Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

Abstract

Abstract. A simple method is described for highresolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.F LUORESCENT labeling techniques have become increasingly popular in many areas of cell biology, particularly due to the expanded use of laser scanning confocal microscopy. Immunofluorescent localization of proteins and fluorescence-based in situ hybridization detection of specific nucleic acid sequences in cells and tissues have several advantages over other labeling procedures including relative simplicity, shorter processing times, high sensitivity, and good spatial resolution. One of the major disadvantages of fluorescent labeling has been the inability to use this technique for correlated light and electron microscopic studies in cases requiring better than light microscopic resolution. Several methods have been introduced in an attempt to overcome this limitation. These include the use of anti-fluorophore antibodies, e.g., anti-fluorescein, conjugated to peroxidase (Peters et al., 1990) and the process of fluorescence photooxidation of diaminobenzidine (DAB)? This latter method, first described by Maranto (1982), involves the use of fluorescent dyes to oxidize DAB into an insoluble reaction product.Photooxidation represents a relatively simple method to render fluorescently labeled preparations suitable for elec- tron nficroscopic examination. Briefly, a fluorescent dye is excited in the presence of DAB and gradually an oxidized DAB reaction product forms. The reaction product is highly insoluble and easily visible with ordinary transmitted light microscopy. Since the DAB reaction product is osmiophilic, this technique can be used for correlative light and electron microscopic studies using a variety of fluorescent compounds including Lucifer yellow, DiI, Bodipy ceramide, fluorogold, and fluororuby (Pagano et al., 1989;Bentivoglio and Su, 1990;yon Bartheld et al., 1990;Balercia et al., 1992;Lubke, 1993;Papadopoulos and Dori, 1993;Schmued and Snavely, 1993;Takizawa et al., 1993). A poten...

The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications

“…Labeled or tagged molecules are dynamic within cells (Deerinck et al, 1994) and it is extremely difficult to deliver reagents into the cells since this is a diffusion-based process. In addition, heat is generated during the photooxidation process and warming and subsequent re-freezing of the tissue would likely cause ice crystal damage.…”

“…In addition, heat is generated during the photooxidation process and warming and subsequent re-freezing of the tissue would likely cause ice crystal damage. Current protocols use 2-3% of glutaraldehyde as either a primary fixation, as in the case of genetically tagged fluorescent proteins, (Gaietta et al, 2002) or as a secondary fixation after immunolabeling (Deerinck et al, 1994) or small molecule labeling (Capani et al, 2001). Since specimens cannot be maintained with current light microscope stages at the low temperatures required for frozen samples and temperature dependent diffusion processes will limit DAB infiltration, cryofixation of photoconverted material is impractical.…”

“…Several neurons were injected in each slice. These dye-injected slices were then photooxidized according to procedures described in (Deerinck et al, 1994) and recently updated in (Gaietta et al, 2006).…”

A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed Nanogold-labeled molecules

“…Other methods involve attachment of labels that are visualized after photooxidation of diaminobenzidine (DAB) and metal deposition (e.g., peroxidase (Rodewald, 1980) and fluorescent labels for correlative light and electron microcopy (Deerinck et al, 1994;Gaietta et al, 2002)). None of these commonly used labels is well-suited for high resolution studies of dynamic events in intracellular trafficking.…”

Enhancing Serial Block-Face Scanning Electron Microscopy to Enable High Resolution 3-D Nanohistology of Cells and Tissues

“…MiniSOG has quantum yields for fluorescence and singlet oxygen of 0.30 and 0.47 respectively. It can be genetically fused to the target protein of interest for both fluorescence imaging and efficient photooxidation of diaminobenzidine into an osmiophilic polymer for subsequent electron microscopy [3]. Since miniSOG is genetically encoded and all other reactants (O 2 , diaminobenzidine, OsO 4 ) are permeant small molecules, there is no need to compromise chemical fixation to preserve protein epitopes or to permeabilize with detergents, which further degrade cellular ultrastructure.…”