FRET acceptor photobleaching.In vitro FRET were evaluated by the FRET acceptor photobleaching method using AZD1152-HQPA confocal laser-scanning microscope (LSM 510 meta; Carl Zeiss, Inc.) equipped with a Plan-Apochromat 63×/1.4 oil objective. ECFP and EYFP images were sequentially collected prior to photobleaching of the acceptor. ECFP was excited at 405 nm at moderate laser power, and emissions were detected using a 420–480 nm bandpass emission filter. EYFP was excited at 514 nm at moderate laser power, and emission was detected using a 530–600 nm bandpass emission filter. After image collection, EYFP was bleached at 514 nm at 100% laser power.
Results and discussion
Theory and experimental design concept
A unique feature of the FRET assay is the enhanced emission of the acceptor and quenching of the donor emission, which results from the energy transfer governed by the donor–acceptor interaction in response to the excitation of the donor [19]. In theory, it is important to note that the FRET efficiency (E) is inversely proportional to the sixth power of the distance between the two fluorophores, as determined by the following Eq. (1):
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