Super-resolution imaging using fluorescence-lifetime emission-difference

The diffraction of light limits the ability of light microscopes to resolve biological structures and intracellular processes with nanoscale spatial information. Fluorescence emission difference (FED) improves image contrast and effective spatial resolution of confocal microscopy by alternating the excitation of the sample with a Gaussian and donut-shaped laser beam. Improvement in image resolution is obtained by subtracting images captured with both excitation modes. The combination of this technique with fluorescence lifetime imaging microscopy (FLIM) has remained limited. Here, we propose a super-resolution fluorescence-lifetime emission-difference (FLED) microscopy approach obtained through a multi-image weighted-subtraction algorithm that combines images reconstructed from multiple temporal bins. A high-throughput implementation was demonstrated using a commercial time-resolved stimulated-emission depletion microscope (STED). Through simulations of fluorescent beads and experimental validation on biological samples, we demonstrated a 1.6× resolution enhancement corresponding to 0.22λ (154 nm). The proposed FLED method enables simultaneous imaging of multiple fluorescent targets and, unlike conventional super-resolution methods, requires lower excitation power and is compatible with a wide range of fluorescent dyes.