Click-EM for imaging metabolically tagged nonprotein biomolecules

Abstract

EM has long been the main technique for imaging cell structures with nanometer resolution but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce click-EM, a labeling technique for correlative light microscopy and EM imaging of nonprotein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal 'click chemistry' ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of click-EM in imaging metabolically tagged DNA, RNA and lipids in cultured cells and neurons and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes.

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Figure 1: Click-EM imaging of EdU-labeled HeLa cells.
Figure 2: SBEM imaging of EdU-labeled HEK293 cells.
Figure 3: Imaging of nascent transcripts using EU.
Figure 4: Click-EM imaging of AzCho labeled HeLa cells.
Figure 5: Click-EM imaging of PG in L. monocytogenes.
Figure 6: Click-EM imaging of wild-type and ramoplanin-treated L. monocytogenes.

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Acknowledgements

We gratefully acknowledge S. Phan for assistance with SBEM image processing, C. Woodford for assistance with NMR, L. Gross for assistance with MS and C. Hill for providing wild-type and lmo2754::tn EGD-e L. monocytogenes. Funding for this work was provided by NIH GM086197 (to R.Y.T. and M.H.E.), NIH GM058867 (to C.R.B.), and NIH AI051622 (to C.R.B.). The work described herein was carried out using shared research resources at the National Center for Microscopy and Imaging Research (NCMIR) at UCSD supported by the NIH under award number P41 GM103412 (to M.H.E.). F.R. was supported by a Ford Foundation Predoctoral Fellowship and a Chancellor's fellowship from UC Berkeley.

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J.T.N., S.R.A., T.J.D., D.B., F.R.-R. and S.F.P. performed experiments. J.T.N., S.R.A., T.J.D., D.B., F.R.-R., S.F.P., C.R.B., M.H.E. and R.Y.T. analyzed data and prepared the manuscript.

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Correspondence to Roger Y Tsien.

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Ngo, J., Adams, S., Deerinck, T. et al. Click-EM for imaging metabolically tagged nonprotein biomolecules. Nat Chem Biol 12, 459–465 (2016). https://doi.org/10.1038/nchembio.2076

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