Multi-Parametric Microfluidic Screening and Sorting for Simultaneous Evolution of Photophysical Parameters of Fluorescent Proteins

Our understanding of molecular and cellular biology has been tremendously advanced by genetically encoded fluorescent proteins (FPs). FPs with excellent properties including high brightness and photostability that cover a wide spectral range are now available - but visualization of individual FPs in environments with significant background fluorescence such as fixed and live cells remains challenging. To overcome this, new technologies are under development and commonly utilize dark states that are ubiquitous in FPs. Enhanced image contrast is obtained by selectively modulating the FP fluorescence while holding the background level constant which enables background subtraction, resulting in increased signal-to-background ratios. As an example, SAFIRe has been shown to yield 100-fold improvements in probe visibility, allowing for single molecule detection even with high background fluorescence levels present. A common bottleneck is limited modulation depth as current generation FPs are not optimized for favorable dark state kinetics.We present a microfluidic cell sorter that serves as a platform to evolve properties not accessible by traditional fluorescence-activated cell sorters. The key advantage of microfluidic platforms is that dark states and photobleaching can be characterized in-flow as typical cell velocities enable measurements on the timescale of their kinetics. Furthermore, multi-beam experiments are accessible and allow for multiple measurements on the same cell. Our sorter is capable of multiparameter screening and sorting including properties such as fluorescence intensity, photostability, dark state kinetics and fluorescence lifetime. We demonstrate its capabilities through directed evolution experiments, evolving an array of novel red-fluorescent proteins using mCherry as a parent. Generated FPs exhibit properties including higher brightness, increased conversion efficiency into dark states upon photoexcitation as well as fluorescent lifetimes twice as long as their parent and are possible candidates for use in frequency domain imaging techniques.