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Abstract
Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by transmission 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 which limit its utility. Accordingly, a photoconversion method was devised that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. It was confirmed that photoconversion times were dramatically reduced when using a confocal laser scanning microscope in the photoconversion process. It was also demonstrated that the region of interest scanning capabilities of a confocal laser scanning microscope equipped with an acousto-optical tunable filter represented a unique tool to facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This specificity was shown to be far superior to conventional widefield microscopy-based photoconversion where target selectivity directly mirrors objective aperture size. Moreover, region of interest scanning greatly reduced the area of the cell exposed to light energy, ameliorating the ultrastructural damage common to this process when widefield microscopes are employed. The potential of this new methodology extends beyond the neurosciences to any scientific modality which requires ultrastructure analysis of fluorescently-labeled specimens, especially those where discrete labeling on a cellular or subcellular basis could be beneficial.
