Stimulated emission depletion with sub-ns pulses

We have designed a new stimulated emission depletion microscope that applies longer STED pulses (>100 ps) than commonly used (5-10 ps). It is found that longer pulses have a positive effect on photostability of the fluorescent molecules. Utilizing ultrafast shutters, the recovery of the fluorescence signal after shutting the STED beam off was determined on timescales between 100 ns and 10 ms. We examined a number of dyes that could be used in biological applications, e.g. to measure changes in pH.