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.
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