Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells.
Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations.
Clayton
Andrew H A
AH
Ludwig Institute for Cancer Research, Royal Melbourne Hospital, P.O. Box 2008, 3050 Parkville, Victoria , Australia. Andrew.Clayton@ludwig.edu.au
Klonis
Nectarios
N
Cody
Stephen H
SH
Nice
Edouard C
EC
D000818:Animals; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000378:metabolism*; D003208:Concanavalin A / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*
D003208:Concanavalin A