Although they have become important tools in biological microscopy, fluorescent proteins (FPs) are dimmer and less photostable than small-molecule fluorophores. These limitations thus limit the signal output needed for next-generation single molecule measurements. The goal of this project is to generate new red fluorescent proteins (RFPs) with higher photostability and a reduced tendency to convert to transient dark states. Our approach to developing new RFPs involves constructing targeted genetic libraries based on existing RFPs, expressing these libraries in mammalian cells and screening the photophysical properties of each cell at high rates in a microfluidic flow cytometer.
To this end, we have developed an innovative microfluidic cytometer for rapidly sorting FP-expressing mammalian cells based on the selective measurement of photostability, magnitude of dark-state conversion, or the combined effects of both. In the cytometer (shown schematically in the figure), eight elliptically shaped laser beams intersecting a hydrofocused stream in a microfluidic channel interrogate each cell. Subsequently, a piezo-steered infrared trapping beam deflects cells for sorting using optical forces. The multi-beam sequence provides millisecond timescales of excitation and dark intervals to separate the effects of reversible dark-state conversion and irreversible photobleaching. We are also in the process of adding single-cell excited-state lifetime measurements to this instrument, to enable screening and sorting on the basis of multiple parameters, including fluorescence lifetime and photobleaching. This technology is uniquely capable of sorting genetic libraries of cell-based FPs on the basis of their excited-state dynamics. Its ability to investigate the various processes that limit the photon output of FPs position this method as a versatile and powerful new tool for characterizing FP photophysics and engineering new FPs.
This project is a collaboration with Amy Palmer's laboratory, whose group is designing various genetic libraries of RFPs. For example, one set of libraries contains sequence diversity vicinity of the chromophore to alter the steric bulk, hydrogen bonding, and local dielectric properties. These libraries are being investigated to uncover relations between mutations in the chromophore pocket and the propensity for triplet state formation, cis-trans isomerization, and excited state proton transfer, all of which which lead to dark state formation. Other libraries with sequence diversity more distant from the chromophore pocket will also be investigated in an attempt to improve barrel rigidity and reduce permeability to molecular oxygen, which is likely to be important in reducing irreversible photobleaching. In general this work will provide insight into the triad of structure-function-dynamics which control the dynamics of excited electronic states in proteins.