Light- field tomographic fluorescence lifetime imaging microscopy

Fluorescence lifetime imaging microscopy (FLIM) is a powerful imaging technique that enables the visualization of biological samples at the molecular level by measuring the fluorescence decay rate of fluorescent probes. This provides critical information about molecular interactions, environmental changes, and localization within biological systems. However, creating high- resolution lifetime maps using conventional FLIM systems can be challenging, as it often requires extensive scanning that can significantly lengthen acquisition times. This issue is further compounded in three- dimensional (3D) imaging because it demands additional scanning along the depth axis. To tackle this challenge, we developed a computational imaging technique called light- field tomographic FLIM (LIFT- FLIM). Our approach allows for the acquisition of volumetric fluorescence lifetime images in a highly data- efficient manner, significantly reducing the number of scanning steps required compared to conventional point- scanning or line- scanning FLIM imagers. Moreover, LIFT-FLIM enables the measurement of high- dimensional data using low- dimensional detectors, which are typically low cost and feature a higher temporal bandwidth. We demonstrated LIFT-FLIM using a linear single- photon avalanche diode array on various biological systems, showcasing unparalleled single- photon detection sensitivity. Additionally, we expanded the functionality of our method to spectral FLIM and demonstrated its application in high- content multiplexed imaging of lung organoids. LIFT-FLIM has the potential to open up broad avenues in both basic and translational biomedical research.