The push for new fluorophores in single molecule imaging is motivated by sensitivity constraints, especially in biological samples. Normally detrimental, blinking limits emission rates by transitioning to photoaccessible dark states. We have developed the capability to optically rescue fluorophores from these dark states to dynamically modulate detected fluorescence. Using photoreversible isomerizations, we have identified specific green fluorescent mutants capable of optical modulation and signal recovery from high background. Single molecule and bulk spectroscopy agree with Monte Carlo simulations and rate analyses to explain chromophore dynamics influencing modulation depth and frequency. Signals from cells are readily recovered with >10-fold improvements in signal:noise. These coupled experimental and model-based studies provide a way to increase sensitivity in high background bulk and single molecule bioimaging, while simultaneously yielding bulk spectroscopic methods to yield dark state kinetic parameters. These studies offer another dimension enabling dark state lifetime-based discrimination of fluorophores with otherwise identical photophysics.

Optically modulated GFPs for single molecule and bulk bioimaging

243rd National Meeting (2012)

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