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An ultrastructural readout of fluorescence recovery after photobleaching using correlative light and electron microscopy.
journal contribution
posted on 2023-06-08, 04:50 authored by Kevin J Darcy, Kevin StarasKevin Staras, Lucy M Collinson, Yukiko GodaFluorescence 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.
History
Publication status
- Published
Journal
Nature ProtocolsISSN
1754-2189External DOI
Volume
1Page range
988-994Pages
7.0Department affiliated with
- Neuroscience Publications
Notes
Joint first and corresponding author. Devised methodology, participated in all experiments and wrote paper.Full text available
- No
Peer reviewed?
- Yes
Legacy Posted Date
2012-02-06Usage metrics
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