You are on page 1of 59

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. brackets and other devices can be added to a mount to position a specimen. risers. . shims.

Specimen sitting in plasma glow discharge .Clean specimens is especially important. Plasma cleaning can destroy surface organics that interfere with imaging and analysis.

soap and water. such as oil or grease. . alcohol. and plasma.Surface contaminants. should be removed with a solvent (acetone).

Sputter coating with metal Coating with carbon .Specimens need to be electrically conductive. Insulators must be coated with a conductive layer.

• The coating must provide a path to ground. • 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 charging leads to variations in surface potential.• Insulating specimens build up an electrostatic charge. .

– Line of sight.• Coats specimen with thin. . electrically conductive film. the finer the coating. – Multidirectional – Two types for SEM: diode and triode. – The higher the metal’s melting point and the higher the vacuum. • Sputter Coating – Erosion of metal atoms by an energetic plasma. • Thermal Evaporation – Metal wire or particle may be resistance heated and evaporated under vacuum. – Carbon can be evaporated through resistance heating.

.

. Gold and silver form larger crystals than niobium. Small crystals may grow. platinum. or chromium.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.

Pt and Ag are common targets. • Metal atoms are ejected from the target and attracted to the specimen. • Plasma is ionized Ar or N.• Erosion of atoms from a metal target by an energetic plasma. Au/Pd. • Positive ions accelerated toward negative target. but Au has too large of a grain for high resolution SEM. • Electrons collide with Ar atoms leaving positive ions. • Thin coating forms on specimen. • Electrons are ejected from a negative target (glow discharge). • Au. .

Specimen remains cool.Metal target (high voltage) Magnet Iron pole piece Vacuum jar Gold atoms Gas ions Cooled stage • • • • Plasma-Magnetron Sputtering Annular cathode. Iron pole piece Target (cathode) O-ring seal High voltage line . Center permanent magnet deflects high-energy electrons away from specimen.

soft specimens. .• An alternative to polishing is the slicing with an ultramicrotome. • Good for small.

• A small specimen is placed in the bottom.• We begin with a mold such as a Beam capsule. . and the mold is filled with a resin that can be polymerized.

• The specimen should be in the tapered end. • An identification label should be embedded with the specimen.• The polymerized resin block is removed from mold. .

• The end of the block is sliced with a diamond knife in the ultramicrotome to expose a sectioned specimen surface. .

Mount with tape Silt spread on weight boat Silt on mount .• Powders can be stuck onto carbon tape by inverting the mount and pressing it on the distributed powder.

This technique reduces clumping and provides a more even distribution. A small amount of powder.• Fine powders can also be distributed using the shotgun approach. . placed in the end of a cocktail straw. is blown onto the carbon tape.

Unwashed sample Washed sample .Structures in a dissolved substance must be washed free of the solute before it is dried on a sample mount.

Copper block cooled with liquid nitrogen Dewar of liquid nitrogen .Cryofracturing is a way of preparing a specimen for examination of its interior.

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. The transition liquid turns into a gas with little disruption of sample walls . At the point of critical temperature and pressure interfacial tensions are low.

Critical Point of CO2 • Critical Point: The combination of temperature and pressure at which the density of the liquid phase of a material (e. 31.073 PSI .g. CO2) equals the density of the vapor phase.1o C 1.

Under these conditions the drying occurs without specimen distortion. the pressure will increase as the temperature is raised and the compound will pass through its critical point. The carbon dioxide gas is gradually released and the specimen is dried. Specimen must be dehydrated. If this replacement process takes place in a pressurized container. wet materials such as polymers may be critical point dried to retain undistorted structure. Specimen is placed in a pressure vessel or “bomb” that is filled with liquid carbon dioxide. Transitional fluid is usually liquid carbon dioxide. At the critical point. usually in ethanol. The most convenient compound to use is carbon dioxide. the phase boundary between the gas and liquid not longer exists and surface tension is zero.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. The liquid carbon dioxide combines with the ethanol. • • • • • • • Biological tissues or soft. The temperature is raised until the critical point is reached. The critical point drying process is carried out in specialized apparatus. .

