Developing Red Fluorescent Proteins – Tools and Strategies*

Fluorescent proteins have become an indispensable tool for biological and biomedical research owing to their genetic encodability. Red Fluorescent Proteins (RFPs) are advantageous for live-cell imaging due to low optical attenuation and phototoxicity for excitation beyond 550 nm. We are devoted to developing novel strategies and tools to improve RFPs with multiple desirable photophysical properties. The most commonly adopted strategy to enhance molecular brightness of RFPs is to reduce the nonradiative decay pathways, which lengthens the excited state lifetime and possibly reduces photostability due to the longer time spent in the excited state. We propose to simultaneously improve molecular brightness and photostability of RFPs by increasing the radiative decay rate. We also develop microfluidic sorters capable of selections based on multiple photophysical properties. We use these instruments to accelerate and visualize the evolution of RFPs towards desirable photophysical properties for library sizes on the order of 10⁶ mutants.