Improved Fluorescent Protein Contrast and Discrimination by Optically Controlling Dark State Lifetimes

Abstract

Modulation and optical control of photoswitchable fluorescent protein (PS-FP) dark state lifetimes drastically improves sensitivity and selectivity in fluorescence imaging. The dark state population of PS-FPs generates an out-of-phase fluorescence component relative to the sinusoidally modulated 488nm laser excitation. Because this apparent phase advanced emission results from slow recovery to the fluorescent manifold, we hasten recovery and, therefore, modulation frequency by varying co-illumination intensity at 405nm. As 405nm illumination regenerates the fluorescent ground state more rapidly than via thermal recovery, we experimentally demonstrate that secondary illumination can control PS-FPs dark state lifetime to act as an additional dimension for discriminating spatially and spectrally overlapping emitters. This experimental combination of out of phase imaging after optical modulation (OPIOM) and synchronously amplified fluorescence image recovery (SAFIRe) optically controls the fluorescent protein dark state lifetimes for improved time resolution, with the resulting modulation-based selective signal recovery being quantitatively modeled. The combined experimental results and quantitative numerical simulations further demonstrate the potential of SAFIRe-OPIOM for wide-field biological imaging with improved speed, sensitivity, and optical resolution over other modulation-based fluorescence microscopies.

Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

“…To test this hypothesis, future experiments should probe the performance of Enhancer-bound rsGreens in RESOLFT-type imaging compared to the unbound rsGreens and other existing labels. Additionally, the fact that binding of a protein partner changes the switching kinetics opens up the possibility for Enhancer binding to serve as a contrast mechanism for advanced imaging techniques which can discern fluorescent labels with different photochromic behavior, such as lock-in detection [ 34 , 35 , 36 ], multitau (mt)-pcSOFI [ 37 ] or -RESOLFT [ 38 ]. A highly interesting prospect in this direction is that modulation of photochromism through binding with interaction partners could revolutionize biosensor design by providing a (ratiometric and super-resolution) readout mechanism, which is easy to multiplex with other fluorescence measurements due to limited usage of the visible spectrum [ 39 , 40 ].…”

Quantification of the Depolarization and Anisotropy of Fluorophore Stokes-Shifted Fluorescence, On-Resonance Fluorescence, and Rayleigh Scattering

“…Existing research on the fluorophore optical properties has focused predominantly on its photon absorption and the Stokes-shifted fluorescence (SSF) in which emitted photons have longer wavelengths than that of the excitation photon. There are many SSF-based analytical methods that are based on SSF intensities, − lifetimes, − and anisotropies. − There is scant information on fluorophore photon scattering and on-resonance fluorescence (ORF) in which the scattered or the emitted photons have the same wavelength as the excitation photons. This is in spite of the fact that molecular ORF has been depicted in the standard Jablonski energy level diagram for decades, and photon scattering is a universal material property because all materials have nonzero polarizability.…”

Optically Activated Delayed Fluorescence

“…32 As endogenous emitters are not modulatable, only the signal of interest is recovered at the modulation frequency to simultaneously improve signal sensitivity and discrimination. 26, 32 …”