Critical Point Drying .

Agarose gel 2.0% 0.2% .• Critical Point Drying .

50 60. 80. 100 100% EtOH . 90. 70.• Critical Point Drying .Agarose gel • Dehydrate in ethyl alcohol series – 30. 100.

.Agarose gel • Dehydrate using a rotator.• Critical Point Drying .

• Critical Point Dried Agarose Gel .

• Critical Point Dried Agarose Gel .

The potted. polished mount .

polished mount Mixing the resin Molds for specimen encapsulation .The potted.

polished mount Cutting mount with diamond saw Sanding to expose sample .The potted.

The potted. polished mount Mount clamped in weight Weight with mount inverted in vibratory polisher .

polished mount Different polishing slurries to get mirror finish Examination on metallograph microscope .The potted.

4.Why do we have to clean the specimen surface? • To remove contaminants that may have an adverse effect on secondary electron emission.60). Care should always be taken to handle specimens and specimen holders with gloves to avoid introducing volatile compounds from fingerprints into the vacuum system. It is important to avoid introducing volatile compounds into the SEM (see Fig. This problem can be minimized by using traps cooled with liquid nitrogen to condense hydrocarbon vapors. Because the electron beam can cause cracking of hydrocarbons. • • • . Therefore. resulting in the deposition of carbon and other breakdown products on the specimen during examination. Residual hydrocarbons from the diffusion pump’s oil can also produce contamination under the influence of the beam. Contamination during operation frequently can be detected in the form of a “scan square”. A dirty specimen rather than a dirty vacuum system can also course contamination.

A weak-contrast mechanism such as electron channeling is frequently impossible to detect in the presence of a strongcontrast mechanism such as topographic contrast. 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? • To eliminate specimen topography when we desire to work with weak-contrast mechanism. 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.

.

Why quantitative x-ray analysis requires a perfectly smooth surface (continue)? • The polished layer will completely eliminates electron-channeling contrast. As we can see. SEM specimen preparation. remains an art. Mechanical polishing to produce a flat surface followed by brief electropolishing or a chemical treatment to remove the damaged layer often give optimum results. in general. However. with each material presenting a different problem to the investigator. The interface chemistry may also be modified. 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. chemical or electrochemical polishing may attacks phase boundaries. • • . such a residual stress layer must be avoided.

a track of conductive paint should be applied from the specimen to the stub to ensure good electrical contact. using an ultrasonic cleaner if the specimen can withstand ultrasonic vibration without losing important surface material.Specimen Preparation for Surface Topography • • To cut large specimens to fit the specimen holder as needed. The sample should then be dried in a clean. or conductive double-sticky tape. It is important to ensure that the solvent does not compromise the integrity of the surface. To degrease the specimen in a solvent such as clean acetone. • • • . The specimen should never be “pumped dry” in the SEM chamber. A final wash with methanol removes any remaining surface film. low temperature (75 °C) oven. The cleaned specimen can then be mounted onto a specimen stub either mechanically with a clamp or with conductive paint. If a nonconductive glue is used.

Coating equipment such as evaporator and sputter coater. Slicing and wafering equipment Cut-off saws. 5. wire saws. Conductive paint such as silver paint or colloidal graphite. 8. 9.Bulk Specimens for SEM and X-Ray Microanalysis Prepare metallic. etching supplies). etc. ceramic. . and biological specimens: Equipment 4. 7. polishing compounds. 6. Metallographic/petrographic polishing equipment (lapping wheels. polymeric. electropolishing equipment. diamond wheel wafering saws. Facilities for mounting small specimens in polymeric compounds. polishing wheels.

conductive paint. An ultrasonic cleaner is useful. or sticky tape. A final wash with methanol will remove any remaining surface film. Ensure that the solvent does not compromise the integrity of the surface. Read all safety and disposal information pertaining to the solvent. 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. Run a track of conductive paint from the specimen to the stub to insure good electrical contact. The key consideration is to insure that the specimen surface to be clean and undamaged. Mount specimen on specimen stub either mechanically or with glue.Specimens for Surface Topography Analysis These specimens are among the easiest to prepare for examination in the SEM. Warning: Flammable solvents in an ultrasonic cleaner may be hazardous. • .

