Time alignment optimization of pulsed STED microscopy

Stimulated emission depletion (STED) microscopy implemented with both pulsed excitation and STED beams (pulsed STED) is especially suitable in biological superresolution imaging as it reduces photobleaching rates and provides better resolution. Proper time alignment between the excitation and STED pulse can improve resolution. To get the optimal time alignment the fluorescence depletion effect was analysed under different time alignments in theory first. Then, the relationship between the time alignment and the position of two moveable mirrors used for changing the excitation optical path length was established. Finally, the depletion experiments were performed under different time alignments. By both simulation and experimentation, the optimal time alignment was got which was about 210 ps when the widths of excitation and STED pulses were both 120 ps. STED image with 60 nm resolution was achieved based on the optimal time alignment. The method provides a reliable way to optimize the time alignment of pulsed STED microscopy.