Advanced Instrumentation and Methodology Related to Cryoultramicrotomy: A Review

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

Cryofixation and Cryosubstitution for Routine Work in Transmission Electron Microscopy

After a brief review of the present state of the theory of cryofixation, methods and instruments as well as criteria for the application of cryofixation and cryosubstitution in daily routine work in cell biology and medicine are described. Good results are obtainable using liquid nitrogen for impact freezing on highly polished copper surfaces or by plunging into liquefied propane. Based on these results a versatile and safe system for routine plunging and impact freezing for the majority of biomedical objects has been developed. In order to enable ultramicrotomy at ambient temperature a cryosubstitution system according to the Edelmann principle has been de signed and applied

A New Versatile System for Freeze-Substitution, Freeze-Drying and Low Temperature Embedding of Biological Specimens

A universal system for freeze-substitution (FS), freeze-drying (FD) and low temperature embedding (LTE) has been developed, suited to perform standardized procedures of cryoprocessing biological and medical specimens as well as systematic studies of dehydration and embedding at various low and high temperatures. In a 35 I Dewar vessel with 110 mm neck diameter an aluminum tube is mounted to the bottom of the liquid nitrogen (LN2x) reservoir and extends to the lower part of the cylindrical neck. At its top an aluminum plate serves as a contact surface for either the FS chamber or the FD chamber. FS and subsequent LTE are carried out in an environment of dry cold nitrogen gas provided by evaporating nitrogen from the dewar. Different capsules and moulds may be used for cryodehydration and LTE. FD of bulk specimens or cryosections takes place in an absolutely clean vacuum provided by a cryosorption pump integrated in the FD apparatus. Most of the Hp molecules from the frozen specimen are trapped by large cold surfaces inside the drying chamber. Due to the low LN2 consumption during FS or FD (3-4 l LN2/day) both procedures may be carried out for 8-10 days without refilling the dewar. A few representative results show that well frozen biological material is stabilized by prolonged FS or FD at temperatures of about -80°C without use of chemical fixatives like OsO4 in the substitution medium during FS or by OsO4 vapor fixation after FD