Thermally defined cryomicroscopy and some applications on human leucocytes

Abstract

SUMMARY A freezing‐stage has been developed for use on a standard light‐microscope, which can provide reproducible, precisely linear cooling and warming rates in the range from 0·1 to 10,000 K/min. Biological cells in aqueous solutions can be observed during the freeze‐thaw cycle; the volume loss due to osmotic efflux of water and the intracellular crystallization of water are detected by video‐monitoring. The temperature field generated in the observed samples is comparable to extended cylindrical probes and allows the transfer of cryomicroscopic data to technically used vial geometries. Lymphocytes and granulocytes were observed during freezing using the system described. They were separated and washed, and then frozen on the cold stage of the cryomicroscope at cooling rates ranging from 2 to 500 K/min. Shrinkage of the cells was observed up to 100 K/min and intracellular ice formation could be detected starting at 10 K/min. The results show that human leucocytes show excessive shrinkage up to 36% of their initial volume; the probability of intracellular ice formation exhibits a sharp increase from 10 to 100 K/min where nearly all cells contain ice.

Low temperature light microscopy and its application to study freezing in aqueous solutions and biological cell suspensions

“…1. Portions of this setup have already been described earlier (Scheiwe & Korber, 1982aKorber et al, 1984) and will hence only be outlined briefly in the following. Freezing stage.…”

“…The freezing stage used has a circular observation hole of 1 mm diameter yielding a dendritic solidification pattern under those conditions. As reported previously (Scheiwe & Korber, 1982a, individual shrinkage curves may vary considerably from cell to cell even in one experiment, and data points are usually averaged from between about ten and fifty cells for obtaining smoother curves. These can then be correlated to curves calculated numerically from water transport models (e.g.…”

“…0 1986 The Royal Microscopical Society *Presently at Nordmark-Werke, D-2082 Uetersen. Molisch (1897), Rapatz & Luyet (1960), Diller & Cravalho (1971), Reid (1978), McGrath & Khompis (1981), Schwartz & Diller (1982), , Scheiwe & Korber (1982a, Thom & Matthes (1984) and Steponkus et al (1984a). The development has led from merely qualitative studies to instrumentation generating a wide range of well-defined cooling and warming rates and thus permitting quantitative evaluation.…”

An optical differential scanning calorimeter cryomicroscope

“…This instrument integrates the fundamental features of the cryomicroscope and DSC instruments. The principles of the design and operation of light cryomicroscopy systems have been discussed on many occasions (Diller, 1982; Scheiwe & Körber, 1982; Körber et al ., 1986; Diller & Aggarwal, 1987; Walcerz & Diller, 1991) and therefore will not be repeated. The characteristic feature of all DSC systems is differential energy measurement between a matched set of specimen and reference holders.…”