.
Insulators must be coated with a conductive layer.
Sputter coating with metal
Coating with carbon
• Insulating specimens build up an electrostatic
charge.
• The charging leads to variations in surface
potential.
• Results in:
–
–
–
–
deflected secondary electrons
increased secondary emission
deflection of electron beam
spurious x-ray signals
• One solution is to coat the specimen with a thin
conductive layer.
• The coating must provide a path to ground.
• Coats specimen with thin, electrically
conductive film.
• Thermal Evaporation
– Metal wire or particle may be resistance heated and
evaporated under vacuum.
– Line of sight.
– The higher the metal’s melting point and the higher
the vacuum, the finer the coating.
– Carbon can be evaporated through resistance
heating.
• Sputter Coating
– Erosion of metal atoms by an energetic plasma.
– Multidirectional
– Two types for SEM: diode and triode.
Atom arrives
Migration and
re-evaporation
Collisions and
combinations
Nucleation and
islands of atoms
Islands grow
Islands merge
Continuous
coating
Nucleation sites form which
grow and coalesce into a
continuous film.
Small crystals may grow.
Gold and silver form larger
crystals than niobium,
platinum, or chromium.
• Erosion of atoms from a metal target by an energetic
plasma.
• Au, Au/Pd, Pt and Ag are common targets, but Au has
too large of a grain for high resolution SEM.
• Plasma is ionized Ar or N.
• Electrons are ejected from a negative target (glow
discharge).
• Electrons collide with Ar atoms leaving positive ions.
• Positive ions accelerated toward negative target.
• Metal atoms are ejected from the target and attracted
to the specimen.
• Thin coating forms on specimen.
Metal target
(high voltage)
Magnet
Iron
pole piece
Vacuum
jar
Gold atoms
Gas ions
Cooled stage
•
•
•
•
Plasma-Magnetron Sputtering
Annular cathode.
Center permanent magnet
deflects high-energy electrons
away from specimen.
Specimen remains cool.
Iron pole piece
O-ring seal
Target (cathode)
High voltage
line
• An alternative to polishing is the slicing with an
ultramicrotome.
• Good for small, soft specimens.
• We begin with a mold such as a Beam
capsule.
• A small specimen is placed in the bottom,
and the mold is filled with a resin that can
be polymerized.
• The polymerized resin block is removed from mold.
• The specimen should be in the tapered end.
• An identification label should be embedded with the
specimen.
• The end of the block is sliced with a diamond knife in the
ultramicrotome to expose a sectioned specimen surface.
• Powders can be stuck onto carbon tape by inverting the
mount and pressing it on the distributed powder.
Mount with tape
Silt spread on weight boat
Silt on mount
• Fine powders can also be distributed using the shotgun
approach.
A small amount of powder,
placed in the end of a cocktail
straw, is blown onto the
carbon tape.
This technique reduces
clumping and provides a
more even distribution.
Structures in a dissolved substance must be washed free
of the solute before it is dried on a sample mount.
Unwashed sample
Washed sample
Cryofracturing is a way of preparing a specimen for
examination of its interior.
Copper block cooled with liquid nitrogen
Dewar of liquid nitrogen
Critical Point Drying
Critical point dehydration
makes use of the property of
liquids to change from a
liquid phase to a gaseous
phase without the latent heat
of vaporization or density
change.
At the point of critical
temperature and pressure
interfacial tensions are low.
The transition liquid turns
into a gas with little
disruption of sample walls
Critical Point of CO2
• Critical Point: The
combination of
temperature and
pressure at which
the density of the
liquid phase of a
material (e.g.
CO2) equals the
density of the
vapor phase.
