We optically control residence time in dark states to dynamically modulate fluorescent protein emission. Demodulation of collected fluorescence drastically improves sensitivity and selectivity in fluorescence imaging, rejecting autofluorescence and distinguishing multiple spectrally overlapped fluorophores based on their characteristic dark state lifetimes. Such optically modulated fluorescent proteins (OMFPs) provide tools to study low-abundance species in high background, resolve species separated below the diffraction limit, and study both dynamics and equilibria in living cells, all without interference from heterogeneous background sources. Importantly, recovered OMFP intensities are directly proportional to protein concentration at all points within the cell. By tailoring excitation sources to each OMFP’s unique photophysics, the sensitivity and selectivity improvements of optically modulated fluorescence imaging are readily achieved on commercial microscopes, opening avenues for wider use in biological imaging. These implementations of synchronously amplified fluorescence image recovery (SAFIRe) demonstrate the potential of SAFIRe for wide-field live cell imaging with excellent speed, sensitivity, and optical resolution. Applications to viral tracking and visualizing immobile proteins in a background of mobile fluorophores will be shown, highlighting the advantages of this approach.

Optically modulated fluorescent proteins for improved fluorescence microscopy 1

2019 Spring National Meeting

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