Ratiometric photon reassignment based on fluorescence lifetime to improve resolution in pulse STED microscopy

Depletion beams with long pulse durations (∼600ps) have recently been applied in stimulated emission depletion (STED) microscopy to reduce photobleaching and phototoxicity. Therefore, improving the resolution of pulse STED at lower depletion power for the super-resolution imaging of live biological specimens has attracted increasing interest. Herein, we present a simple method termed ratiometric photon reassignment based on the fluorescence lifetime, in which highly spatially resolved long-lifetime fluorophore components in the center are extracted, and short-lifetime fluorophore components in the periphery are discarded to improve resolution without increasing the depletion power. The experimental results demonstrate improved resolution and signal-to-noise ratio compared with traditional time-gated STED. Our proposed method requires lower budget and data processing because, in contrast to the separation of photons by lifetime tuning), lifetime measurements are not required.