Multiplexed video hyperspectral microendoscopy reveals tumor-immune dynamics in vivo

Tumor-immune interactions evolve dynamically within heterogeneous tumor microenvironments, yet methods for repeatedly measuring multiple cellular phenotypes in vivo remain limited. We developed multiplexed video hyperspectral microendoscopy to image fluorescently labeled tumor and immune cell biomarkers in living tissue at cellular resolution. The platform combines photon-efficient spectral detection with rapid spectral unmixing to resolve cocktails of antibody and ligand probes and quantify multiple tumor and immune biomarkers simultaneously in vivo. In mouse tumor models, longitudinal imaging revealed spatially organized tumor and immune cell phenotypes and quantified treatment-associated changes within individual animals. Wide-field mosaics extended the microendoscopic field of view to map biomarker heterogeneity across tumors. These results establish video hyperspectral microendoscopy as a platform for longitudinal measurement of spatially structured tumor-immune dynamics in vivo.