Superresolution Microscopy


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.


Abbe E (1873) Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Archiv für Mikroskopische Anatomie 9 (1): 413–418.

Backlund MP, Lew MD, Backer AS, et al. (2013) The double‐helix point spread function enables precise and accurate measurement of 3D single‐molecule localisation and orientation. Proceedings of SPIE The International Society for Optical Engineering 8590: 85900.

Balzarotti F, Eilers Y, Gwosch KC, et al. (2016) Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355 (6325): 606–612.

Barak LS and Webb WW (1982) Diffusion of low density lipoprotein‐receptor complex on human fibroblasts. Journal of Cell Biology 95 (3): 846–852.

Bates M, Huang B, Dempse GT, et al. (2007) Multicolor super‐resolution imaging with photo‐switchable fluorescent probes. Science 317 (5845): 1749–1753.

Betzig E (1995) Proposed method for molecular optical imaging. Optics Letters 20 (3): 237–239.

Betzig E, Patterson GH, Sougrat R, et al. (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313 (5793): 1642–1645.

Chen BC, Legant WR, Wang K, et al. (2014) Lattice light‐sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346 (6208): 1257998.

Dempsey GT, Vaughan JC, Chen KH, et al. (2011) Evaluation of fluorophores for optimal performance in localization‐based super‐resolution imaging. Nature Methods 8 (12): 1027–1036.

Dickson RM, Cubitt AB, Tsien RY, et al. (1997) On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388 (6640): 355–358.

Folling J, Bossi M, Bock H, et al. (2008) Fluorescence nanoscopy by ground‐state depletion and single‐molecule return. Nature Methods 5 (11): 943–945.

Gao L, Shao L, Higgins CD, et al. (2012) Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens. Cell 151 (6): 1370–1385.

Giannone G, Hosy E, Levet F, et al. (2010) Dynamic superresolution imaging of endogenous proteins on living cells at ultra‐high density. Biophysical Journal 99 (4): 1303–1310.

Grotjohann T, Testa I, Leutenegger M, et al. (2011) Diffraction‐unlimited all‐optical imaging and writing with a photochromic GFP. Nature 478 (7368): 204–208.

Gustafsson MG, Agard and DA, Sedat JW, (1995) Sevenfold Improvement of Axial Resolution in 3D Wide‐field Microscopy using Two Objective Lenses. IS&T/SPIE's Symposium on Electronic Imaging: Science & Technology, International Society for Optics and Photonics.

Gustafsson MG (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. Journal of Microscopy 198 (Pt 2): 82–87.

Gustafsson MG (2005) Nonlinear structured‐illumination microscopy: wide‐field fluorescence imaging with theoretically unlimited resolution. Proceedings of the National Academy of Sciences of the United States of America 102 (37): 13081–13086.

Habuchi S, Ando R, Dedecker P, et al. (2005) Reversible single‐molecule photoswitching in the GFP‐like fluorescent protein Dronpa. Proceedings of the National Academy of Sciences of the United States of America 102 (27): 9511–9516.

Heilemann M, Margeat E, Kasper R, et al. (2005) Carbocyanine dyes as efficient reversible single‐molecule optical switch. Journal of the American Chemical Society 127 (11): 3801–3806.

Heilemann M, van de Linde S, Schuttpelz M, et al. (2008) Subdiffraction‐resolution fluorescence imaging with conventional fluorescent probes. Angewandte Chemie International Edition in English 47 (33): 6172–6176.

Heintzmann R, Jovin TM and Cremer C (2002) Saturated patterned excitation microscopy–a concept for optical resolution improvement. Journal of the Optical Society of America. A, Optics, Image Science, and Vision 19 (8): 1599–1609.

Hell SW, Stelzer EH, Lindek S, et al. (1994) Confocal microscopy with an increased detection aperture: type‐B 4Pi confocal microscopy. Optics Letters 19 (3): 222.

Hell SW and Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated‐emission‐depletion fluorescence microscopy. Optics Letters 19 (11): 780–782.

Hell SW and Kroug M (1995) Ground‐state‐depletion fluorscence microscopy: a concept for breaking the diffraction resolution limit. Applied Physics B: Lasers and Optics 60 (5): 495–497.

