Scanning Microscopy


Cryotechniques must be employed throughout all preparation and observation steps in order to extract high resolution scanning electron microscopical information from biological material.

Cryoimmobilization, followed by freeze-drying and metal-shadowing at low temperature, yields optimal structural information of T4 polyheads used as a test specimen. Freeze-substitution of frozen T4 polyheads and subsequent freeze-drying renders the substructures recognizable but less crisp than freeze-drying from aequous solutions. Critical point drying of ethanol dehydrated chemically fixed, or freeze-substituted test specimens results in complete loss of discrete polyhead structure.

In-lens field-emission scanning electron microscopes and highly sensitive electron detectors are instrumental prerequisites in achieving transmission electron microscope-like resolution of structural details. Shape and size of fine structures, of the test specimen, are accurately imaged at high acceleration voltage ( > 7 kV). Precise localization of antigenic sites via ultra-small (0.8 nm) colloidal gold marker systems by backscattered electrons also depends on the appropriate choice of acceleration voltage.

Contamination of the specimen surface is a serious problem in high resolution scanning electron microscopy. It can be controlled in practice, by photographing selected areas of cooled specimens during the first scan of the electron beam.

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