Biomedical Significance and Development of “IVCT”

Abstract

Preparation procedures after resection of animal tissues are commonly a series of chemical fixation, alcohol dehydration, paraffin or epoxy resin embedding, thick or thin sectioning, and dye or metal staining steps. During such preparation steps, various kinds of inevitable artifacts always modify their original morphology. Some problems are both molecular movement and structural changes of cells and tissues during the fixation time. Another problem is that dynamic morphological images in vivo are difficult to be captured by the conventional chemical fixation. Then, a strong effort of morphologists has been made to avoid such technical artifacts during the conventional preparation steps. The quick-freezing (QF) method was introduced for biological specimens at the middle of the twentieth century. For the final morphological purpose, various preparation procedures can be chosen after the quick-freezing. One of them is freeze-substitution (FS) fixation, in which the frozen specimens are usually incubated in cooled organic solvents containing chemical fixatives at about −80 °C. However, the freeze-substituted specimens are known to be affected to some extent by the organic solvents. Another deep-etching (DE) replication method has been developed, in which replica membranes of freeze-fractured and deeply etched tissues are obtained by rotary-shadowing with platinum metal at lower temperatures below −100 °C under high vacuum conditions. However, some pieces of tissues have to be always resected and taken out from living animal organs. Thus, the dynamically changing morphology of living animal organs is hardly investigated by the conventional QF method. To overcome these technical problems, it is necessary to avoid the tissue resection step of living animal organs and directly freeze them in vivo under normal blood circulation. The IVCT was an original technique to directly cryofix living animal organs without separating their tissues from blood circulation. The most significant point of IVCT is that normal blood circulation into living animal organs is strictly preserved at the exact moment of freezing.