Preparation of cryosections with a modified Sorvall MT2B ultramicrotome and cryoattachment

Abstract

SUMMARY The Sorvall MT2B ultramicrotome and cryoattachment were modified to extend the duration of cutting and to overcome problems in tissue preparation. Microtome and cryochamber were encased and surrounded by an atmosphere of dry nitrogen gas at room temperature. A specially designed cold block sited within the box close to the microtome enabled tissues to be trimmed at low temperatures in the dry environment. Tissues could be readily visualized both on the trimming block and in the chamber with the existing microscope modified to improve its working distance and lighting system. During transfer from storage in liquid nitrogen, through the trimming procedure, to arrival in the cryochamber, the temperature of tissues as determined using thermocouples embedded in PVP on a stub, never exceeded 140 K. Although there was a considerable standing temperature gradient within the cryochamber, the cutting environment, specimen and edge of the knife were thermally stable. Sections could be routinely cut dry and freeze‐dried within the chamber, if desired.

Optimum conditions for cryoquenching of small tissue blocks in liquid coolants

“…Although most X-ray microanalyses of cryocut sections to date have been done on sections cut from more superficial layers, a growing number of studies is being made of tissues in the presence of ice-crystal damage (Koepsell et al, 1974;Cameron et al, 1979;Bulger et al, 1981). Cryosections (0.5-1 pm) somewhat thicker than can be cut from more superficial regions must be used, but our own preliminary findings confirm that, provided a probe diameter larger than the ice-crystal artefacts is used, intracellular and extracellular ionic concentrations of diffusible inorganic ions are retained (Biddlecombe et al, 1982). Critical X-ray microanalytical studies remain to be done comparing the distribution and concentrations of diffusible elements between superficial and deep sites in cryofixed tissue blocks of a suitable test tissue to assess the known osmotic concentration and 'solution effects' of freezing injury (Mazur, 1977;Meryman et al, 1977).…”

“…Care was taken that the PVP did not come into contact with the area of interest in the tissue sample protruding from the PVP. The stubs are convenient for handling during transfer from quenchant to storage in LNz and they can be inserted directly into the microtome head of a Sorval MT2B ultramicrotome with FTS cryokit (Biddlecombe et al, 1982). Samples were normally quenched 15-20 s after mounting on the stubs.…”

“…0 1982 The Royal Microscopical Society Generally, where structural or chemical examination is the objective and cell survival is not a consideration, very rapid cooling techniques are the chosen methods (Mazur, 1977). Our present interest stems from the desire to apply quantitative X-ray microanalytical methods to the study of the distribution of labile and diffusible substances, and particularly inorganic ions, during physiological processes (Dempster et al, 1980a, b;Nicholson & Dempster, 1980;Biddlecombe et al, 1982).…”

X‐ray microanalysis of freeze‐dried and frozen‐hydrated cryosections

“…Cryotransfer Several cryotransfer devices for frozen specimens have been described in the literature (Biddlecombe et al, 1982;Gupta et al, 1977;Hax and Lichtenegger, 1982;Heide, 1981;Heide and Grund, 1974;Hutchinson et al, 1978;Ross et al, 1981;Talmon et al, 1979;Zierold et al, 1981). All these cryotransfer systems are designed for the transfer of a frozen-hydrated specimen into the electron microscope.…”

Cryotechniques in Biological Electron Microscopy

“…This is performed by means of a transportable cold chamber, which can be attached to the cryoultramicrotome and the electron microscope (Ross et al 1981;Zierold et al 1981;Biddlecombe et al 1982;. For this purpose cryosections have to be transferred into an electron microscope equipped with a cold stage.…”