BPS2026 – Combining ISM and FLIM for high-fidelity lifetime imaging in dense environments

The integration of confocal fluorescence microscopy with single-photon detection and time-correlated single-photon counting (TCSPC) has enabled fluorescence lifetime imaging microscopy (FLIM), providing unique functional, environmental, and metabolic insights by monitoring the excited state lifetime of fluorophores. Despite the continuous development of FLIM-capable cameras, traditional confocal laser scanning approaches utilizing point-detectors still provide the highest quality lifetime data by offering both high spatial resolution and precise optical sectioning. However, FLIM analysis usually is performed on a pixel-by-pixel basis, provides limited spatial resolution (especially when spatial averaging is applied in low signal-to-noise scenarios), and suffers from out-of-focus contributions that contaminate the estimated lifetime values. With the recent introduction of high performance SPAD-arrays, confocal microscopes can be readily upgraded to image scanning microscopes (ISM) for a significant gain in spatial resolution, however, the combination of ISM with FLIM has not been fully realized. Data processing plays a pivotal role in ISM to both improve the lateral resolution by photon reassignment and deconvolution and to provide computational sectioning for two-dimensional images acquired at a single z-position. Here, we present a novel approach to seamlessly integrate the lifetime information into the ISM workflow. Our pipeline extracts spatially deconvolved and computationally sectioned FLIM images on a continuous lifetime support (i.e., no discretization or pattern matching), providing both improved spatial resolution, out-of-focus rejection, and improved FLIM contrast. Importantly, by accounting for the spatial blur of the FLIM information in the deconvolution procedure, the precision of the lifetime estimate is markedly improved. Our approach provides a general strategy to incorporate the fluorescence lifetime dimension into advanced imaging workflows and is readily applicable to volumetric imaging using standard confocal z-stacks or other non-ISM FLIM modalities, such as lightsheet or spinning disc microscopy, when combined with FLIM capable cameras.