FRET-Enabled Optical Modulation for High Sensitivity Fluorescence Imaging

摘要

Abstract: Fluorescence resonance energy transfer is utilized to engineer donor photophysics for facilensignal amplification and selective fluorescence recovery from high background. This is generalized suchnthat many different fluorophores can be used in optical modulation schemes to drastically improvenfluorescence imaging sensitivity. Dynamic, simultaneous, and direct excitation of the acceptor brightensnand optically modulates higher energy donor emission. The externally imposed modulation waveform enablesnselective donor fluorescence extraction through demodulation. By incorporating an acceptor with significant,nspectrally shifted, dark-state population, necessary excitation intensities are quite low and agree well withnsimulated enhancements. Enhancement versus modulation frequency directly yields dark-state lifetimesnin a simple ensemble measurement. Using the long-lived Cy5 dark state in conjunction with Cy3 donors,nwe demonstrate image extraction from a large background to yield 10-fold sensitivity improvements throughnsynchronously amplified fluorescence image recovery (SAFIRe)..