Micro- and Cryo-Techniques Prevent the Loss of Structural Information. EM-Studies on High-Pressure Frozen Tissues, Suspensions and Cell Monolayer.

Extract

Cells are information driven systems. Cellular information is stored in certain molecules, at certain places, in a certain concentration, at a particular time and under given physiological conditions. The goal of biological electron microscopy is to provide this information network, to correlate the cellular ultrastructure and its function. In this sense, it is essential to combine the high resolution of our electron optical instruments with a high information density of the biological system. Most of the structural information is lost in the course of the different preparation steps prior to electron microscopy. For this reason it is necessary that all preparation steps such as: 1. sampling: e.g. excision of tissues, 2. cryoimmobilisation, 3. follow-up procedures: e.g. freeze-fracturing, freeze-substitution and embedding, should have identical high quality levels preventing or minimizing the loss of structural information. To this aim our methodological activities focus on the development of special micro-techniques for the sampling of: a) native tissues, with an automatic fine-needle biopsy technique (1), of b) suspensions, with a special cellulose capillary technique (2), of c) cell monolayer, with a thin film cultivation technique (3) and the application/perfection of cryotechniques (high-pressure freezing (HPF) and freeze-substitution). In particular, the high-pressure freezer (HPM 010, Bal-Tec) has proven to be a highly useful tool for successful cryoimmobilization of almost any kinds of cells and tissues, bulk specimens (< 200 μm in thickness) being included. This freezing technique does not require any cryoprotection, and if combined with our micro-techniques the risk of inducing artefacts as a result of specimen preparation prior to freezing is minimized.