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

Darcy, Kevin J, Staras, Kevin, Collinson, Lucy M and Goda, Yukiko (2006) An ultrastructural readout of fluorescence recovery after photobleaching using correlative light and electron microscopy. Nature Protocols, 1. pp. 988-994. ISSN 1754-2189

Full text not available from this repository.

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.

Item Type: Article
Additional Information: Joint first and corresponding author. Devised methodology, participated in all experiments and wrote paper.
Schools and Departments: School of Life Sciences > Neuroscience
Depositing User: Kevin Staras
Date Deposited: 06 Feb 2012 20:04
Last Modified: 26 Mar 2012 08:11
URI: http://sro.sussex.ac.uk/id/eprint/23840
📧 Request an update