Photooxidation technology for correlated light and electron microscopy

Abstract

Summary The combination of the capabilities of light microscopical techniques with the power of resolution of electron microscopy along with technical advances has led to a gradual decline of the gap between classical light and electron microscopy. Among the correlative techniques using the synergistic opportunities, photooxidation methods have been established as valuable tools for visualizing cell structures at both light and electron microscopic level. Fluorescent dyes are used to oxidize the substrate diaminobenzidine, which in its oxidized state forms fine granular precipitates. Stained with osmium, the diaminobenzidine precipitates are well discernible in the electron microscope, thus labelling and defining the cellular structures, which at light microscopy level are recorded by fluorescent probes. The underlying photooxidation reaction is based on the excitation of free oxygen radicals that form upon illumination of fluorochromes; this is a central step in the procedure, which mainly influences the success of the method. This article summarizes basic steps of the technology and progresses, shows efforts and elaborated pathways, and focuses on methodical solutions as to the applicability of different fluorochromes, as well as conditions for fine structural localizations of the reaction products.

Quantification of Photosensitized Singlet Oxygen Production by a Fluorescent Protein

“…[26,27] This protein has excellent photophysical and biochemical properties, including high brightness, long lifetime, complete chromophore maturation and high pH stability. [13] More specifically, and in comparison with other FPs used for CALI such as EGFP, its longer excitation wavelength will minimize non-specific photodamage.…”

Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells

“…Photooxidation allows the localization of molecules of interest by conversion of light microscopic fluorescent signals by illumination with high energetic light into stable, electron dense signals by oxidation of 3,3´-diaminobenzidine tetrahydrochloride (DAB) (Meißlitzer-Ruppitsch et al, 2009). For this, fluorescently labeled and fixed samples are pre-incubated with DAB and illuminated, which causes fluorophores to emit reactive oxygen species, resulting in DAB oxidation and precipitation.…”

The ceramide-enriched trans-Golgi compartments reorganize together with other parts of the Golgi apparatus in response to ATP-depletion

“…As central step, upon illumination of proper Xuorophores high energetic light triggers the release of free oxygen radicals, which rapidly oxidize DAB. Oxidized DAB then forms networks, which are insoluble in water and after osmiWcation appear as Wne granular precipitates at the sites of the former Xuorescent signals (for review Meisslitzer-Ruppitsch et al 2009). The DAB-photooxidation technology was used to visualize ceramide and its metabolites at the ultrastructural level within the Golgi apparatus and along the secretory pathway (Pagano et al 1989 and further to deWne details of trans-Golgi compartments (Ladinsky et al 1994).…”