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An ultrastructural readout of fluorescence recovery after photobleaching using correlative light and electron microscopy

Nature Protocols volume 1, pages 988–994 (2006)Cite this article

Abstract

Fluorescence recovery after photobleaching (FRAP) provides an important quantitative readout of the mobility of fluorescently tagged structures in live tissue. Here we present a protocol for visualizing FRAP signal at the ultrastructural level, permitting the nature of recovered fluorescence signal to be studied at greater resolution than afforded by conventional light microscopy. Specifically we use FRAP, fixation, photoconversion and correlative light and electron microscopy (CLEM) to examine the ultrastructural organization of mobile FM1-43-labeled vesicles in synapses of cultured hippocampal neurons. At photobleached synapses, the FRAP signal can be visualized as photoconverted electron-dense vesicles. The combination of FRAP and CLEM provides a powerful tool for examining the specific localization of imported vesicles in relation to synaptic architecture. Moreover, with the increasing availability of photoconvertible fluorophores, this approach should be readily applicable to other systems where an ultrastructural characterization of FRAP signal is desirable. After cultures are prepared and ready to use, this protocol takes 2–3 days.

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Figure 1: Marking coverslip to assist in relocating ROI during EPON embedding.
Figure 2: Tracking neurons through the different stages of CLEM.
Figure 3: Direct visualization of FM1-43FX labeled vesicles at the ultrastructural level.
Figure 4: Preventing the formation of an FM1-43FX photoconversion product by photobleaching.
Figure 5: An ultrastructural readout of FRAP.

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Acknowledgements

This work was supported by the UK Medical Research Council, the US National Institutes of Health and the National Alliance for Research on Schizophrenia and Depression (NARSAD) in association with the Sidney Baer Trust.

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  1. Kevin J Darcy and Kevin Staras: These authors contributed equally to this work.

Authors and Affiliations

  1. MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, Gower Street, London, WC1E 6BT, UK

    Kevin J Darcy, Kevin Staras & Yukiko Goda

  2. Electron Microscopy Facility, MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, Gower Street, London, WC1E 6BT, UK

    Lucy M Collinson

  3. Department of Pharmacology, University College London, Gower Street, London, WC1E 6BT, UK

    Yukiko Goda

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  1. Kevin J Darcy
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Correspondence to Kevin Staras.

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Darcy, K., Staras, K., Collinson, L. et al. An ultrastructural readout of fluorescence recovery after photobleaching using correlative light and electron microscopy. Nat Protoc 1, 988–994 (2006). https://doi.org/10.1038/nprot.2006.146

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