A key method to gather data from dynamic neural networks in living model organisms is two-photon microscopy together with genetically-encoded fluorescent proteins. There is a need to optimize fluorescent proteins for the two-photon regime, as some desirable characteristics (e.g. brightness) do not translate from one-photon optimization. This project aims to fill that gap with the directed evolution of a two-photon bright green fluorescent protein. Multiple constructs containing this fluorescent protein will be created for use in two-photon imaging. The directed evolution will be implemented using a new optical setup that can collect two-photon excited fluorescence from individual E. coli colonies on an agar plate. The brightest mutants from ~8 rounds of evolution will be crystallized, and one will be made into a tandem dimer. The dimer will be incorporated into four different constructs: 1) with a DREADD receptor, 2) with a channelrhodopsin, 3) in a histone H2B protein fusion, and 4) in a Cre-FLEX-switch expression vector. All constructs will be tested in HEK cells.
Discovering brighter fluorescent proteins for two-photon imaging would enable more high quality information to be extracted from living neural circuits of model organisms. This will enhance the understanding of how the brain works and how to alleviate and prevent neurological disease.
