Two-dimensionally expanded flow cytometry with an array of time-gated single photon avalanche diodes.

Conventional flow cytometry typically relies on narrow fluidic channels and optical filters, which can limit the handling of varied sample types and increase system complexity. Here we present two-dimensionally expanded flow cytometry (2DFC) employing an array of time-gated single-photon avalanche diodes (SPADs) to enable parallelized, high-throughput fluorescence detection of single cells and cell clusters of various sizes. In 2DFC, individual time-gated SPADs serve as interrogation points of the flow cytometer, enabling two-dimensional expansion of the interrogation area through an array of SPADs integrated into the bottom surface of a wide microfluidic channel. By temporally gating the SPADs following pulsed laser excitation, fluorescence signals are separated from excitation light in the time domain, enabling direct detection of fluorescence from cells flowing over the SPAD array without the need for optical filters. Leveraging the scalable architecture of the time-gated SPAD array, 2DFC successfully detects both individual cells and multicellular clusters. By tuning the accumulation count for SPAD detection, 2DFC successfully resolves cellular fluorescence signals at a cell concentration of 1 × 106 cells/mL and a volumetric flow rate of up to 1 mL/min without fluidic focusing mechanisms. These results demonstrate the potential of 2DFC as a scalable platform for high-throughput analysis of heterogeneous cell suspensions in biomedical research and clinical diagnosis.