A Stimulated Emission Depletion (STED) Microscope of All Trades
Lukas Finzel and Matthias Reuss abberior Instruments GmbH, Göttingen, Germany
info@abberior-instruments.com
Abstract: Super-resolution microscopy gives researchers invalu- able opportunities and continues to make great strides in terms of performance and applicability. Clever developments in stimulated emission depletion (STED) microscopy have pushed the doors open wider for many applications. Here, we discuss three examples: first, how time-resolved detection unlocks new information; then, live-cell imaging enabled by intelligent illumination schemes; and finally, deep tissue imaging with dynamic aberration correction. As an outlook, we examine MINFLUX as an approach for molecular resolution with fluorescence.
Keywords: super-resolution microscopy, STED microscopy, live-cell imaging, tissue imaging, MINFLUX microscopy
Introduction Te basic approach behind stimulated emission deple-
tion (STED) is straightforward [1]. It starts where confocal microscopy ends: a small region is excited by a focused laser beam (Figure 1A). However, “small,” by the laws of physics, is not an arbitrary term but happens to be about half of the wavelength used. At a diameter of about 200 nanometers, this is still much larger than a molecule, so that individual fluo- rophores cannot be singled out. Essentially, it’s like probing a vinyl disk with a broomstick—you’re missing the groove. Now, STED brings a second light beam into play. Tis light beam switches off excited fluorophores through a basic molecular process called stimulated emission (hence the name). It is shaped like a ring with a hole, oſten referred to as a donut, and thus deliberately silences fluorescent molecules in the outskirts of the diffraction-limited focus region, effec- tively making it much smaller than the limitation dictated by basic optics (Figure 1B). What is leſt to fluoresce is a tiny area in the dark center of the donut with a minute diameter way below 200 nm. Even better, this concept can be extended to three dimensions [2] (Figure 1C). STED unlocks the direct imaging of structures much
smaller than the diffraction limit and helps in the understand- ing of biological processes on a sub-cellular scale in a very direct, visual way. Because of this, it has grown into an essential tool in fields such as neurobiology [3], genomics [4], and virol- ogy [5], to name a few.
All Tools Under a Single Roof In the past decade, the focus of STED development has
shiſted from improving the approach itself to making it avail- able for imaging of a wide range of samples from living cells to complex tissue samples. STED has now matured into a tech- nically refined method. It not only overcomes the diffraction barrier in a proof-of-principle way, but integrates the com- plex demands of microscopic imaging as a basic research tool providing:
26 doi:10.1017/S155192952200089X
• A comprehensive imaging station with microscopes designed around a standard light microscope frame. Today’s imaging systems offer everything from brightfield microscopy over state-of-the-art confocal laser scanning to super-resolution STED in one package. Existing light microscope setups can oſten be upgraded with packages such as the STEDYCON to provide a super-resolution microscope.
• Superior performance: At the same time, there should be no compromises on efficacy. Breakthrough discoveries require exceptional tools.
• Sample protection: Pulsed lasers are preferred as they sig- nificantly reduce the destructive effects of light on the sam- ple [6,7]. Intelligent adaptive illumination schemes apply laser light only and exactly when it is needed, thus avoiding unnecessary exposure.
• Extraordinary sensitivity: Avalanche photodiodes (APD) have the highest quantum efficiency on the market. Other detector types miss many of the photons that are emitted from the sample. High-sensitivity detectors mean faster acquisition especially of dim specimens and save the sample from unnecessary excitation [8].
• Optics and fluorophores from a single vendor: In addition to pioneering hardware, fluorescent dyes and labeling solu- tions can be purchased from abberior. Tis means that all components of the imaging process are perfectly matched for the best results.
Figure 1: Narrowing of the effective emission area by stimulated emission depletion. (A) Diffraction-limited PSF. (B) Donut-shaped STED beam narrows PSF diameter. (C) 3D-STED enables axial super-resolution.
www.microscopy-today.com • 2022 July
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