31.1o C
1,073 PSI
Critical Point Drying
Critical point drying works on the principle that the dehydrating solvent
which remains in the specimen is replaced with a compound which is
liquid at high pressures and room temperatures and turns to a gas as the
temperature is raised slightly. If this replacement process takes place in a
pressurized container, the pressure will increase as the temperature is
raised and the compound will pass through its critical point. At the
critical point, the phase boundary between the gas and liquid not longer
exists and surface tension is zero. Under these conditions the drying
occurs without specimen distortion. The most convenient compound to
use is carbon dioxide. The critical point drying process is carried out in
specialized apparatus.
•
•
•
•
•
•
•
Biological tissues or soft, wet materials such as polymers may be critical point
dried to retain undistorted structure.
Transitional fluid is usually liquid carbon dioxide.
Specimen must be dehydrated, usually in ethanol.
Specimen is placed in a pressure vessel or “bomb” that is filled with liquid
carbon dioxide.
The liquid carbon dioxide combines with the ethanol.
The temperature is raised until the critical point is reached.
The carbon dioxide gas is gradually released and the specimen is dried.
Critical Point Drying
• Critical Point Drying - Agarose gel
2.0%
0.2%
• Critical Point Drying - Agarose gel
• Dehydrate in ethyl alcohol series
– 30, 50 60, 70, 80, 90, 100, 100 100% EtOH
• Critical Point Drying - Agarose gel
• Dehydrate using a rotator.
• Critical Point Dried Agarose Gel
• Critical Point Dried Agarose Gel
The potted, polished mount
The potted, polished mount
Mixing the resin
Molds for specimen encapsulation
The potted, polished mount
Cutting mount with diamond saw
Sanding to expose sample
The potted, polished mount
Mount clamped in weight
Weight with mount inverted in vibratory polisher
The potted, polished mount
Different polishing slurries
to get mirror finish
Examination on metallograph microscope
Why do we have to clean the specimen surface?
•
To remove contaminants that may have an adverse effect on
secondary electron emission. Because the electron beam can
cause cracking of hydrocarbons, resulting in the deposition of
carbon and other breakdown products on the specimen during
examination.
•
Contamination during operation frequently can be detected in the
form of a “scan square”. It is important to avoid introducing
volatile compounds into the SEM (see Fig. 4.60).
•
Residual hydrocarbons from the diffusion pump’s oil can also
produce contamination under the influence of the beam. This
problem can be minimized by using traps cooled with liquid
nitrogen to condense hydrocarbon vapors.
•
A dirty specimen rather than a dirty vacuum system can also
course contamination. Therefore, Care should always be taken to
handle specimens and specimen holders with gloves to avoid
introducing volatile compounds from fingerprints into the vacuum
system.
Why quantitative x-ray analysis requires a
perfectly smooth surface?
•
To eliminate specimen topography when we desire to work with
weak-contrast mechanism.
•
A weak-contrast mechanism such as electron channeling is
frequently impossible to detect in the presence of a strongcontrast mechanism such as topographic contrast.
•
Chemical polishing or electro-polishing can produce a mirror
surface nearly free from topography in metal specimens.
Metallographic mechanical polishing also removes topography
and gives a high-quality mirror surface, but such mechanical
polishing results in the formation of a shallow layer (~100nm) of
intense damage in most metals.
Why quantitative x-ray analysis requires a
perfectly smooth surface (continue)?
•
The polished layer will completely eliminates electron-channeling
contrast. In magnetic materials the residual stresses in the layer
result in the formation of surface magnetic domains characteristic
of that particular stress state. If we are interested in domains
characteristic of the bulk state of the material, such a residual
stress layer must be avoided. Mechanical polishing to produce a flat
surface followed by brief electropolishing or a chemical treatment to
remove the damaged layer often give optimum results.
•
However, chemical or electrochemical polishing may attacks phase
boundaries. The interface chemistry may also be modified.
•
As we can see, SEM specimen preparation, in general, remains an
art, with each material presenting a different problem to the
investigator.
Specimen Preparation for Surface Topography
•
To cut large specimens to fit the specimen holder as needed.
