Microfluidic selection strategy targeting the excited-state dynamics of fluorescent proteins

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Venue:Academic Health Center 3-205

Abstract:

Genetically encoded fluorescent proteins (FPs) have enabled explorations of cellular dynamics with unprecedented spatio-temporal resolution. Compared to the best fluorescent dyes, however, the complex excited-state dynamics of FPs result in ~100-fold accelerated photobleaching and dark state conversion. These properties limit the imaging duration and signal output in fluorescence microscopy and compromise the widespread use of FPs in single-molecule or low-copy fluorescence imaging. We developed a cell-based library screening method to improve the photophysics of FPs with a microfluidic cell sorter that measures fluorescence photobleaching characteristics of individual cells. Viable cells retrieved from a sort can be used either in subsequent rounds of screening, or these clones may be mutated further (i.e. enabling “directed evolution.”)

We demonstrate this selection approach on a >105 member saturation mutagenesis library of the mCherry chromophore environment in a region where computational studies predict the presence of thermally accessible pathways for O2 entry to the chromophore. Multiple rounds of selection on this library have enabled the isolation of new clones with improved photostability. Finally, we have integrated phase-fluorometry capabilities into the instrument, which will enable cell sorting on the basis of both photobleaching and fluorescence lifetime. Our approach will not only enable the creation of new generations of FPs with excited-state dynamics optimized for brighter, long-duration imaging, but it can also be extended to enable development of FPs for novel imaging modalities.