Photoconversion of Diaminobenzidine

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.ref 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.ref 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.ref Punctate background staining can reportedly be eliminated without affecting the DAB photoconversion signal by treating the sample with the peroxisomal catalase inhibitor aminotriazole.ref

Electron micrograph 


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.

Eosin Probes

In 1994, Deerinck and colleagues reported a simple method for eosin-mediated photoconversion of DAB.ref Halogenated derivatives of fluorescein dyes are known to be effective photosensitizers and singlet oxygen generators.ref 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.ref Furthermore, the small size of eosin promotes exceptional penetration into tissues resulting in increased resolution for electron microscopy.ref 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:ref