Development and Application of Avalanche Photodiode-triggered Spectral Nano-Flow Cytometry System Based on Field-Programmable Gate Array

Spectral nano-flow cytometry enables multiparameter characterization of nanoscale biological particles, such as viruses and extracellular vesicles, at single-particle level. However, its performance is limited by the conventional photomultiplier tube (PMT)-triggered electron-multiplying charge-coupled device (EMCCD) exposure strategy, which suffers from a high lower-size detection limit and significant background noise. To address these limitations, a high-sensitivity spectral nano-flow cytometry system based on a field-programmable gate array (FPGA)-controlled single photon counting avalanche photodiode (APD) triggering mechanism was developed in this work. Key innovations included replacing the PMT with a high-quantum-efficiency APD combined with optical spatial filtering to substantially suppress background noise, implementing an FPGA-based high-speed signal processing chain that achieved precise identification and synchronous triggering of APD pulse signals within a 0.2–30 MHz frequency range using equal-precision measurement and threshold comparison algorithms, and optimizing the spectral detection module to enhance fluorescence collection efficiency and spectral resolution. Experimental results demonstrated that the system clearly distinguished a mixture of fluorescent silica beads with molecules of equivalent soluble fluorochrome (MESF) values of 574 and 1438. When detecting Di-8-ANEPPS-labeled red blood cell-derived extracellular vesicles (RBC EVs), the false-trigger rate was reduced from >70% (with PMT triggering) to