PROJECT SUMMARY/ABSTRACT Isolating individual cells from mixed populations offers a powerful way to study molecular underpinnings of diseases and unravel new targets for drug development. Single-cell isolation is also important for directed evolution screens for engineering biological systems. Despite the growing interest in single-cell isolation techniques from both industry and academia, current methods have a critical limitation. They fail to isolate individual interesting target cells from tissue or 3D cultures. Addressing this problem would enable research in a more physiological environment, providing a more accurate assessment of gene, protein, or cell behaviors and functions. We have recently reported SPOTlight, a versatile all-optical method for isolating single cells within live cultures based on spatial and temporal properties. In SPOTlight, we optically tag target cells exhibiting interesting phenotypes through fluorescence photoactivation using a specialized light-patterning device connected to a microscope. These photoactivated cells, distinguishable by their bright red or green fluorescence, are then isolated with a FACS machine. However, the current SPOTlight setup relies on a 1-photon widefield microscopy setup, limiting its utility for 3D samples. The overall objective of this project is to create a 2-photon (2P) microscopy version of SPOTlight, dubbed 2P-SPOTlight. Our commercialization plan is to provide 2P-SPOTlight as a service to our customers in industry and academia. In Phase I, we will develop the necessary reagents to allow optical labeling of cells within 3D cultures using 2-photon light. We will also develop a 2P-SPOTlight assay to isolate spatially distinct subpopulations of tumor-infiltrating T-cells. We believe this project will satisfy the unmet need to isolate cells from 3D cultures to conduct downstream spatiotemporal omics.