Reducing Photobleaching in STED Microscopy with Higher Scanning Speed

Abstract

Photobleaching is a major limitation of super-resolution STED microscopy. Using our custom-built resonant-scanning STED microscope with a large (50 x 50 µm) field of view, we now show that the photobleaching rate in STED microscopy can be slowed down by scanning with high linear speed. The effect of scanning speed on photobleaching is more remarkable at higher levels of depletion laser irradiance. With a depletion irradiance of 0.4 GW/cm2 (time average) and a 7-fold faster scanning (speed increased from 0.17 m/s to 1.38 m/s), we were able to slow down the photobleaching of the Atto 647N dye by 80%. Photobleaching is primarily caused by the depletion light acting upon the fluorophores in the excited states. Experimental data qualitatively agree with a theoretical model. Our results encourage to increase linear scanning speed further or to reduce continuous light exposure time to significantly reduce photobleaching in STED microscopy.