•
To degrease the specimen in a solvent such as clean acetone, using
an ultrasonic cleaner if the specimen can withstand ultrasonic
vibration without losing important surface material.
•
A final wash with methanol removes any remaining surface film. It is
important to ensure that the solvent does not compromise the
integrity of the surface.
•
The cleaned specimen can then be mounted onto a specimen stub
either mechanically with a clamp or with conductive paint, or
conductive double-sticky tape. If a nonconductive glue is used, a
track of conductive paint should be applied from the specimen to the
stub to ensure good electrical contact.
•
The sample should then be dried in a clean, low temperature (75 °C)
oven. The specimen should never be “pumped dry” in the SEM
chamber.
Bulk Specimens for SEM and X-Ray Microanalysis
Prepare metallic, ceramic, polymeric, and biological specimens:
Equipment
4.
5.
6.
7.
8.
9.
Slicing and wafering equipment
Cut-off saws, wire saws, diamond wheel wafering saws, etc.
Facilities for mounting small specimens in polymeric
compounds.
Metallographic/petrographic polishing equipment (lapping
wheels, polishing wheels, polishing compounds,
electropolishing equipment, etching supplies).
Conductive paint such as silver paint or colloidal graphite.
Coating equipment such as evaporator and sputter coater.
Specimens for Surface Topography Analysis
These specimens are among the easiest to prepare for examination in
the SEM. The key consideration is to insure that the specimen surface
to be clean and undamaged. The following steps will serve as a guide:
•
Cut large specimens to fit specimen holder.
•
Degrease the specimen in a solvent such as clean acetone. An
ultrasonic cleaner is useful. A final wash with methanol will
remove any remaining surface film. Ensure that the solvent does
not compromise the integrity of the surface. Warning: Flammable
solvents in an ultrasonic cleaner may be hazardous. Read all
safety and disposal information pertaining to the solvent.
•
Mount specimen on specimen stub either mechanically or with
glue, conductive paint, or sticky tape. Run a track of conductive
paint from the specimen to the stub to insure good electrical
contact.
(d) Dry specimen in a clean, low-temperature (75 °C) oven. The
sample should never be “pumped dry” in a SEM chamber or
airlock.
(e) Coat specimens that are electrical insulators with a thin
conductive layer using either a sputter coater or an evaporator.
Specimens for Microstructural Morphology Studies
Metallic Specimens:
•
Slice specimen into pieces small enough to be placed into an
appropriate 1-1.5” metallurgical mount. If the cut surface is the
one to be ultimately polished and examined, the cut should be
made with a slow speed diamond saw or slurry wire saw.
•
Mount using standard metallographic practice. Either an epoxy,
cold mount or in some cases fusible metal alloy should be
employed. Although a specimen itself may be conducting, it may
be useful to mount the specimen in a conductive epoxy to
prevent charging during examination.
•
Polish using standard metallographic practice and appropriate
polishing compounds. A typical grinding and polishing sequence
might be 320, 400, 600 SiC papers followed by 3 μm, 0.25 μm
alumina or diamond polishing compounds. This work may be
done by hand or by using rotating wheels. Be sure to carefully
clean the entire mount in soap and water before moving to the
next smaller polishing compound.
4. Etch the surface to bring out the phase structure using
standard chemical, electrochemical, or ion etching
procedures. Rinse and dry thoroughly. Heavy etching may
generate artifacts which could be confused with the true
microstructure. Even without etching, the phase structure
may be apparent in backscatter images as a result of
atomic number contrast. Resolution in BSE imaging mode
(100-300 nm) is generally inferior to that obtainable with
secondary electrons on etched specimens, but still better
than that obtainable by most light optical metallography
(500 nm). Etched specimens should never be used for x-ray
microanalysis.
Polymers, plastics, and other nonhydrated or partially
hydrated organic specimens
They require special handing for examination in the SEM
Equipment:
5.
