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Fluorescence recovery after photobleaching and photoconversion in multiple arbitrary regions of interest using a programmable array microscope
Author(s) -
Hagen Guy M.,
Caarls Wouter,
Lidke Keith A.,
De Vries Anthony H.B.,
Fritsch Cornelia,
Barisas B. George,
ArndtJovin Donna J.,
Jovin Thomas M.
Publication year - 2009
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.20686
Subject(s) - photobleaching , fluorescence recovery after photobleaching , fluorescence microscope , confocal microscopy , microscopy , microscope , confocal , fluorescence , biophysics , optical sectioning , chemistry , fluorescence lifetime imaging microscopy , photoactivated localization microscopy , membrane , super resolution microscopy , optics , biochemistry , biology , microbiology and biotechnology , physics
Photomanipulation (photobleaching, photoactivation, or photoconversion) is an essential tool in fluorescence microscopy. Fluorescence recovery after photobleaching (FRAP) is commonly used for the determination of lateral diffusion constants of membrane proteins, and can be conveniently implemented in confocal laser scanning microscopy (CLSM). Such determinations provide important information on molecular dynamics in live cells. However, the CLSM platform is inherently limited for FRAP because of its inflexible raster (spot) scanning format. We have implemented FRAP and photoactivation protocols using structured illumination and detection in a programmable array microscope (PAM). The patterns are arbitrary in number and shape, dynamic and adjustable to and by the sample characteristics. We have used multispot PAM–FRAP to measure the lateral diffusion of the erbB3 (HER3) receptor tyrosine kinase labeled by fusion with mCitrine on untreated cells and after treatment with reagents that perturb the cytoskeleton or plasma membrane or activate coexpressed erbB1 (HER1, the EGF receptor EGFR). We also show the versatility of the PAM for photoactivation in arbitrary regions of interest, in cells expressing erbB3 fused with the photoconvertible fluorescent protein dronpa. dronpa. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.