LFC-plus: simultaneous multicolour volume cytometry for high-throughput single-cell analysis

Imaging flow cytometry demands a careful balance between spatial resolution, spectral multiplexing, throughput, and system complexity. Here, we present LFC-plus, a next-generation light-field cytometry platform that enables multiparametric, simultaneous multi-color, and volumetric single-cell analysis. The system integrates model-based image restoration, custom-designed light-field optics, and spectral aperture partitioning, achieving subcellular resolution in all three dimensions, a near-millimeter-scale imaging cross-section, and an analytical imaging throughput of nearly 200 000 cells per second. We validate its performance across diverse biological applications, including chemotherapy response profiling, PEG-mediated cell fusion, and stiffness-based flow migration. These results establish LFC-plus as a robust and scalable platform for high-content volumetric cytometry, with broad implications spanning fundamental biology and translational diagnostics.