Abstract
A complete portrait of a cell requires a detailed description of its molecular topography: proteins must be linked to particular organelles. Immunocytochemical electron microscopy can reveal locations of proteins with nanometer resolution but is limited by the quality of fixation, the paucity of antibodies and the inaccessibility of antigens. Here we describe correlative fluorescence electron microscopy for the nanoscopic localization of proteins in electron micrographs. We tagged proteins with the fluorescent proteins Citrine or tdEos and expressed them in Caenorhabditis elegans, fixed the worms and embedded them in plastic. We imaged the tagged proteins from ultrathin sections using stimulated emission depletion (STED) microscopy or photoactivated localization microscopy (PALM). Fluorescence correlated with organelles imaged in electron micrographs from the same sections. We used these methods to localize histones, a mitochondrial protein and a presynaptic dense projection protein in electron micrographs.
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Acknowledgements
We thank H. Hess and E. Betzig (Janelia Farm) for access to the PALM microscope for proof-of-principle experiments; R. Fetter for sharing protocols, reagents and encouragement; M. Davidson (Florida State University), G. Seydoux (Johns Hopkins Univeristy), S. Eimer (European Neuroscience Institute), R. Leube (Universität Aachen), K. Nehrke (University of Rochester), C. Frøkjær-Jensen (Utah), A. Ada-Nguema (Utah) and M. Hammarlund (Yale University) for DNA constructs. We thank Marine Biological Laboratory for equipment and funding for pilot experiments and Carl Zeiss Inc. for providing access to a beta version of the PAL-M. This research was supported by the US National Institutes of Health (NS034307), National Science Foundation (0920069) and Marine Biology Laboratory fellowship. (The Dart Neuroscience Scholars Program in Learning and Memory).
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S.W. and E.M.J. conceived and designed experiments. G.H., R.J.H. and M.W.D. provided strains and advice. S.W. optimized the methods, prepared the samples and performed PALM imaging. A.P. and K.I.W. performed STED imaging. S.W., S.W.H. and E.M.J. wrote the manuscript. S.W.H. and E.M.J. provided funding.
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Watanabe, S., Punge, A., Hollopeter, G. et al. Protein localization in electron micrographs using fluorescence nanoscopy. Nat Methods 8, 80–84 (2011). https://doi.org/10.1038/nmeth.1537
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DOI: https://doi.org/10.1038/nmeth.1537
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