Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy

TL;DR

This study demonstrates that high-speed resonant scanning with a large field of view in STED microscopy significantly reduces photobleaching and increases fluorescence yield, especially at high depletion laser irradiance levels.

Contribution

The paper introduces a quantitative analysis showing that high linear scanning speeds in a custom resonant-scanning STED microscope decrease photobleaching and enhance fluorescence, supported by experimental data and a theoretical model.

Findings

Photobleaching rate decreases with higher scanning speed.

Fluorescence yield increases by approximately 80%.

Effects are more pronounced at higher depletion irradiance.

Abstract

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.