Strategies to maximize performance in STimulated Emission Depletion (STED) nanoscopy of biological specimens

Methods. 2020 Mar 1:174:27-41. doi: 10.1016/j.ymeth.2019.07.019. Epub 2019 Jul 22.

Abstract

Super-resolution fluorescence microscopy has become an important catalyst for discovery in the life sciences. In STimulated Emission Depletion (STED) microscopy, a pattern of light drives fluorophores from a signal-emitting on-state to a non-signalling off-state. Only emitters residing in a sub-diffraction volume around an intensity minimum are allowed to fluoresce, rendering them distinguishable from the nearby, but dark fluorophores. STED routinely achieves resolution in the few tens of nanometers range in biological samples and is suitable for live imaging. Here, we review the working principle of STED and provide general guidelines for successful STED imaging. The strive for ever higher resolution comes at the cost of increased light burden. We discuss techniques to reduce light exposure and mitigate its detrimental effects on the specimen. These include specialized illumination strategies as well as protecting fluorophores from photobleaching mediated by high-intensity STED light. This opens up the prospect of volumetric imaging in living cells and tissues with diffraction-unlimited resolution in all three spatial dimensions.

Keywords: Fluorescence microscopy; Live imaging; Nanoscopy; Optical imaging; Protected STED; STED; Stimulated emission depletion microscopy; Super-resolution microscopy.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Color
  • Equipment Reuse
  • Fluorescence
  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / radiation effects
  • Image Processing, Computer-Assisted / methods*
  • Lighting / methods
  • Microscopy, Fluorescence / methods*
  • Optical Imaging / methods
  • Photobleaching
  • Scientific Experimental Error
  • Time Factors

Substances

  • Fluorescent Dyes