Mapping the molecular architecture of proteins within a cell is essential for understanding their function. Fluorescence microscopy has been widely used for this purpose. However, the diffraction limit of light limits this approach, since fluorophores that are within ~200 nm of each other cannot be resolved. Several microscopy techniques have been developed to break this limit, including photo-activated localization microscopy (PALM)[1] and related techniques (STORM[2] and fPALM[3]). PALM allows one to precisely pinpoint the location of a single molecule in a cell to within 10 nm. However, precise localization in a field of black is not useful. Where is that protein in the context of cellular structure? PALM images need to be overlaid on the structures visualized by electron microscopy. Therefore, a method to preserve fluorophores in tissues fixed and embedded in plastic needs to be developed. To preserve fluorescence in plastic, three conditions are required: hydration, limited oxidation from fixatives, and neutral pH. We found that use of 5% water, a less oxidative fixative (KMnO4), and hydrophilic plastic (GMA - pH8), were sufficient to preserve signals and morphology. To test the feasibility of fEM using PALM, we tagged histone, TOM-20, and liprin, with photo-convertible fluorescent proteins. We showed that histone, TOM-20, and liprin were successfully localized to the expected organelles - nucleus, mitochondrial membrane, and dense projection, respectively[4]. We also have mapped the
a2d subunits of voltage-gated calcium channels in the nerve ring of C. elegans. We found the subunits to be exclusively localized to dense projections, supporting the previous localization pattern by immuno-EM[5]. This result suggests that the fEM is feasible for protein localization, and many proteins can be localized to their cellular structures if they can be tagged by fluorophores. References [1]Betzig, E. et al. Science 313, 1642-1645 (2006). [2]Rust, M.J., Bates, M. & Zhuang, X. Nat. Methods 3, 793-795 (2006). [3]Hess, S.T., Girirajan, T.P.K. & Mason, M.D. Biophys. J 91, 4258-4272 (2006). [4]Watanabe, S. et al. Nat. Methods 8, 80-84 (2011). [5]Gracheva, E.O., Hadwiger, G., Nonet, M.L. & Richmond, J.E. Neurosci. Lett 444, 137-142 (2008).