PROJECT SUMMARY/ABSTRACT Fluorescent proteins are ubiquitous reagents in the biomedical sciences for reporting gene expression, protein and nucleic acid localization, cell shape, and cellular activity. However, fluorescent proteins (FPs) become progressively dimmer — they photobleach — with repeated or prolonged illumination. Photobleaching limits multiple types of biological experiments where photostability is essential, such as single-molecule biophysics and timelapse imaging of cellular activity during development, learning, and aging. Photobleaching often cannot simply be addressed by increasing the excitation light, as high illumination power can induce membrane blebbing, nuclear fragmentation, alterations in the cell cycle, changes to the concentration of intracellular calcium, and, ultimately, cell death. While over two decades of FP engineering has led to a toolbox of bright FPs, less attention has been devoted to improving photostability because of the greater difficulty and lower throughput endured when screening for photostable FPs. Moreover, few studies have attempted to improve photophysical properties under two-photon illumination — a method of choice for deep-tissue imaging — because of technical challenges associated with screening under this imaging modality. The overall objective of this research proposal is, therefore, to develop and apply a color palette of bright and photostable FPs for one- and two-photon imaging in mammalian cells. Our proposal leverages two specialized and synergistic approaches to FP discovery and engineering: (1) SPOTlight, a new all- optical screening approach developed in Dr. St-Pierre's lab that circumvents technical hurdles and enables rapid screening of both brightness and photostability at the single-cell level under one- and two-photon illumination; and (2) transcriptomic and metagenomic mining for novel FPs from marine invertebrates, a technique pioneered by Dr. Shaner’s lab. SPOTlight relies on light patterning technology to selectively illuminate individual cells labeled with fluorophores that can be photoactivated from a dim to a bright state. The cells are therefore tagged with a unique fluorescence signature that can then be distinguished and retrieved using Fluorescence Activated Cell Sorting (FACS). SPOTlight thus enables screening in dense mixed cultures with single-cell resolution, thereby eclipsing the throughput of traditional well-based approaches. Mining for novel FPs in marine invertebrate transcriptomes and metagenomes will allow us to rapidly identify and characterize hundreds of novel FPs. From this pool of new FPs, we will select the most photostable for engineering with the SPOTlight pipeline. We will also model their structures to guide site-directed mutagenesis. We propose to leverage these new technologies and assays to develop FPs of different colors that are bright, monomeric, and sufficiently photostable for long-term imaging experiments. We also propose to apply these ne...