Super-resolution imaging flow cytometry

Organelle architecture is a key determinant of cellular state. However, its heterogeneity remains difficult to quantify at population scale because conventional imaging flow cytometry is diffraction-limited. Here we present super-resolution imaging flow cytometry (SR-IFC), a high-throughput structured-illumination platform combining stroboscopic excitation, microfluidic hydrodynamic focusing and AI-assisted reconstruction. From three phase-shifted frames acquired along the flow axis, SR-IFC reconstructs images with approximately 118-nm isotropic in-plane resolution, about two-fold beyond the diffraction limit, at up to approximately 720 cells per second. In microbiology, SR-IFC resolves individual lipid droplets in nitrogen-starved Euglena gracilis and reveals spatial reorganization during lipid biogenesis missed by diffraction-limited imaging. In immunology, it maps activation-associated mitochondrial remodeling across primary human CD8 ⁺ T-cell differentiation, including a transient networked state. SR-IFC thus provides a broadly applicable platform for population-scale super-resolution phenotyping across diverse areas of biology.