Click-EM for imaging metabolically tagged nonprotein biomolecules


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Stirling, J.W. Immuno- and affinity probes for electron microscopy: a review of labeling and preparation techniques. J. Histochem. Cytochem. 38, 145–157 (1990).

    Article  CAS  Google Scholar 

  2. 2

    Deerinck, T.J. et al. Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy. J. Cell Biol. 126, 901–910 (1994).

    Article  CAS  Google Scholar 

  3. 3

    Shu, X. et al. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 9, e1001041 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Gaietta, G. et al. Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–507 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Martell, J.D. et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143–1148 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Lam, S.S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nat. Methods 6, 331–338 (2009).

    Article  CAS  Google Scholar 

  8. 8

    Laughlin, S.T., Baskin, J.M., Amacher, S.L. & Bertozzi, C.R. In vivo imaging of membrane-associated glycans in developing zebrafish. Science 320, 664–667 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Salic, A. & Mitchison, T.J. A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. USA 105, 2415–2420 (2008).

    Article  Google Scholar 

  10. 10

    Jao, C.Y. & Salic, A. Exploring RNA transcription and turnover in vivo by using click chemistry. Proc. Natl. Acad. Sci. USA 105, 15779–15784 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  11. 11

    Jao, C.Y., Roth, M., Welti, R. & Salic, A. Metabolic labeling and direct imaging of choline phospholipids in vivo. Proc. Natl. Acad. Sci. USA 106, 15332–15337 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  12. 12

    Ngo, J.T. & Tirrell, D.A. Noncanonical amino acids in the interrogation of cellular protein synthesis. Acc. Chem. Res. 44, 677–685 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Rostovtsev, V.V., Green, L.G., Fokin, V.V. & Sharpless, K.B. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Edn Engl. 41, 2596–2599 (2002).

    Article  CAS  Google Scholar 

  14. 14

    Tornøe, C.W., Christensen, C. & Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057–3064 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Wang, Q. et al. Bioconjugation by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125, 3192–3193 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Agard, N.J., Prescher, J.A. & Bertozzi, C.R. A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126, 15046–15047 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Jewett, J.C. & Bertozzi, C.R. Cu-free click cycloaddition reactions in chemical biology. Chem. Soc. Rev. 39, 1272–1279 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Gandin, E., Lion, Y. & Van de Vorst, A. Quantum yield of singlet oxygen production by xanthene derivatives. Photochem. Photobiol. 37, 271–278 (1983).

    Article  CAS  Google Scholar 

  19. 19

    Usui, Y. Determination of quantum yield of singlet oxygen formation by photosensitization. Chem. Lett. 7, 743–744 (1973).

    Article  Google Scholar 

  20. 20

    Kishimoto, S. et al. Evaluation of oxygen dependence on in vitro and in vivo cytotoxicity of photoimmunotherapy using IR-700-antibody conjugates. Free Radic. Biol. Med. 85, 24–32 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Capani, F. et al. Phalloidin-eosin followed by photo-oxidation: a novel method for localizing F-actin at the light and electron microscopic levels. J. Histochem. Cytochem. 49, 1351–1361 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Chibisov, A.K., Zakharova, G.V. & Görner, H. Effects of substituents in the polymethine chain on the photoprocesses in indodicarbocyanine dyes. J. Chem. Soc., Faraday Trans. 92, 4917–4925 (1996).

    Article  CAS  Google Scholar 

  23. 23

    Görner, H. & Chibisov, A.K. in CRC Handbook of Organic Photochemistry and Photobiology (eds. Horspool, W. & Lenci, F.) 36.1–36.21 (2004).

  24. 24

    Velapoldi, R.A. & Tønnesen, H.H. Corrected emission spectra and quantum yields for a series of fluorescent compounds in the visible spectral region. J. Fluoresc. 14, 465–472 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Fleming, G.R., Knight, A.W.E., Morris, J.M., Morrison, R.J.S. & Robinson, G.W. Picosecond fluorescence studies of xanthene dyes. J. Am. Chem. Soc. 99, 4306–4311 (1977).

    Article  CAS  Google Scholar 

  26. 26

    Dimitrova, D.S. & Berezney, R. The spatiotemporal organization of DNA replication sites is identical in primary, immortalized and transformed mammalian cells. J. Cell Sci. 115, 4037–4051 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Chagin, V.O., Stear, J.H. & Cardoso, M.C. Organization of DNA replication. Cold Spring Harb. Perspect. Biol. 2, a000737 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Iborra, F.J., Pombo, A., Jackson, D.A. & Cook, P.R. Active RNA polymerases are localized within discrete transcription “factories” in human nuclei. J. Cell Sci. 109, 1427–1436 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Papantonis, A. & Cook, P.R. Transcription factories: genome organization and gene regulation. Chem. Rev. 113, 8683–8705 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Bensaude, O. Inhibiting eukaryotic transcription: Which compound to choose? How to evaluate its activity? Transcription 2, 103–108 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  31. 31

    Li, C. et al. Practical labeling methodology for choline-derived lipids and applications in live cell fluorescence imaging. Photochem. Photobiol. 90, 686–695 (2014).

