Three-dimensional forward-scattering imaging flow cytometry system for single-cell analysis.

Label-free single-cell 3D imaging is essential for accurate phenotyping by minimizing perturbations to native cellular states and facilitating downstream molecular analyses. Among the available modalities, 3D forward-scattering (dark-field) imaging offers the richest subcellular details but faces challenges from strong transmitted-beam interference. We present a high-throughput 3D forward-scattering imaging flow cytometry system employing optical needle-beam illumination, linear micro-mirror arrays for axial scatter detection, and spatiotemporal deconvolution algorithms. Experimental validations with microstructures, hydrogel beads, and HEK-293 cells confirm the method's capability for robust subcellular resolution at ∼400 cells/s, enabling advanced label-free cellular diagnostics and analysis. This platform enables label-free, high-content cellular analysis and is readily adaptable to integrated cell sorting and AI-driven classification, offering broad potential for biomedical research and diagnostics.