title: Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.
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 can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a 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 ≥6 GW∙cm(-2) depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 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 the linear scanning speed for photobleaching reduction in STED microscopy.
authors.affiliation: Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.|Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.
authors.forename: Xundong
authors.initials: X
authors.lastname: Wu
authors.affiliation: Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.
authors.forename: Rong
authors.initials: R
authors.lastname: Lu
authors.affiliation: Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.|Department of Molecular &Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.|Department of Molecular &Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.|Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.
authors.forename: Ligia
authors.initials: L
authors.lastname: Toro
authors.affiliation: Division of Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.|Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.|Cardiovascular Research Laboratory, David Geffen School of Medicine, University of California, Los Angeles, CA, 90095, USA.
authors.forename: Enrico
authors.initials: E
authors.lastname: Stefani
mesh_terms: D000465:Algorithms; D005453:Fluorescence; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence / Q000379:methods*; D008962:Models, Theoretical; D038761:Photobleaching
chemical_list: D005456:Fluorescent Dyes