Superresolution Microscopy

Abstract

More than a century after Ernst Abbe defined the optical diffraction limit and thus seemed to rule out the accurate visualisation of subcellular details below its threshold forever, several approaches now achieve resolutions significantly below it. Structured illumination microscopy, stimulated emission depletion microscopy and single‐molecule localisation‐based methods use different strategies to separate and visualise the cellular and molecular details that until now were lost in the blur of diffraction. This opens up new possibilities in the observation of biological processes at the smallest scales. The new field is only developing and still poses exciting challenges that require new instrumentation and new labeling strategies.

Key Concepts

  • Resolution in light microscopy is not limited anymore by diffraction even though the optical diffraction limit still exists.
  • Superresolution microscopy requires local contrast between structures that can be achieved through different mechanisms.
  • All superresolution concepts are based on the control of ‘on’ and ‘off’ states of fluorescence.
  • Label properties contribute to the achievable resolution gain, and labelling is an essential part of the superresolution result.
  • Most superresolution images require postprocessing, and care needs to be taken to avoid image artefacts.

Keywords: superresolution microscopy; stimulated emission depletion microscopy; localisation‐based microscopy methods; structured illumination microscopy; fluorophore properties

Figure 1. Schematics of the three main forms of superresolution light microscopy. (a) In structured illumination microscopy, a sinusoidal pattern of illumination is projected into the focal plane of the microscope image. The interaction of the illumination pattern with the sample generates Moiré effects that are displayed in lower optical frequencies than the details that they encode. Phase shifting and rotating the illumination pattern generates an image series that can be used to calculate an image with twice the optical resolution by taking into account the known illumination pattern to extract the image components from the Moiré fringes. (b) Lightpath of a stimulated emission depletion (STED) microscope with a fluorescence excitation beam (green) that is overlaid with a depletion beam (red) at a wavelength that can induce stimulated emission of the fluorophore. The depletion lightpath contains a phase plate that generates a central minimum in the focal plane (shown in the depletion cross section). As seen in the cross section of the focal plane, excitation and depletions overlap in a manner that detectable fluorescence (orange) is only generated in the centre of the diffraction‐limited beams. (c) For localisation‐based microscopy methods, series of sparse single‐molecule signals are transformed into a list of highly accurate coordinates for every detected event that can be combined into calculated image based on the coordinates and not on the diffraction patterns of the molecules.
Figure 2. Comparison of a confocal and a deconvolved STED image of Histone2B labelled with a SNAP‐tag and Silicon‐Rhodamine (SiR)‐SNAP in a fixed Hela cell nucleus. The STED images were taken with pulsed 775 nm depletion with a water immersion objective.
Figure 3. dSTORM/GSDIM images of a fixed nucleus with Histone2B labelled with a SNAP‐tag and TMR‐STAR‐SNAP. Image (a) is the diffraction‐limited sum of all fluorescence signals collected during the acquisition of the blinking events and Image (b) is the superresolution image reconstituted from the list of localisations. (c) The superresolution image is overlaid in yellow over the grey diffraction‐limited image.
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Further Reading

Hell SW, Sahl SJ, Bates M, et al. (2015) The 2015 super‐resolution microscopy roadmap. Journal of Physics D: Applied Physics 48: 443001.

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Zimmermann, Timo(Jul 2017) Superresolution Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0025317]