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Preparing samples for scanning electron microscopy

Scanning electron microscopy is typically done in a high vacuum, as gas molecules interfer with the electron beam and with the emitted secondary and backscattered electrons used for imaging. There are particular instances when a low-vacuum or environmental SEM can be used, or is required, but these instruments necessarily trade off resolution to be able to work with gas in the sample chamber.

Specimens for SEM must then be prepared for the high-vacuum imaging environment. The preparation procedures depend upon both the samples being examined and the aim of the study. Biological specimens, such as cells and tissues or tissue components, must first be fixed to preserve their native structure. Some samples, such as hard tissues like bone or teeth, and organisms with a tough exoskeleton, such as some arthropods, can be studied without any preparation, but these are the exception.

Fixation is done either by chemical or physical means. Chemical fixation is the standard with which most people are familiar, and typically uses formalin or glutaraldehyde of varying per cent concentrations in a buffer of a specific pH and osmolarity. Physical fixation may be by heat (such as boiling an egg), but is more commonly done by freezing. Freeze-fixation (=cryofixation) is done in several different ways, but the best ways are by plunging the specimen into slush nitrogen -- liquid nitrogen cooled to its freezing point -- or by high-pressure freezing.

Hydrated samples, like most biological and some materials specimens, must first be dehydrated before placing the specimen in the SEM sample chamber. This is typically done by passing the specimens through a graded series of ethanol-water mixtures to 100% EtOH, and then drying the samples by the critical-point method. Freeze-fixed samples may also be freeze-dried (but not in a lyophilizer).

The details of the different fixation, dehydration, and drying procedures, how big the samples shoud be, and so on, are all dependent on the samples and the questions being asked. Please feel free to make an appointment with BBPIC staff for a no-charge consultation on specimen preparation before beginning your study

The BBPIC has several instruments for preparing samples for SEM. These are service instruments for use by any group that needs them. Sample preparation can be done by BBPIC staff, or investigators may be trained for independent use.

Bal-Tec HPF-010 high-pressure freezer
This instrument freeze-fixes small (about TEM grid-sized) samples at 2100 bars pressure and liquid nitrogen temperatures. This produces the greatest depth of vitreous freezing, up to 500 micrometers in ideal cases.

Slush-nitrogen plunge freezing
Slush nitrogen is made by pulling a vacuum on a container of liquid nitrogen. This is easily done in any laboratory with a vacuum desiccator and a high-speed vacuum pump (a 2-stage rotary pump is the minimum needed). Users can do this using BBPIC equipment, or after training, in their own laboratory.

Bal-Tec MED-010 cryocoater
Freeze-fixed samples may then be coated in the cryocoater. The upper few micrometers of water are first sublimated, and then the specimen is coated with a thin layer of platinum (or other metal) to provide conductivity and some protection from beam damage. The samples are held in a Gatan cryostage for the coating and examination in the Hitachi S-900 FESEM. Freeze-fixed samples may also be freeze-dried, coated in one of the other coaters, and then examined at room temperature in the Hitachi LaB6 SEM.

Lab-built freeze dryer
Freeze-fixed specimens may be dried by sublimation of the frozen water in a vacuum at -90 to -60 degrees Celsius. This procedure removes the fluid from the samples without exposing them to any surface tension forces. Surface tension is what turns a grape into a raisin on drying, and freeze drying one way to avoid this problem.

Tousimis Samdri 780 and lab-built critical point dryers (CPD)
After chemical fixation and ethanol dehydration, specimens may then dried by the critical-point method. This procedure removes the fluid from the samples without exposing them to any surface tension forces. Drying by the critical point procedure is another way to avoid subjecting samples to surface tension forces.

SeeVac Auto conductavac IV sputter coater
Many biological and materials specimens are insulators. This means that when they are bombarded by the electron beam in a SEM, the electrons have no where to go and cause serious imaging problems. Further, the electron beam may damage the sample. These problems are reduced or eliminated by coating the specimen with a thin layer of metal, such as gold, or a 60/40 gold/palladium alloy (as used by the BBPIC). The layer deposited is typically 10 to 20 nanometers thick, and nearly evenly coats the surface of the specimen, faithfully reflecting the surface morphology.

VCR IBS/TM200S ion-beam coater
Some samples to be studied at high resolution - over 50,000X and up to 400,000X - are instead coated using the ion-beam coater. This produces much thinner layers, down to 1 nm thickness, and uses platinum, which gives a finer coat. This is also useful for samples such as cells labeled with gold-conjugated antibodies. The thin layer of platinum allows the gold particles conjugated to the antibodies to be easily seen. (See images on the S-900 FESEM and colloids gallery pages.)

Training & scheduling instrument use:
Please contact Ralph Albrecht or Joe Heintz for further information.