Electron microscopic analysis of fluorescent neuronal labeling after photoconversion

Ultrastructural visualization of non-electron-dense fluorescent retrograde neuronal labeling was attempted by means of photo-oxidation. This procedure was used to convert the fluorescence of neurons labeled by the tracers propidium iodide, rhodamine latex microspheres and fluorogold into a stable diaminobenzidine reaction product. The ultrastructural study revealed an accumulation of electron-dense material in these cells both within lysosomes and scattered in the cytoplasmic matrix. Comparison with several different sets of control samples indicated that this material, on the basis of its amount, electron density and appearance, specifically represents the photoconversion reaction product. The effects of the intensity of the fluorescent labeling and of a prolonged photoconversion on the fine structural features of the reaction product are also described and discussed. The present findings indicate that photoconversion can be effectively applied to ultrastructural study of fluorescent retrogradely labeled neurons. The specificity of the photoconversion reaction product should be tested routinely for each fluorochrome and tissue sample.