DAB photo-oxidation as a tool for detecting low amounts of free and membrane-bounded fluorescent molecules at transmission electron microscopy

Abstract

DAB photo-oxidation is a well-established cytochemical technique, originally proposed in light microscopy to transform the fluorescence signals into stable reaction products. The electron-density of oxidized DAB made it possible to detect the precise location of the fluorescent probes at the high resolution of electron microscopy. Especially in the last decade, this technique has been extensively used for correlative light and electron microscopy to investigate the molecular composition of subcellular structures as well as dynamic cell processes. In the present article, we summarize the results we obtained by DAB photo-oxidation experiments performed using different fluorescent molecules under variable experimental conditions. In our experience, DAB photo-oxidation proved to be a very sensitive technique, and allowed to locate even small amounts of fluorophores irrespective of their localization either within membrane-bounded organelles or vesicles, or free in the cytosol.