  Photobleaching is a major limitation of superresolution Stimulated Depletion
Emission (STED) microscopy. Fast scanning has long been considered an effective
means to reduce photobleaching in fluorescence microscopy, but a careful
quantitative study of this issue is missing. In this paper, we show that the
photobleaching rate in STED microscopy is slowed down and fluorescence yield is
enhanced by scanning with high linear speed, enabled by the large field of view
in our custom-built resonant-scanning STED microscope. The effect of scanning
speed on photobleaching and fluorescence yield is more remarkable at higher
levels of depletion laser irradiance, and virtually disappears in conventional
confocal microscopy. With a depletion irradiance of >0.2 GW$\cdot$cm$^{-2}$
(time average), we were able to extend the fluorescence survival time of the
Atto 647N dye by ~80% with an 8-fold wider field of view. We confirm that STED
Photobleaching is primarily caused by the depletion light acting upon the
excited fluorophores. Experimental data agree with a theoretical model. Our
results encourage further increasing linear scanning speed for photobleaching
reduction in STED microscopy.
