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.
Hermann, René and Müller, Martin
"Prerequisites of High Resolution Scanning Electron Microscopy,"
Scanning Microscopy: Vol. 5
, Article 8.
Available at: https://digitalcommons.usu.edu/microscopy/vol5/iss3/8