FRET-Enabled Optical Modulation for High Sensitivity Fluorescence Imaging

Abstract

Fluorescence resonance energy transfer is utilized to engineer donor photophysics for facile signal amplification and selective fluorescence recovery from high background. This is generalized such that many different fluorophores can be used in optical modulation schemes to drastically improve fluorescence imaging sensitivity. Dynamic, simultaneous, direct excitation of the acceptor brightens and optically modulates higher energy donor emission. The externally-imposed modulation waveform enables selective donor fluorescence extraction through demodulation. By incorporating an acceptor with significant, spectrally-shifted, dark state population, necessary excitation intensities are quite low and agree well with simulated enhancements. Enhancement vs. modulation frequency directly yields dark state lifetimes in a simple ensemble measurement. Using the long-lived Cy5 dark state in conjunction with Cy3 donors, we demonstrate image extraction from a large background to yield ≫10-fold sensitivity improvements through Synchronously Amplified Fluorescence Image Recovery (SAFIRe).

Optical Imaging with Dynamic Contrast Agents

“…The same strategy has recently been applied toward donor–acceptor dye pairs used in fluorescence energy resonance transfer (FRET), coupling dynamic contrast enhancement with biomolecular signaling. [83] Fluorescence signal demodulation by FT resulted in frequency-selective images with enhanced signal quality and image contrast, similar to the examples discussed above involving MM modulation. [48,49]…”

All-optical fluorescence image recovery using modulated stimulated emission depletion

“…As the secondary laser is of longer wavelength than is the collected fluorescence, the STED experiment becomes directly analogous to SAFIRe, 20–22 using the fluorescent level as the “dark” state analog of previous efforts (Figure 1). Essential for modulation, when the depletion laser is on, fluorescence is reliably suppressed, but the modulation depth tolerance enables working with lower intensities than necessary for super-resolution imaging.…”

“…The corresponding amplitude at the modulation frequency of each pixel was used to recover the demodulated image. 20–22 …”

“…34 Complementary to fluorescence modulation experiments, 20–22 secondary laser induced partial and repetitive optical depletion of the fluorescent state should also lead to strongly modulated fluorescence. 33 Thus, by implementing Stimulated Emission Depletion (STED) for fluorophore modulation, one should be able to repetitively suppress fluorescence at any desired modulation frequency.…”

“…Recent efforts have extended these lessons in Ag nanodot modulation to a series of xanthene dyes, 21 and most recently by optically inhibiting FRET between donor and acceptor through acceptor saturation. 22 The modulation frequency is limited by the lifetimes of the states involved, making long-lived photoswitches 3,5 or chemically induced transitions 23 possible alternative schemes for slower signal recovery applications.…”