Drying the Specimen

Before specimens can be viewed in the conventional SEM, they must be completely dried because the high vacuum conditions in the SEM chamber will cause hydrated specimens to boil, thereby destroying the integrity of the specimen surface. Specimens may be dried in a variety of ways, depending on the nature of the specimen (e.g., intact whole organisms, cell suspensions, excised portions). If there is any doubt as to the proper procedure to follow, especially with an important specimen, critical point drying is the safest method to use.

3.5.1. Air Drying

Air-drying may be used with some unfixed, hardy specimens such as insects or botanical specimens such as seeds or pollen (see Fig. 5). Some bacteria, such as the thick-walled Gram-positive organisms, may also be air-dried following chemical fixation and dehydration in ethanol. Air-drying can be achieved several ways:

• Put unfixed specimens in a drying oven at 30 to 40°C for several days to weeks. This works well with insects with exoskeletons, some botanical specimens, and possibly some bacteria and fungi.

• Air-dry certain chemically fixed and ethanol-dehydrated specimens using a solvent with high vapor pressure such as hexamethyldisilazane.

Fig. 5. Pollen from day lily. Pollen was shaken onto cover glass, air-dried and sputter coated as described in Subheading 3.5.1. Bar = 120 pm. (Courtesy of Steve Schmitt).

3.5.1.1. Basic Steps for SEM Preparation of Air-Dried Specimens

1. Clean specimen surfaces and fix in glutaraldehyde/osmium fixatives following the standard schedule presented earlier in this chapter (see Subheading 3.3.1.).

2. Dehydrate in ethanol series up to absolute ethanol.

3. Transfer specimen into hexamethyldisilazane for 5 to 10 min.

4. Air dry specimen in dust free environment at room temperature or in a drying oven.

5. Mount specimen on SEM stubs, coat with heavy metal, and examine in SEM.

6. Store specimens in dry, dust free environment.

3.5.2. Critical Point Drying (CPD)

CPD is the method most commonly used to complete the drying of chemically fixed and dehydrated specimens (see Note 12).

1. Transfer small specimens into holding devices (see Fig. 6) to prevent loss during CPD process. This step may be accomplished at any point after fixation in osmium tetroxide.

2. After the final change of absolute ethanol, place 10 to 15 mL of absolute ethanol in the prechilled chamber of the critical point dryer and quickly transfer specimens into the chamber.

Fig. 6. Small holders used to protect small specimens during the critical point drying procedure. Inset, top left corner, shows holder fashioned from polypropylene embedding mold used in transmission electron microscopy. Large, rectangular container is a microscope slide mailer that has been modified to hold slides and cover slips. The three sets of stainless-steel mesh holders are commercially available.

Fig. 6. Small holders used to protect small specimens during the critical point drying procedure. Inset, top left corner, shows holder fashioned from polypropylene embedding mold used in transmission electron microscopy. Large, rectangular container is a microscope slide mailer that has been modified to hold slides and cover slips. The three sets of stainless-steel mesh holders are commercially available.

3. Seal the chamber and fill with liquid CO2, and maintain the recommended cold temperature, usually 0°C.

4. After several changes of liquid CO2, to completely displace ethanol, raise the temperature of the CPD device to the critical point. For liquid CO2, the critical point is 31.1°C at 1073 PSI.

5. After the liquid CO2 is converted to gas, release the pressure while maintaining the elevated temperature to prevent re-condensation of the liquid CO2.

6. Remove the fragile specimens, mount on specimen stubs, coat with heavy metal and view in the SEM.

7. After viewing, store specimens in dry, dust free environment.

3.5.3. Freeze-Drying

Freeze-drying is occasionally used on specimens that would be damaged by the CPD procedure. Although chemically fixed specimens are often used, this

Fig. 7. Adhesives used to hold specimens on scanning electron microscopy stubs. These include conductive paints containing carbon or silver, conductive tapes containing carbon, sticky tabs (shown in lower left) that transfer a dab of adhesive material onto the stub, and carbon, double-stick disks.

is normally not needed since rapid freezing preserves the ultrastructure to a depth of 5 to 10 pm below the surface.

1. Rapidly freeze the specimen by plunging into isopentane or absolute ethanol that is chilled in liquid nitrogen to approx -85°C.

2. Transfer the specimen into liquid nitrogen (for storage) and then onto the -75°C cold stage of the freeze drying apparatus and activate the vacuum.

3. After several hours or days, depending on the mass of the specimen, warm the specimen stage to room temperature.

4. Remove specimens, mount on stubs, coat with heavy metal and view in the SEM. Store specimens in a dry, dust free environment.

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