Impact of freeze substitution on biological electron microscopy

Abstract

Considering the increasing necessity for improved preparation techniques in biological electron microscopy as a basis for the identification and localization of cellular substances within the compartments of the cell, this review is focussed on the method of freeze substitution as an important link between the cryofixation (ultrarapid freezing) and resin embedding of biological specimens. The theory and practice of freeze substitution is summarized with particular interest in the physical and thermodynamic as well as in the chemical basis of this technique. A survey of practical aspects of the technical process of freeze substitution concerning the equipment and various protocols successfully applied in biological systems is also given. The main advantage of freeze substitution versus conventional chemical fixation is seen in the maintenance of the hydration shell of molecules and macromolecular structures. This results in an improved fine structural preservation, superior retention of the antigenicity of proteins and decreased loss of unbound, diffusible cellular components. Examples of excellent visualization of the ultrastructure of macromolecular complexes (nucleic acids, extracellular material, membranes etc.), small organisms (bacteria, algae, cyanobacteria and fungi) and large biological samples such as plant and animal tissue as well as the plant-pathogen (fungus) interface and infection structures are presented. Recent data on the molecular characterization of freeze-substituted biological tissue are exemplified with special emphasis on the subcellular detection of soluble components (elements, lipids, proteins and drugs) and the inter-/intracellular localization of proteins including foreign proteins in transgenic plants. The molecular analysis of freeze-substituted specimens is achieved by the combination of low temperature preparation techniques in biological electron microscopy with various detection methods such as X-ray microanalysis, immunocytochemistry and high resolution autoradiography.

A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution

“…During the freezing process internal and external cellular constituents, including their soluble components, are immobilized in their native structure and position in just a few milliseconds. During the FS step an organic solvent (methanol or acetone) is then substituted for the vitreous ice at −90°C, which minimizes the surface tension-mediated collapse and shrinkage that occurs when water is rapidly and progressively removed at room temperature (see Hippe-Sanwald 1993). By incorporating a fixative into the FS denydrant, the structure, location, and spatial relationships of components are preserved from distortions that occur in conventional preparations owing to a fixation front that sweeps through the cell at room temperature.…”

“…This HFP/FS approach has many advantages over conventional fixation protocols. Not only does it minimize the displacement, extraction, and coagulation of molecules that occur as a front of buffered chemical fixative sweeps through a cell, but it also minimizes the structural distortions produced in the specimen during dehydration due to changes in surface tension (Hippe-Sanwald 1993).…”

Biochemical and molecular studies using human autopsy brain tissue

“…Although tissue prepared by this method may be used for molecular and ultrastructural studies (Scopsi et al 1992;Hippe-Sanwald 1993;Schafer and Kaufmann 1999), rapid freezing causes considerable damage to membrane structures within the cell (Koenig 1964;Walder and Vrensen 1972;Scott and Lew 1986). As a result, synaptosomes prepared after fast tissue freezing are generally inactive on metabolic and functional indices .…”

The intercellular biotrophic leaf pathogen Cymadothea trifolii locally degrades pectins, but not cellulose or xyloglucan in cell walls of Trifolium repens

“…Hippe- Sanwald, 1993;Berg, 1994;Hippe-Sanwald et al, 1994;Mims et al, 2002). Yet few plant pathologists actually use these techniques (Mims et al, 2002).…”