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> “GFP” for electron microscopy
“GFP” for electron microscopy
Electron microscopy (EM) once revolutionized cell biology by revealing subcellular anatomy at resolutions of tens of nanometers, well below the diffraction limit of light microscopy. Over the past two decades, light microscopy has been revitalized by the development of spontaneously fluorescent proteins, which allow nearly any protein of interest to be specifically tagged by genetic fusion. EM has lacked comparable genetic tags that are generally applicable. Here, we introduce “miniSOG”, a small (106-residue) fluorescent flavoprotein that efficiently generates singlet oxygen when illuminated by blue light. In fixed tissue, photogenerated singlet oxygen locally polymerizes diaminobenzidine into a precipitate that is stainable with osmium and therefore can be readily imaged at high resolution by EM. Thus miniSOG is a versatile label for correlated light and electron microscopy of genetically tagged proteins in cells, tissues, and organisms including intact nematodes and mice. As a demonstration of miniSOG’s capabilities, controversies about the localization of synaptic cell adhesion molecules are resolved by EM of miniSOG fusions in neuronal culture and intact mouse brain.
Link to original article in PLoS Biology
Discussion of this article in BioTechniques