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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.

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


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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).

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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.

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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)

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

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