Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission.

Saturated stimulated-emission depletion (STED) of a fluorescent marker has been shown to break the diffraction barrier in far-field fluorescence microscopy and to facilitate spatial resolution down to a few tens of nanometers. Here we investigate the photostability of a fluorophore that, in this concept, is repeatedly excited and depleted by synchronized laser pulses. Our study of bacteria labeled with RH-414, a membrane marker, reveals that increasing the duration of the STED pulse from approximately 10 to 160 ps fundamentally improves the photostability of the dye. At the same time the STED efficiency is maintained. The observed photobleaching of RH-414 is due primarily to multiphoton absorption from its ground state. One can counteract photobleaching by employing STED pulses that range from 150 ps to approximately half of the lifetime of the excited state. The results also have implications for multiphoton excitation microscopy.