ABSTRACT The systems-wide analysis of biomolecules in time and space is key to our understanding of cellular function and heterogeneity in health and disease 1 . Remarkable technological progress in microscopy and multi-omics technologies enable increasingly data-rich descriptions of tissue heterogeneity 2,3,4,5 . Single cell sequencing, in particular, now routinely allows the mapping of cell types and states uncovering tremendous complexity 6 . Yet, an unaddressed challenge is the development of a method that would directly connect the visual dimension with the molecular phenotype and in particular with the unbiased characterization of proteomes, a close proxy for cellular function. Here we introduce Deep Visual Proteomics (DVP), which combines advances in artificial intelligence (AI)-driven image analysis of cellular phenotypes with automated single cell laser microdissection and ultra-high sensitivity mass spectrometry 7 . DVP links protein abundance to complex cellular or subcellular phenotypes while preserving spatial context. Individually excising nuclei from cell culture, we classified distinct cell states with proteomic profiles defined by known and novel proteins. AI also discovered rare cells with distinct morphology, whose potential function was revealed by proteomics. Applied to archival tissue of salivary gland carcinoma, our generic workflow characterized proteomic differences between normal-appearing and adjacent cancer cells, without admixture of background from unrelated cells or extracellular matrix. In melanoma, DVP revealed immune system and DNA replication related prognostic markers that appeared only in specific tumor regions. Thus, DVP provides unprecedented molecular insights into cell and disease biology while retaining spatial information.

AI-driven Deep Visual Proteomics defines cell identity and heterogeneity