ADVANTAGES AND APPLICATIONS OF LOW TEMPERATURE SCANNING ELECTRON MICROSCOPY

Authors: WERGIN, WILLIAM; YAKLICH, ROBERT; ERBE, ERIC

Submitted to: Focus on Modern Microscopy
Publication Type: Monograph
Publication Acceptance Date: 5/28/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Plant disease-causing organisms cause billions of dollars of crop losses each year in the United States. Microscopic examination of these organisms can reveal new information that can be used to develop new measures of controlling them. Before biological material can be observed in a powerful microscope known as a scanning electron microscope (SEM), the samples must be subjected to many chemical processing procedures. Although these procedures are widely used, they cause artifacts by failing to preserve some structures, dissolving fats and carbohydrates, and causing shrinkage and distortion. These artifacts have now been avoided by placing the specimens on a cold stage, which can be used at temperatures below -300F. This new procedure enabled specimens to be rapidly frozen and then observed and photographed in their frozen state. The procedure permitted observation of samples that normally would be dissolved, injured, separated, or altered by either the water or the time required for normal preparation. The SEM, on which this stage is installed, can magnify samples more than 100,000x. This technique is a valuable new research tool that will enable research scientists to gain new useful information about the structure of disease organisms that are important in agriculture, information that will be used to develop new strategies of plant disease control.

Technical Abstract: Low temperature scanning electron microscopy (LTSEM), which enables observations of frozen hydrated samples, permits imaging of some specimens that cannot be processed or properly preserved for ambient temperature SEM studies. Because LTSEM specimen preparation merely consists of cryofixation, artifacts arising from chemical fixation, solvent dehydration and critical point drying are averted. The cryotechnique is particularly useful for observing specimens that are i) soluble in aqueous fixatives or dehydrating solvents, ii) impermeable to chemical fixatives, iii) delicate and thereby subject to mechanical damage, iv) loosely associated or easily dislodged, or v) physiologically altered by either the water or the time that is required for conventional chemical fixation. LTSEM also allows comparisons of freeze-fractured and freeze-etched faces of a specimen; this advantage can be used to determine the location of the water in the specimen and to distinguish aqueous areas from naturally occurring air pockets and non-etchable solids. In the past, the resolution of frozen hydrated samples was limited by conventional SEM; however, use of cryotechniques on a field emission SEM enables imaging of macromolecular structures such as membrane particles which measure less than 10 nm.