Imaging flow cytometry using linear array spot excitation

Imaging flow cytometry is a powerful tool for single-cell analysis or sorting, combining high-throughput flow cytometry and high-content microscopy. However, its scalability and utility are limited by the fundamental trade-off between throughput, sensitivity, and resolution when cells are flowing quickly. To overcome these limitations, linear array spot excitation (LASE) sequentially illuminates vertical profiles of single cells at successive horizontal positions using a line of equally spaced dots. The cell images are subsequently decoded from signals detected by single-pixel photomultipliers (PMTs) to reconstruct label-free and fluorescence images of cells. A LASE imaging flow cytometer was built with two lasers and five imaging channels, achieving a theoretical maximum throughput of >5,000 cells/s and a resolution of 1.3 μm. Moreover, we conceptionally demonstrated a spectral imaging flow cytometer with 32 hyperspectral imaging channels for the first time. With its simplicity, scalability, and compatibility with conventional flow cytometers, LASE shows great promise for widespread biomedical applications.