Guiding Principles of Specimen Preservation for Confocal Fluorescence Microscopy

Abstract

Traditionally, biologists have been confined to electron microscopy and light microscopy in order to correlate biochemical and molecular data with morphology. Electron microscopy provides fine ultrastructural detail, but tends to limit one to the study of cellular structures that react with electron-dense stains deposited in fixed specimens. Immunogold labeling allows one to study non electron-dense material, but the EM sections must be relatively thin, and even then there are problems with penetration of the labelled antibodies. Another problem is the necessity of making thin sections to prevent scattering of the electron beam. The electron microscopist is forced to live like a citizen of Flatland. Flatland is a story about beings that existed entirely within a plane, and were trying to figure out what the various two dimensional shapes appearing on their “world” were (Abbott, 1884). The shapes were sections of three dimensional objects passing through their plane of being. Similarly, biologists using an electron micrsoscope attempt to reconstruct the three dimensional structure of a cell based on the two dimensional cuts through the specimen.