Optical Lock-In Detection of FRET Using Synthetic and Genetically Encoded Optical Switches
- Mao, Shu
- Benninger, Richard K.P.
- Yan, Yuling
- Petchprayoon, Chutima
- Jackson, David
- Easley, Christopher J.
- Piston, David W.
- Marriott, Gerard
DOIAbstract
AbstractThe Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a ...
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