Living specimens being prepared for scanning electron microscopy first need to be killed and fixed. This is usually done using a chemical fixative, such as glutaraldehyde and osmium. When applied in combination, typically glutaraldehyde fixation is used first. Concentrations of glutaraldehyde typically vary between 2% and 5% and are usually mixed with a buffer that operates at physiological pH. Two common choices, are phosphate buffers and cacodylate buffers, which are typically used at a concentration of approximately 0.05M and a pH of between 6.8 and 7.4. The goal of buffering the fixative is to provide an isotonic solution and to protect the biological tissue from becoming acidic, which frequently occurs during glutaraldehyde cross-linking. Killing may occur within the first several minutes of incubation in glutaraldehyde, but cross-linking of proteins through covalent bonds typically requires several hours to reach saturation in; therefore overnight fixation is not unusual. The specimen is then typically rinsed several times in their respective buffer, and transferred to buffered osmium tetroxide. Typically the same buffer and pH is used. Osmium fixation is usually conducted on ice or in the refrigerator for a shorter period of time, typically two hours. Use of warm osmium over a period of several hours can actually be used to extract some materials and expose internal surfaces by removing membranes. Following osmium fixation, water is chemically extracted from the specimen using a graded series of ethanol. Typically, the concentrations of ethanol used begin at 30% and proceed at 20% steps up to 70% followed by 10% changes to 100% ethanol. The final changes of ethanol must be conducted using anhydrous ethanol. Typically three changes of anhydrous ethanol are used.
A semi-automated Tousimis critical point drying system in our lab. Specimens are dried without a phase change boundary
The ZEISS 960 accepts conventional specimens that are mounted on stubs, which are 1 cm wide discs that have a pin-mount on the base of the disc. Specimens are mounted on to the stub before being made conductive -- usually by sputter coating the specimens with gold/palladium. Because of the palladium content, specimens typically appear silverish. Although the coating is coarse by TEM or JSM-880 standards, the grains cannot be resolved in the DSM-960.
Below are some examples of stubs used for the DSM-960 from the Oklahoma Uglybug Contest which is an annual outreach program for OU's SRNEML, as well as other EM labs around the State. Specimens are kept in a desiccator under vacuum and often with silica beads to keep them dry so that they do not change or decay. Mothballs are used as an effective deterrent for biological activity when sheets of specimens are too large to allow storage in a vacuum.
Specimen storage for conventional SEM is facilitated by using commercially available Plexiglass sheets with holes for the specimen pins. Hundreds of specimens can be mounted, indexed and stored on such a sheet.
Here are some insects from the Oklahoma Uglybug contest at higher magnification.
The JEOL JSM-880 consists of an SEM electronic console that is mated with a TEM column. Accordingly, the specimen carriers must be small enough to fit within the objective lens. Instead of stubs, "boats" are used that are made of bent copper strips that are about an inch long and a cm wide. The working distance is frequently -1 to -3 mm, which limits the space available for imaging.
Below are some examples of boats used for the JSM-880. Because of its high resolution, including material may be visualized on the surface of the specimen, increasing the difficulty of preparing artifact free coatings. The specimen carrier of the JSM-880 is interchangable with that of the JEOL-2000FX TEM, so the same specimen may easily be examined in both microscopes. Boats are a specialty item that is not made by routine EM supply houses and therefor are recycled in the SRNEML.
The maximum size of the area that can be imaged is 1 cm long by 3 mm wide and up to 2 mm thick.
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