Advanced instrumentation and methodology related to cryoultramicrotomy: a review.

Abstract

This review is concerned with the considerable progress in the field of cryo-ultramicrotomy (cryofixation, cryosectioning, investigation and analysis of cryosections) during recent years. This progress includes both more efficient instrumentation and methodology. The article is mainly directed to the investigation and analysis of frozen-hydrated sections in the low dose cryo-transmission electron microscopy (TEM) and cryo-energy filtered TEM (EFTEM). A general survey is followed by an evaluation of the different relevant procedures. Both cryo-ultramicrotomy for macromolecular cytochemistry (Tokuyasu technique) and cryo-ultramicrotomy for element analysis are only shortly mentioned without discussion of the chemical and analytical approach. Because of lack of first hand experience, cryo-sectioning for X-ray microanalysis in the frozen-hydrated state according to Hall and Gupta is not included into this review. The methods and instruments required for ultrathin sectioning at low temperatures are described and discussed in detail. This concerns the preceding cryofixation, the cryosectioning itself with special emphasis to the required stability and precision of the cryo-ultramicrotome, the characteristics of the knives, the charging phenomena due to sectioning and the subsequent TEM investigation including EFTEM with electron spectroscopic imaging (ESI) and the available accessories for digital low dose registration of signals.