Microtome, razor blades and scalpels
6.
Fine forceps.
7.
Glassware and chemicals found in a general electron microscopy
preparation laboratory.
Both dehydration in vacuum and electron beam damage can severely
alter the morphology of a polymer surface. At low SEM magnifications
both problems can be avoided by coating the specimen with a thick
(>20 nm) self-supporting layer of gold or Au-Pd. While the basic surface
morphology is preserved in the gold casing, all fine details of the
surface are lost. To retain fine surface details the following steps will be
helpful:
(a) Expose the interior surface for examination. Method for this
include the following:
Fracturing – Brittle polymers will fracture along a surface of least
resistance. The specimen can be trimmed by repeated, more
precise fracturing. Soft materials must be cooled well below their
glass transition temperature to allow them to be fractured. This is
best achieved by immersing specimens in liquid nitrogen and
fracturing by impact. Tough polymers may be fractured and peeled
back along their long axis.
Polished Bulk Specimens – Hard polymers and plastics can often
be polished. If the specimen is very small, it may first need
embedding in an epoxy resin. It is important to check the solubility
of the materials in the resin. The hardened material is cut withy a
diamond saw to produce a flat surface. This surface is then
polished by using standard metallographic procedures.
Sectioning – The aim of this procedure is to produce sections of the
specimen which are thin enough to be examined by a transmitted
beam of electrons or to create a flat surface. Sections may be cut
dry or wet using metal, glass, or diamond knives. Soft plastics,
emulsions, elastomers, and polymers which absorb water, may be
cut by using cryomicrotomy methods. The specimen in the chuck
from which the sections have been cut has been planed to a smooth
finish and may be observed in the SEM, while the sections
themselves may be observed in the TEM.
Etching – There are physical and/or chemical procedures which
selectively remove one or more of the components in a polymer
mixture. Whole molecules of material may be removed by
dissolution. The physical procedures involving plasma, ion, and
electron beam etching are generally less satisfactory than chemical
procedures since they cause many uncontrollable artifacts. The
effectiveness of solvent etching will depend largely on the polymer
or plastic being studies and there is no one general method witch
can be recommended.
(b) Dry the polymer to remove water. Be careful about polymer solubility
in organic fluids.
(C) Mount the specimen on an SEM stub with silver epoxy, conductive
paint, or double-sided adhesive tape painted with a drop of
conductive paint. Be careful that solvents in these preparations do
not rise up onto the specimen by capillary action and degrade the
surface to be imaged.
(d) Coat the specimen with a thin metal film to provide a conductive
path to electrical ground. Since many polymers are heat sensitive,
they should not be exposed to excessive heat during the coating
processes. This is a particular problem with carbon evaporation. An
Au-Pd sputter coater with a cold stage is often useful.
Since the average atomic numbers of various polymeric materials
(containing largely carbon, nitrogen, oxygen, and hydrogen) are very
similar, second phases that contain different bonding arrangements of
these same atoms cannot be distinguished by atomic number contrast.
While heavy metal staining of the second phase is possible, second
phases are most often observed in the SEM as morphological features
in fractures surfaces.
Biological Specimens
Very few bulk biological specimens may be placed directly into the
high vacuum SEM. As with other bulk SEM specimens, biological
specimens must be free of foreign particles, stable in vacuum, stable
in the electron beam, electrically conductive, and must be unaltered in
chemistry and morphology. It is difficult to meet all these criteria at the
same time. Therefore, the following guide does not intend to be all
inclusive.
Necessary Equipment:
10. Freeze dryer, critical point dryer.
11. General glassware and chemicals associated with an electron
microscope laboratory.
(a)
Mount the nonliving specimen on a specimen stub. For insects
allow the legs to touch the stub surface that has been precoated
with a thin layer of glue or conductive paint. For a sliver of wood
or paper join the specimen to the stub with a layer of conductive
paint.
