Paired single-cell imaging of calcium and expression to map niches of identity and function

Cellular identity is often inferred from molecular markers, while function is measured independently, obscuring how these dimensions align at single-cell resolution. In cardiomyocytes, this disconnect is especially limiting, as calcium dynamics and subtype markers are typically assessed in bulk or separate cells then averaged across populations. In human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), this gap has limited our ability to determine whether heterogeneity in electrophysiology and calcium handling reflects noise, maturation, or structured biological states. This lack of clarity is due in part to the lack of methods that directly link live functional measurements with molecular identity at single-cell resolution. Here, we introduce CARBONITE (Calcium Recordings Before Identification by Expression), a scalable single-cell imaging framework that pairs live calcium dynamics with protein expression and spatial phenotyping. Using high-content imaging in 96-well plates, CARBONITE integrates per-cell calcium transient features with immunofluorescent identification applied to cardiomyocyte subtype markers, enabling quantitative functional-molecular mapping within the same cells. Applying CARBONITE to mixed human induced pluripotent stem cell derived cardiomyocyte (iPSC-CM) populations reveals heterogeneity in calcium transient dynamics and marker expression within individual wells. Canonical atrial and ventricular markers capture only a subset of functional variability. Notably, calcium transient shape segregates cells into two discrete functional states that exhibit perinuclear ANP (atrial marker) enrichment and nucleation state (i.e., mono- vs. binucleation). Notably, these groups show know relationship to MYL2 (ventricular marker) expression. Binucleated cells are more likely to exhibit a spike-like calcium transient, identifying nucleation as a dominant and previously underappreciated axis of cardiomyocyte identity influencing calcium function. Together, these results establish CARBONITE as a functional multimodal single-cell platform that reveals organizational principles of cell identity, providing a foundation for dissecting functional niches in development and disease.