Deep phenotypic profiling of small clinical samples through MultiSpectral Imaging Cytometry (MuSIC) in nanowell arrays

Many disease relevant immune processes occur within tissues, making them challenging to monitor clinically using standard blood based assays. Comprehensive phenotyping of the scarce cells acquired directly from tissue samples is limited by inefficiencies in sample handling and data collection inherent in current flow-based profiling methods. To overcome this barrier, we developed Multi-Spectral Imaging Cytometry (MuSIC), a method that combines an array of sub-nanoliter wells (nanowells) for efficient cell capture with multi-spectral imaging to resolve up to 16 fluorescent markers on ~10 3 –10 5 cells. Importantly, the cells remain viable for further functional characterization, enabling linkage of deep cell surface phenotypes with functional activity. Using MuSIC in combination with a single-cell protein secretion assay, we were able to identify unique T cell populations present in human cervical biopsies. MuSIC provides an effective and accessible means to acquire deep phenotypic profiles of leukocytes from small clinical samples.