(b) Coat the specimen with a relatively thick layer of gold or Au-Pd
for low magnification observation. Other hard tissues may be
prepared in a manner similar to polymers or ceramics.
Soft Tissue Preparation
Considerably more effort is involved in preserving a soft tissue
specimen that may require fixation and removal of water. Typical
preparation of this type consists of the following:
(8)
Selection and Cleaning – Most specimens may be cut, sliced,
sawed, or fractured, and in addition to reducing specimens to a
suitable size. These procedures also provide one of the best
ways of exposing a clean surface which has the characteristics
of the bulk specimen. The natural or artificially exposed surface
may require further cleaning.
(2) Structural Stabilization – This forms the central part of the
procedures used in preparing most biological and hydrated
material for microscopy and analysis. For structural studies,
the aim is to preserve the macromolecular architecture of the
specimen and methods based on chemical fixation usually
provide the best results. For analytical studies it is necessary
to retain the complete chemical identity of the specimen and
the best results are obtained using low-temperature fixation
which avoids the use of disruptive chemicals. There are many
different recipes and those methods which work well for a
particular specimen examined in the TEM will usually work
equally well for the SEM. Chapter 11 and 12 provide more
details.
(3) Drying – Nearly all biological specimens will need drying
before they may be examined in the electron microscope. The
principal liquid which has to be removed is water, although
some specimens may have other organic liquids. Unless the
specimen is very tough and rigid, e.g., wood, bone, some
seeds, air drying should not be used. Because water has a high
surface tension and as the last traces of the liquid are removed
the surface tension forces which develop will seriously distort
soft and pliable surfaces, therefore, the specimens should be
dried by solvent drying or by critical point drying.
•
Warning – It is important that one is familiar with the operating
procedures for the particular critical point dryer to be used. It is a
potentially dangerous procedure as it involves the use of gases at
pressures up to 1200 psi. Be sure to read the safety instruction
supplied by the manufacturer.
(4) Coating the specimen to prevent charging.
Particles and Fibers
Equipment
8.
Conductive paints (I.e., silver, aluminum, carbon).
9.
Particle dispersant equipment (evaporative fluid., freon).
10. Coating equipment (evaporator, sputter coater).
For large particles specimens:
A simple but effective technique for entrapping free standing
particles is to place a drop of carbon paint on a carbon
substrate and spread the drop to form a layer. The solvent of the
carbon paint is then allowed to evaporate to near dryness. While
the paint is still lightly tacky, the particles or fibers are simply
dropped on the surface. The momentum form falling will embed
the particle into the carbon paint. Alternatively, particles and
fibers may be placed on the surface using fine forceps or an
eyelash probe viewed through a binocular microscope. It is
important that the carbon paint should not be so wet that the
solvent and colloidal carbon can wick up onto the particle
surfaces. This method works best with large particles
(dimensions > 10 mm).
For small particle specimens
For very small (<5 µm diameter) particles, there is an alternative but
still fairly simple approach for mounting the particles on carbon
planchets. For particles smaller than about 40 µm in diameter, the
adhesion due to surface charge in addition to the adhesion provided
by the carbon coat is adequate to keep most particles in place. A
small amount of specimen of fine particles can be dispersed on a
carbon planchet by:
•
placing the particles in a very dilute suspension in a fast-evaporating
solvent such as Freon;
•
ultrasonicating the solution to keep the particles in suspension;
•
pipeting a small aliquot of the particle suspension onto the carbon
planchet and allowing the solution to evaporate.
The faster the solution evaporates, the less particle aggregation will occur. You
can assist evaporation by placing the planchet and particle suspension under a
heat lamp, although this may damage organic and biological specimens. When
the planchet is completely dry, it should be carbon coated. The planchet should
be made of high-purity graphite and polished to as smooth a surface as
possible to act as fine particle substrate. Pyrolitic graphite planchets are
particularly good as substrates for particles.