(e) Coat specimens that are electrical insulators with a thin conductive layer using either a sputter coater or an evaporator.(d) Dry specimen in a clean. . The sample should never be “pumped dry” in a SEM chamber or airlock. low-temperature (75 °C) oven.

the cut should be made with a slow speed diamond saw or slurry wire saw. 0. it may be useful to mount the specimen in a conductive epoxy to prevent charging during examination. Although a specimen itself may be conducting. • Mount using standard metallographic practice. 600 SiC papers followed by 3 μm. Be sure to carefully clean the entire mount in soap and water before moving to the next smaller polishing compound.25 μm alumina or diamond polishing compounds. If the cut surface is the one to be ultimately polished and examined. A typical grinding and polishing sequence might be 320. Polish using standard metallographic practice and appropriate polishing compounds.5” metallurgical mount. cold mount or in some cases fusible metal alloy should be employed. Either an epoxy. This work may be done by hand or by using rotating wheels.Specimens for Microstructural Morphology Studies Metallic Specimens: • Slice specimen into pieces small enough to be placed into an appropriate 1-1. • . 400.

. Heavy etching may generate artifacts which could be confused with the true microstructure. or ion etching procedures. 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. Even without etching. electrochemical.4. Etch the surface to bring out the phase structure using standard chemical. the phase structure may be apparent in backscatter images as a result of atomic number contrast. Rinse and dry thoroughly.

Fine forceps. Glassware and chemicals found in a general electron microscopy preparation laboratory. Microtome. While the basic surface morphology is preserved in the gold casing. 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. and other nonhydrated or partially hydrated organic specimens They require special handing for examination in the SEM Equipment: 5. Both dehydration in vacuum and electron beam damage can severely alter the morphology of a polymer surface. all fine details of the surface are lost. 7. plastics.Polymers. To retain fine surface details the following steps will be helpful: . razor blades and scalpels 6.

This surface is then polished by using standard metallographic procedures. it may first need embedding in an epoxy resin.(a) Expose the interior surface for examination. It is important to check the solubility of the materials in the resin. more precise fracturing. Method for this include the following: Fracturing – Brittle polymers will fracture along a surface of least resistance. . If the specimen is very small. Polished Bulk Specimens – Hard polymers and plastics can often be polished. Tough polymers may be fractured and peeled back along their long axis. The hardened material is cut withy a diamond saw to produce a flat surface. This is best achieved by immersing specimens in liquid nitrogen and fracturing by impact. Soft materials must be cooled well below their glass transition temperature to allow them to be fractured. The specimen can be trimmed by repeated.

Soft plastics. and electron beam etching are generally less satisfactory than chemical procedures since they cause many uncontrollable artifacts. elastomers. . and polymers which absorb water. ion. 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. The physical procedures involving plasma. Whole molecules of material may be removed by dissolution. glass. or diamond knives. while the sections themselves may be observed in the TEM. Sections may be cut dry or wet using metal. 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. may be cut by using cryomicrotomy methods. Etching – There are physical and/or chemical procedures which selectively remove one or more of the components in a polymer mixture.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. emulsions.

Be careful that solvents in these preparations do not rise up onto the specimen by capillary action and degrade the surface to be imaged. An Au-Pd sputter coater with a cold stage is often useful.(b) Dry the polymer to remove water. Since many polymers are heat sensitive. . (d) Coat the specimen with a thin metal film to provide a conductive path to electrical ground. This is a particular problem with carbon evaporation. oxygen. While heavy metal staining of the second phase is possible. (C) Mount the specimen on an SEM stub with silver epoxy. and hydrogen) are very similar. nitrogen. Be careful about polymer solubility in organic fluids. Since the average atomic numbers of various polymeric materials (containing largely carbon. or double-sided adhesive tape painted with a drop of conductive paint. they should not be exposed to excessive heat during the coating processes. second phases that contain different bonding arrangements of these same atoms cannot be distinguished by atomic number contrast. second phases are most often observed in the SEM as morphological features in fractures surfaces. conductive paint.

