Nonconductive specimens collect charge when scanned by the electron beam, and especially in secondary electron imaging mode, this causes scanning faults and other image artifacts. For conventional imaging in the SEM, specimens must be electrically conductive, at least at the surface, and electrically grounded to prevent the accumulation of electrostatic charge. Metal objects require little special preparation for SEM except for cleaning and conductively mounting to a specimen stub. Non-conducting materials are usually coated with an ultrathin coating of electrically conducting material, deposited on the sample either by low-vacuum sputter coating or by high-vacuum evaporation. Conductive materials in current use for specimen coating include gold, gold/palladium alloy, platinum, iridium, tungsten, chromium, osmium, and graphite. Coating with heavy metals may increase signal/noise ratio for samples of low atomic number (Z). The improvement arises because secondary electron emission for high-Z materials is enhanced.
Fixation is usually performed by incubation in a solution of a buffered chemical fixative, such as glutaraldehyde, sometimes in combination with formaldehyde and other fixatives, and optionally followed by postfixation with osmium tetroxide. The fixed tissue is then dehydrated. Because air-drying causes collapse and shrinkage, this is commonly achieved by replacement of water in the cells with organic solvents such as ethanol or acetone, and replacement of these solvents in turn with a transitional fluid such as liquid carbon dioxide by critical point drying. The carbon dioxide is finally removed while in a supercritical state, so that no gas–liquid interface is present within the sample during drying.
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