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 high photostability and brightness as well as 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 yeast 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 yeast cells based on the selective measurement of photostability, fluorescence lifetime, or the combined effects of both. In the cytometer (shown schematically in the figure), ten elliptically shaped laser beams intersecting a hydrofocused stream in a microfluidic channel interrogate each cell. Fluorescence lifetime and the initial fluorescence intensity of each cell is measured in the first beam. Cells then traverse eight beams in which the cells are repeatedly excited to introduce low photostability due to photobleaching. In the last beam, the cell’s fluorescence intensity is measured again to assess the degree of photodamage, measured as the ratio of pre- and post-bleaching fluorescence intensities, that occurred to the FP molecules in the cell. Subsequently, a piezo-steered infrared trapping beam deflects cells with desirable properties using optical forces. In our measurements, 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 have also added 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.
In this project, which is a part of a collaboration with Amy Palmer's group at the CU BioFrontiers Institute, we have examined a wide range of genetic libraries based on multiple 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.
Characterization of dark state conversion in fluorescent proteins
Fluorescent proteins are characterized with rich photo-dynamics due to the existence of several non-fluorescent or “dark” states. Upon excitation, FPs get trapped in these dark states via a process called dark state conversion (DSC) and subsequently relax back to the ground states. Recovery of the molecules from these dark states to the ground state (Ground State Recovery, GSR) has profound impact on in-vivo imaging and super-resolution microscopies. We study the dynamics of DSC/GSR and molecular origin of the dark states using various spectroscopic techniques.