Multifocal Pixel/Photon-Reassignment FLIM (MPPR-FLIM): A Super-Resolution Analytical Tool for Characterizing...

With the rapid progress of biomedical research, fluorescence lifetime imaging microscopy (FLIM) has been increasingly applied as a powerful tool to probe the microenvironmental properties of cells and tissues. However, conventional FLIM suffers from intrinsically limited spatial resolution, making it difficult to resolve the microenvironmental heterogeneity at the subcellular level. Although several approaches have been proposed to enhance the spatial resolution of FLIM, they often require custom-made optical components that hinder their practical implementation and widespread adoption. In this study, we present an alternative super-resolution FLIM technique termed multifocal pixel/photon-reassignment FLIM (MPPR-FLIM). This method employs a wide-field detector commonly used in super-resolution fluorescence intensity imaging, combined with a single-point detector and a time-correlated single-photon counting (TCSPC) module, both of which are standard components in conventional FLIM systems (no custom fabrication required). By simultaneously acquiring structural and lifetime information from the sample using a specially designed synchronization strategy, coupled with a postacquisition data remapping algorithm integrating both pixel reassignment and photon reassignment, MPPR-FLIM achieves a ∼2.1-fold improvement in spatial resolution compared with conventional TCSPC-FLIM systems. Furthermore, through the imaging of lysosomes in live cells, we demonstrate the potential of MPPR-FLIM for characterizing subcellular compartment-specific fluorescence lifetime variations. This capability may facilitate investigations into the potential relationship between subcellular lifetime heterogeneity and cellular functions, offering a practical and accessible super-resolution analytical tool for biomedical and life science research.