Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Near-infrared branding efficiently correlates light and electron microscopy


The correlation of light and electron microscopy of complex tissues remains a major challenge. Here we report near-infrared branding (NIRB), which facilitates such correlation by using a pulsed, near-infrared laser to create defined fiducial marks in three dimensions in fixed tissue. As these marks are fluorescent and can be photo-oxidized to generate electron contrast, they can guide re-identification of previously imaged structures as small as dendritic spines by electron microscopy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Defined induction of fiducial marks by NIRB.
Figure 2: NIRB allows re-identification and electron microscopic reconstruction of dendritic spines.

Similar content being viewed by others


  1. Lichtman, J.W. & Fraser, S.E. Nat. Neurosci. 4 (Suppl), 1215–1220 (2001).

    Article  CAS  Google Scholar 

  2. Bourne, J.N. & Harris, K.M. Annu. Rev. Neurosci. 31, 47–67 (2008).

    Article  CAS  Google Scholar 

  3. Rhodin, J.A. & Fujita, H.J. Submicrosc. Cytol. Pathol. 21, 1–34 (1989).

    CAS  Google Scholar 

  4. Polishchuk, R.S. et al. J. Cell Biol. 148, 45–58 (2000).

    Article  CAS  Google Scholar 

  5. Mironov, A.A. & Beznoussenko, G.V. J. Microsc. 235, 308–321 (2009).

    Article  CAS  Google Scholar 

  6. Grabenbauer, M. et al. Nat. Methods 2, 857–862 (2005).

    Article  CAS  Google Scholar 

  7. Sosinsky, G.E., Giepmans, B.N., Deerinck, T.J., Gaietta, G.M. & Ellisman, M.H. Methods Cell Biol. 79, 575–591 (2007).

    Article  CAS  Google Scholar 

  8. Knott, G.W., Holtmaat, A., Trachtenberg, J.T., Svoboda, K. & Welker, E. Nat. Protoc. 4, 1145–1156 (2009).

    Article  CAS  Google Scholar 

  9. Li, J., Erisir, A. & Cline, H. Neuron 69, 273–286 (2011).

    Article  CAS  Google Scholar 

  10. Darcy, K.J., Staras, K., Collinson, L.M. & Goda, Y. Nat. Neurosci. 9, 315–321 (2006).

    Article  CAS  Google Scholar 

  11. Watanabe, S. et al. Nat. Methods 8, 80–84 (2011).

    Article  CAS  Google Scholar 

  12. Mishchenko, Y. et al. Neuron 67, 1009–1020 (2010).

    Article  CAS  Google Scholar 

  13. Kerschensteiner, M., Schwab, M.E., Lichtman, J.W. & Misgeld, T. Nat. Med. 11, 572–577 (2005).

    Article  CAS  Google Scholar 

  14. Denk, W. & Horstmann, W. PLoS Biol. 2, e329 (2004).

    Article  Google Scholar 

  15. Knott, G., Marchman, H., Wall, D. & Lich, B. J. Neurosci. 28, 2959–2964 (2008).

    Article  CAS  Google Scholar 

  16. Feng, G. et al. Neuron 28, 41–51 (2000).

    Article  CAS  Google Scholar 

  17. Jung, S. et al. Mol. Cell. Biol. 20, 4106–4114 (2000).

    Article  CAS  Google Scholar 

  18. Misgeld, T., Nikic, I. & Kerschensteiner, M. Nat. Protoc. 2, 263–268 (2007).

    Article  CAS  Google Scholar 

  19. Majewska, A., Yiu, G. & Yuste, R. Pflugers Arch. 441, 398–408 (2000).

    Article  CAS  Google Scholar 

  20. Bishop, D., Misgeld, T., Walsh, M.K., Gan, W.B. & Lichtman, J.W. Neuron 44, 651–661 (2004).

    Article  CAS  Google Scholar 

  21. Fiala, J.C. J. Microsc. 218, 52–61 (2005).

    Article  CAS  Google Scholar 

Download references


We thank L. Godinho for critical reading of the manuscript. This work was supported by grants to M.K. and T.M. from the Dana Foundation and the Hertie Foundation and a grant to T.M. and D.B. from the Christopher and Dana Reeve Foundation. D.B. is supported by the US National Institutes of Health. Work in M.K.'s laboratory is financed through grants from the Deutsche Forschungsgemeinschaft (Emmy-Noether Program, SFB 571 and SFB 870), the German Federal Ministry of Research and Education (Competence Network Multiple Sclerosis) and the 'Verein Therapieforschung für MS-Kranke e.V.' T.M. is supported by the Institute of Advanced Study (Technische Universität München), the Deutsche Forschungsgemeinschaft (SFB 596), the Bundesministierium für Bildung und Forschung (ERA-Net 'two-photon imaging'), the Alexander von Humboldt Foundation and the Center for Integrated Protein Science (Munich).

Author information

Authors and Affiliations



D.B., M.K. and T.M. conceived the experiments. I.N., S.P., M.K. and T.M. performed in vivo imaging and near-infrared branding experiments. M.B., S.K. and D.B. performed correlated serial electron microscopy. M.K., T.M. and D.B. wrote the paper.

Corresponding authors

Correspondence to Martin Kerschensteiner or Thomas Misgeld.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 (PDF 569 kb)

Supplementary Video 1

Two-photon stack of the NIRB marks burned at different depths (letters N and R were burned at a depth of 15 μm and the letters I and B at a depth of 30 μm) in a cortex section derived from a wild-type mouse as shown in Figure 1a–c. (AVI 2373 kb)

Supplementary Video 2

Time-lapse of NIRB marking in a cortex section derived from a wild-type mouse using a line scan and interspersed scanning with a second laser (trans-illumination image with superimposed NIRB fluorescence, orange). (AVI 2198 kb)

Supplementary Video 3

Movie sequence of the dendritic spine (shown in Figure 2) that illustrates the correlation between the confocal light microscopic image and ssTEM. (WMV 5016 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bishop, D., Nikić, I., Brinkoetter, M. et al. Near-infrared branding efficiently correlates light and electron microscopy. Nat Methods 8, 568–570 (2011).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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