It is difficult to meet all these criteria at the same time. Therefore. 11. As with other bulk SEM specimens. stable in vacuum. critical point dryer. General glassware and chemicals associated with an electron microscope laboratory. Freeze dryer. biological specimens must be free of foreign particles. stable in the electron beam. Necessary Equipment: 10. and must be unaltered in chemistry and morphology. electrically conductive.Biological Specimens Very few bulk biological specimens may be placed directly into the high vacuum SEM. . the following guide does not intend to be all inclusive.

(a) Mount the nonliving specimen on a specimen stub. and in addition to reducing specimens to a suitable size. For insects allow the legs to touch the stub surface that has been precoated with a thin layer of glue or conductive paint. These procedures also provide one of the best ways of exposing a clean surface which has the characteristics of the bulk specimen. (b) Coat the specimen with a relatively thick layer of gold or Au-Pd for low magnification observation. or fractured. . sliced. The natural or artificially exposed surface may require further cleaning. Typical preparation of this type consists of the following: (8) Selection and Cleaning – Most specimens may be cut. sawed. Soft Tissue Preparation Considerably more effort is involved in preserving a soft tissue specimen that may require fixation and removal of water. Other hard tissues may be prepared in a manner similar to polymers or ceramics. For a sliver of wood or paper join the specimen to the stub with a layer of conductive paint.

Chapter 11 and 12 provide more details. 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.(2) Structural Stabilization – This forms the central part of the procedures used in preparing most biological and hydrated material for microscopy and analysis. 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. 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. .

Unless the specimen is very tough and rigid. e. therefore. 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.g. some seeds. the specimens should be dried by solvent drying or by critical point drying. The principal liquid which has to be removed is water. although some specimens may have other organic liquids.. .(3) Drying – Nearly all biological specimens will need drying before they may be examined in the electron microscope. air drying should not be used. bone. wood.

Be sure to read the safety instruction supplied by the manufacturer. carbon).. silver. .. sputter coater).e. Particles and Fibers Equipment 8. (4) Coating the specimen to prevent charging. aluminum. Conductive paints (I. Coating equipment (evaporator. 9. freon). Particle dispersant equipment (evaporative fluid. It is a potentially dangerous procedure as it involves the use of gases at pressures up to 1200 psi.• Warning – It is important that one is familiar with the operating procedures for the particular critical point dryer to be used. 10.

. Alternatively. the particles or fibers are simply dropped on the surface.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. This method works best with large particles (dimensions > 10 mm). 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. While the paint is still lightly tacky. particles and fibers may be placed on the surface using fine forceps or an eyelash probe viewed through a binocular microscope. The momentum form falling will embed the particle into the carbon paint. The solvent of the carbon paint is then allowed to evaporate to near dryness.

.For small particle specimens For very small (<5 µm diameter) particles. the less particle aggregation will occur. When the planchet is completely dry. • ultrasonicating the solution to keep the particles in suspension. The planchet should be made of high-purity graphite and polished to as smooth a surface as possible to act as fine particle substrate. For particles smaller than about 40 µm in diameter. The faster the solution evaporates. • pipeting a small aliquot of the particle suspension onto the carbon planchet and allowing the solution to evaporate. Pyrolitic graphite planchets are particularly good as substrates for particles. there is an alternative but still fairly simple approach for mounting the particles on carbon planchets. You can assist evaporation by placing the planchet and particle suspension under a heat lamp. it should be carbon coated. 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. although this may damage organic and biological specimens. the adhesion due to surface charge in addition to the adhesion provided by the carbon coat is adequate to keep most particles in place.