    Article  CAS  Google Scholar 

  32. 32

    Debets, M.F. et al. Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition. Chem. Commun. (Camb.) 46, 97–99 (2010).

    Article  CAS  Google Scholar 

  33. 33

    van Meer, G., Voelker, D.R. & Feigenson, G.W. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112–124 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Siegrist, M.S. et al. D-Amino acid chemical reporters reveal peptidoglycan dynamics of an intracellular pathogen. ACS Chem. Biol. 8, 500–505 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Cava, F., de Pedro, M.A., Lam, H., Davis, B.M. & Waldor, M.K. Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids. EMBO J. 30, 3442–3453 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Siegrist, M.S., Swarts, B.M., Fox, D.M., Lim, S.A. & Bertozzi, C.R. Illumination of growth, division and secretion by metabolic labeling of the bacterial cell surface. FEMS Microbiol. Rev. 39, 184–202 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Pittman, J.R. et al. Effect of stressors on the viability of listeria during an in vitro cold-smoking process. Agric. Food Anal. Bacteriol. 2, 195–208 (2012).

    Google Scholar 

  38. 38

    Korsak, D., Vollmer, W. & Markiewicz, Z. Listeria monocytogenes EGD lacking penicillin-binding protein 5 (PBP5) produces a thicker cell wall. FEMS Microbiol. Lett. 251, 281–288 (2005).

    Article  CAS  Google Scholar 

  39. 39

    Fang, X. et al. The mechanism of action of ramoplanin and enduracidin. Mol. Biosyst. 2, 69–76 (2006).

    Article  CAS  Google Scholar 

  40. 40

    Littau, V.C., Allfrey, V.G., Frenster, J.H. & Mirsky, A.E. Active and inactive regions of nuclear chromatin as revealed by electron microscope autoradiography. Proc. Natl. Acad. Sci. USA 52, 93–100 (1964).

    Article  CAS  Google Scholar 

  41. 41

    Gupta, B.L., Moreton, R.B. & Cooper, N.C. Reconsideration of the resolution in EM autoradiography using a biological line source. J. Microsc. 99, 1–25 (1973).

    Article  Google Scholar 

  42. 42

    Iborra, F.J. & Cook, P.R. The size of sites containing SR proteins in human nuclei. Problems associated with characterizing small structures by immunogold labeling. J. Histochem. Cytochem. 46, 985–992 (1998).

    Article  CAS  Google Scholar 

  43. 43

    Paez-Segala, M.G. et al. Fixation-resistant photoactivatable fluorescent proteins for CLEM. Nat. Methods 12, 215–218, 4, 218 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Perkovic, M. et al. Correlative light- and electron microscopy with chemical tags. J. Struct. Biol. 186, 205–213 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Devaraj, N.K., Hilderbrand, S., Upadhyay, R., Mazitschek, R. & Weissleder, R. Bioorthogonal turn-on probes for imaging small molecules inside living cells. Angew. Chem. Int. Edn Engl. 49, 2869–2872 (2010).

    Article  CAS  Google Scholar 

  46. 46

    Shieh, P. et al. CalFluors: a universal motif for fluorogenic azide probes across the visible spectrum. J. Am. Chem. Soc. 137, 7145–7151 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Sanman, L.E. & Bogyo, M. Activity-based profiling of proteases. Annu. Rev. Biochem. 83, 249–273 (2014).

    Article  CAS  Google Scholar 

  48. 48

    tom Dieck, S. et al. Direct visualization of newly synthesized target proteins in situ. Nat. Methods 12, 411–414 (2015).

    Article  CAS  Google Scholar 

  49. 49

    Robinson, P.V., de Almeida-Escobedo, G., de Groot, A.E., McKechnie, J.L. & Bertozzi, C.R. Live-cell labeling of specific protein glycoforms by proximity-enhanced bioorthogonal ligation. J. Am. Chem. Soc. 137, 10452–10455 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Dieterich, D.C. et al. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat. Neurosci. 13, 897–905 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Bowers, P.G. & Porter, G. Triplet state quantum yields for some aromatic hydrocarbons and xanthene dyes in dilute solution. Proc. R. Soc. Lond. A 299, 348–353 (1967).

    Article  CAS  Google Scholar 

  52. 52

    Pal, P. et al. Phototoxicity of some bromine-substituted rhodamine dyes: synthesis, photophysical properties and application as photosensitizers. Photochem. Photobiol. 63, 161–168 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Mujumdar, R.B., Ernst, L.A., Mujumdar, S.R., Lewis, C.J., & Waggoner, A.S. Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl esters. Bioconj. Chem. 4, 105–111 (1993).

    Article  CAS  Google Scholar 

  54. 54

    Caputo, G. & Della, C.L. Symmetric, monofunctionalised polymethine dyes labelling reagents. European patent 1221465A1 (2002).

Download references


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.

Author information




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.

Corresponding author

Correspondence to Roger Y Tsien.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ngo, J., Adams, S., Deerinck, T. et al. Click-EM for imaging metabolically tagged nonprotein biomolecules. Nat Chem Biol 12, 459–465 (2016).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing