Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2

We offer a variety of fluorescent probes for photoconverting diaminobenzidine (DAB), enabling researchers to take advantage of an important development in correlated fluorescence, transmitted and electron microscopy. In 1982, Maranto first described the use of the fluorophore lucifer yellow for DAB photoconversion. When a fluorophore is exposed to light of an appropriate wavelength, excitation from the electronic ground state to a higher singlet state occurs. Instead of emitting a photon, the excited state of the fluorophore may undergo intersystem crossing to the triplet state. Transfer of energy to ground state triplet oxygen (³O₂) generates toxic and highly reactive singlet oxygen (¹O₂), which is capable of causing damage to lipids, proteins and nucleic acids. However, the reactive potential of ¹O₂ can also be harnessed to oxidize diaminobenzidine (DAB) into an electron-opaque osmiophilic precipitate within cells (Figure 1). The resulting DAB reaction product exhibits exceptionally uniform, nondiffusible staining properties, making it extremely useful for subsequent electron microscopy investigation of cellular ultrastructure. Punctate background staining can reportedly be eliminated without affecting the DAB photoconversion signal by treating the sample with the peroxisomal catalase inhibitor aminotriazole.

Figure 1. Electron micrograph of a 80 nm–thick section of formaldehyde-fixed rat soleus muscle, which was first stained with eosin α-bungarotoxin and then used to photoconvert DAB into an insoluble osmiophilic polymer. Photo contributed by Thomas J. Deerinck, University of California, San Diego.

In 1994, Deerinck and colleagues reported a simple method for eosin-mediated photoconversion of DAB. Halogenated derivatives of fluorescein dyes are known to be effective photosensitizers and singlet oxygen generators. Eosin is a brominated analog of fluorescein that has a ¹O₂ yield 19 times greater than fluorescein and is an excellent dye for photoconverting DAB. Furthermore, the small size of eosin promotes exceptional penetration into tissues resulting in increased resolution for electron microscopy. We offer amine- and thiol-reactive eosin derivatives for preparing eosin-labeled bioconjugates (Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5, Thiol-Reactive Probes Excited with Visible Light—Section 2.2). Some other fluorescent tracers that have been used to photoconvert DAB include:

• BODIPY FL C₅-ceramide (D3521, B22650; Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4)
• DiI (D282, Tracers for Membrane Labeling—Section 14.4)
• Fluorescent polystyrene microspheres (Microspheres—Section 6.5)
• Fluoro-ruby dextran (D1817, Fluorescent and Biotinylated Dextrans—Section 14.5)
• Lucifer yellow (L453, L12926; Polar Tracers—Section 14.3)
• Propidium iodide (P1304MP, P3566, P21493; Nucleic Acid Stains—Section 8.1)
• ReAsH (T34562, Thiol-Reactive Probes Excited with Visible Light—Section 2.2)
• Texas Red-X succinimidyl ester (T6134, Long-Wavelength Rhodamines, Texas Red Dyes and QSY Quenchers—Section 1.6)