UNIT 2.15 Confocal Laser Scanning Microscopic Photoconversion: A New Method to Stabilize Fluorescently Labeled Cellular Elements for Electron Microscopic Analysis

Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low-resolution cell targeting. Accordingly, we have developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We have found that this method greatly reduces photoconversion times, as compared to conventional wide field microscopy. Moreover, region-of-interest scanning capabilities of a confocal microscope facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This reduces the area of the cell exposed to light energy, thereby reducing the ultrastructural damage common to this process when widefield microscopes are employed. Curr. Protoc. Neurosci. 58:2.15.1-2.15.12. © 2012 by John Wiley & Sons, Inc.

Keywords: photo-oxidation; diaminobenzidine; ultrastructure; electron microscopy; immunolabeling; confocal microscopy