postfixation

Postfixation is the second-step fixation technique of chemical fixation for biological specimens observed with a TEM.  This technique aims at preservation of lipids or enhancement of a TEM image contrast.  In the preparation of a biological specimen for TEM observation, dehydration and resin embedding are applied.  In these procedures, fixation only with aldehyde (glutaraldehyde, paraformaldehyde, etc.) cannot fix lipids in the specimen.  Thus, the membrane structures that consist of lipids flow out of the specimen when performing dehydration with ethanol or an organic solvent. In addition, aldehyde bounded to the specimen does not contribute to the enhancement of the image contrast because it consists of only light elements.  These problems are solved by postfixation with heavy metal oxide reagents (osmium tetroxide, etc.) which is a subsequent process of prefixation with aldehyde.  Osmium tetroxide fixes lipids by binding it to a double-binding region of unsaturated fatty acid and by constructing a cross-link for preventing outflow and deformation of the lipids.  In addition, osmium is a heavy metal, thus enhancing the image contrast of the binding region.  Osmium tetroxide is an effective reagent, but it has disadvantages that include slow permeation into the specimen and destruction of proteins in the specimen.  To prevent these disadvantageous phenomena, fixation (prefixation) of proteins with aldehyde is required before applying fixation with osmium tetroxide.

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