Single-cell proteomics has the potential to decipher tumor heterogeneity, and a method like single-cell proteomics by mass spectrometry (SCoPE-MS) allows profiling several tens of single cells for >1,000 proteins per cell. This method, however, cannot link the proteome of individual cells with phenotypes of interest. Here, we developed a microscopy-based functional single-cell proteomic-profiling technology, called FUNpro, to address this. FUNpro enables screening, identification, and isolation of single cells of interest in a real-time fashion, even if the phenotypes are dynamic or the cells of interest are rare. We applied FUNpro to proteomically profile a newly identified small subpopulation of U2OS osteosarcoma cells displaying an abnormal, prolonged DNA damage response (DDR) after ionizing radiation (IR). With this, we identified the PDS5A protein contributing to the abnormal DDR dynamics and helping the cells survive after IR.

Linking single-cell genomic or transcriptomic profiles to functional cellular characteristics, in particular time-varying phenotypic changes, could help unravel molecular mechanisms driving the growth of tumour-cell subpopulations. Here we show that a custom-built optical microscope with an ultrawide field of view, fast automated image analysis and a dye activatable by visible light enables the screening and selective photolabelling of cells of interest in large heterogeneous cell populations on the basis of specific functional cellular dynamics, such as fast migration, morphological variation, small-molecule uptake or cell division. Combining such functional single-cell selection with single-cell RNA sequencing allowed us to (1) functionally annotate the transcriptomic profiles of fast-migrating and spindle-shaped MCF10A cells, of fast-migrating MDA-MB-231 cells and of patient-derived head-and-neck squamous carcinoma cells, and (2) identify critical genes and pathways driving aggressive migration and mesenchymal-like morphology in these cells. Functional single-cell selection upstream of single-cell sequencing does not depend on molecular biomarkers, allows for the enrichment of sparse subpopulations of cells, and can facilitate the identification and understanding of the molecular mechanisms underlying functional phenotypes.

Photoactivated genetically encoded voltage indicators (GEVIs) have the potential to enable optically sectioned voltage imaging at the intersection of a photoactivation beam and an imaging beam. We developed a pooled high-throughput screen to identify archaerhodopsin mutants with enhanced photoactivation. After screening ~10⁵ cells, we identified a novel GEVI, NovArch, whose one-photon near-infrared fluorescence is reversibly enhanced by weak one-photon blue or two-photon near-infrared excitation. Because the photoactivation leads to fluorescent signals catalytically rather than stoichiometrically, high fluorescence signals, optical sectioning, and high time resolution are achieved simultaneously at modest blue or two-photon laser power. We demonstrate applications of the combined molecular and optical tools to optical mapping of membrane voltage in distal dendrites in acute mouse brain slices and in spontaneously active neurons in vivo.