Pinhole engineering based enhanced resolution (PEER) for fluorescence lifetime imaging microscopy

In this article, we present a differential confocal microscopy technique based on pinhole engineering that significantly enhances both lateral resolution and axial sectioning depth. Through simulations and experimental validation with a fluorescent calibration slide, we demonstrated a 1.6-fold improvement in lateral resolution and a two-fold increase in axial sectioning capability. Building on these advancements, we integrated an intensity-weighted lifetime imaging strategy to surpass the diffraction limit in fluorescence lifetime measurements. This approach achieved high spatial resolution and quantitative lifetime data comparable to leading super-resolution FLIM (Fluorescence Lifetime Imaging Microscopy) techniques, yet allows for simpler implementation. We further validated the method in subcellular structure lifetime imaging, demonstrating improved resolution and axial depth enhanced lifetime-based multiplexing capacity. This new method provides an accessible route to high-resolution, multiplexed FLIM for advanced biological imaging.