Time-gating improves the spatial resolution of STED microscopy

Abstract

Stimulated-emission depletion (STED) microscopy improves image resolution by encoding additional spatial information in a second stimulated-decay channel with a spatially-varying strength. Here we demonstrate that spatial information is also encoded in the fluorophore lifetime and that this information can be used to improve the spatial resolution of STED microscopy. By solving a kinetic model for emission in the presence of a time-varying STED pulse, we derive the effective resolution as a function of fluorophore lifetime and pulse duration. We find that the best resolution for a given pulse power is achieved with a pulse of infinitesimally short duration; however, the maximum resolution can be restored for pulses of finite duration by time-gating the fluorescence signal. In parallel, we consider time-gating in the presence of a continuous-wave (CW) STED beam and find that time-gating produces theoretically unbounded resolution with finite laser power. In both cases, the cost of this improved resolution is a reduction in the brightness of the final image. We conclude by discussing situations in which time-gated STED microscopy (T-STED) may provide improved microscope performance beyond an increase in resolution.