Processing Biological Tissues for Ultrastructural Study

Abstract

Biological tissues are passed through numerous procedures before they can be studied at the ultrastructural level with the electron microscope. Chemical fixation is widely used as a method for preserving structural detail and can be performed by simple immersion or total body vascular perfusion. A 2 to 4% solution of glutaraldehyde buffered with 0.1 M sodium phosphate, or a combination of similarly buffered glutaraldehyde and paraformaldehyde, can be used successfully to preserve the fine structure of biological tissues. The material next is washed briefly in the buffer vehicle and then secondarily fixed in 1% osmium tetroxide (osmic acid), which also is buffered with sodium phosphate. The tissue then is thoroughly dehydrated in solutions of ethanol at increasing concentrations of 50%, 70%, 95%, and 100%. After dehydration, tissues are infiltrated for a prescribed time interval with an epoxy embedding medium. After infiltration, specimens are transferred into fresh epoxy resin and polymerized at 60 to 70 degrees C for several hours. This orderly process ultimately yields fixed tissues that are encased in hardened blocks that can be thin-sectioned with an ultramicrotome. The thin sections are counterstained with solutions of heavy metals to add contrast. The material then can be subjected to the electron beam in an electron microscope to produce useful images for ultrastructural study. This overall procedure has been used successfully since the advent of biological electron microscopy to define the minute details of cells and tissues.

The creation of geometric three-dimensional models of the inner ear based on micro computer tomography data

“…OsO 4 . Osmium tetroxide is a standard fixing and contrasting substance in electron microscopy (Furness and Hackney, 1986;Mascorro and Bozzola, 2007). Its ability to penetrate deeply into soft tissues allows their visualization.…”

Visual demonstration of transmission electron microscopy for intracellular observation of a single bacterial cell

“…The objective of this step is to halt cellular activity without altering the cellular characteristics, the components that make up the sample or their distribution; to preserve the internal three-dimensional organization, the size and the shape of the sample. The fixation process can be carried out in two different ways, by chemical fixation or by cryofixation (Mascorro and Bozzola, 2007). Several researchers have described a number of chemical methods for staining bacterial cells including the tannic acid-fuchsin method and modifications of this method (Kodaka et al, 1982;Grossart et al, 2000), as well as silver staining methods (Porter et al, 1992).…”

A simple and direct pre-embedding technique for ultrastructure of scarce biological specimens

“…Osmium postfixation is a routine staining procedure for TEM (Mascorro and Bozzola 2007). Processing scarce biological specimens for TEM precludes their osmication, because this would result in loss of the samples during the subsequent steps required to wash out the osmium.…”

“…Results of such studies may be correlated with other investigations, such as biochemical and physiological studies, to unravel the structureÁfunction relations of cells or tissues ). Samples processed for TEM go through multiple steps of dehydration, infiltration and embedding before they can be studied under the microscope Minassian 1987, Mascorro andBozzola 2007). Therefore, processing biological specimens for TEM requires critical amounts of tissues to be processed safely.…”