Technical Merits with “IVCT”

Abstract

The main purpose of in vivo cryotechnique (IVCT) is to make all biological components of cells and tissues of functioning organs promptly frozen in living animals. However, the necessary freezing times are always different at each depth from the frozen tissue surface, because thermal conductance of cooling within cells and tissues is due to the continuous movement of thermal energy. Therefore, only surface tissue layers within certain depths, such as about 10 or 200 μm, are frozen enough to prevent formation of visible ice crystals at electron or light microscopic levels, respectively. With the metal contact freezing method, they are slightly compressed on the cooled copper metal due to crash impact, even though some spacers and cushions are always inserted between specimens and the cooled metal block. To the contrary, with liquid cryogen, such as the isopentane-propane mixture precooled in liquid nitrogen, the frozen tissues can retain their original morphological states without mechanical compression damages. To examine deeper tissue areas from the frozen tissue surface, we always need to cryocut any functioning organs of living animals under anesthesia. When a cryoknife precooled in liquid nitrogen passes through the living animal organ, the exposed tissue surface in direct contact with the cryoknife is first frozen in the same way as the metal contact method. Practically, at an electron microscopic level, well-frozen tissue areas appear to occupy a very narrow band, less than 10 μm deep from the contact tissue surface.