Optical principles of high-speed fluorescence lifetime imaging microscopy for flow cytometry

Abstract Fluorescence lifetime imaging microscopy (FLIM) has emerged as a powerful tool in biology and medicine, as it can circumvent experimental artifacts inherent to conventional fluorescence-intensity-based measurements. However, FLIM has traditionally suffered from limited imaging speed, primarily due to the requirement for time-resolved fluorescence detection. Recently, we developed a high-speed FLIM approach and demonstrated the acquisition of more than 10,000 cell images per second. This review introduces the key optical aspects underlying this high-speed FLIM to a broad readership. Specifically, simultaneous multi-point fluorescence lifetime measurement is achieved by generating and exploiting spatially dispersed, intensity-modulated dual-beam arrays. This unique excitation scheme enables efficient acceleration of FLIM while mitigating frequency-dependent bias arising from multi-component fluorescence lifetimes. The developed high-speed FLIM and FLIM-based flow cytometry are expected to serve as versatile imaging platforms for a wide range of biomedical applications.