Hell SW, Willig KI, Dyba M, et al. (2006) Nanoscale resolution with focused light: STED and other RESOLFT microscopy concepts. In: Pawley JB (ed) Handbock of Biological Confocal Microscopy, 3rd edn, pp. 571–579. New York: Springer.

Hell SW (2007) Far‐field optical nanoscopy. Science 316 (5828): 1153–1158.

Hess ST, Girirajan TP, et al. (2006) Ultra‐high resolution imaging by fluorescence photoactivation localization microscopy. Biophysical Journal 91 (11): 4258–4272.

Huang B, Wang W, Bates M, et al. (2008) Three‐dimensional super‐resolution imaging by stochastic optical reconstruction microscopy. Science 319 (5864): 810–813.

Juette MF, Gould TJ, Lessard D, et al. (2008) Three‐dimensional sub‐100 nm resolution fluorescence microscopy of thick samples. Nature Methods 5 (6): 527–529.

Jungmann R, Avendano MS, Woehrstein JB, et al. (2014) Multiplexed 3D cellular super‐resolution imaging with DNA‐PAINT and Exchange‐PAINT. Nature Methods 11 (3): 313–318.

Keppler A, Gendreizig S, Gronemeyer T, et al. (2003) A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nature Biotechnology 21 (1): 86–89.

Klar TA, Jakobs S, Dyba M, et al. (2000) Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proceedings of the National Academy of Sciences of the United States of America 97 (15): 8206–8210. DOI: 10.1073/pnas.97.15.8206

Lauterbach MA (2012) Finding, defining and breaking the diffraction barrier in microscopy–a historical perspective. Optical Nanoscopy 1 (1): 8.

Lukinavicius G, Umezawa K, Olivier N, et al. (2013) A near‐infrared fluorophore for live‐cell super‐resolution microscopy of cellular proteins. Nature Chemistry 5 (2): 132–139.

Lukinavicius G, Reymond L, et al. (2015) Fluorescent labeling of SNAP‐tagged proteins in cells. Methods in Molecular Biology 1266: 107–118.

McGorty R, Kamiyama D, Huang B, et al. (2013) Active microscope stabilization in three dimensions using image correlation. Optical Nanoscopy 2 (1). DOI: 10.1186/2192-2853-2-3

Nahidiazar L, Agronskaia AV, Broertjes J, et al. (2016) Optimizing imaging conditions for demanding multi‐color super resolution localization microscopy. PLoS One 11 (7): e0158884.

Olivier N, Keller D, Rajan VS, et al. (2013) Simple buffers for 3D STORM microscopy. Biomedical Optics Express 4 (6): 885–899.

Patterson GH and Lippincott‐Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297 (5588): 1873–1877.

Peterson SN and Kwon K (2012) The HaloTag: improving soluble expression and applications in protein functional analysis. Current Chemical Genomics 6: 8–17.

Ries J, Kaplan C, Platonova E, et al. (2012) A simple, versatile method for GFP‐based super‐resolution microscopy via nanobodies. Nature Methods 9 (6): 582–584.

Rust MJ, Bates M and Zhuang X (2006) Sub‐diffraction‐limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3 (10): 793–795.

Sage D, Kirshner H, et al. (2015) Quantitative evaluation of software packages for single‐molecule localization microscopy. Nature Methods 12 (8): 717–724.

Schermelleh L, Carlton PM, Haase S, et al. (2008) Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science 320 (5881): 1332–1336.

Sharonov A and Hochstrasser RM (2006) Wide‐field subdiffraction imaging by accumulated binding of diffusing probes. Proceedings of the National Academy of Sciences of the United States of America 103 (50): 18911–18916.

Urban NT, Willig KI, Hell SW, et al. (2011) STED nanoscopy of actin dynamics in synapses deep inside living brain slices. Biophysical Journal 101 (5): 1277–1284.

Vicidomini G, Moneron G, Han KY, et al. (2011) Sharper low‐power STED nanoscopy by time gating. Nature Methods 8 (7): 571–573.

Waldchen S, Lehmann J, Klein T, et al. (2015) Light‐induced cell damage in live‐cell super‐resolution microscopy. Scientific Reports 5: 15348.

Waterman‐Storer CM, Desai A, Bulinski JC, et al. (1998) Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells. Current Biology: CB 8 (22): 1227–1230.

Zanacchi FC, Lavagnino Z, Perrone Donnorso M et al. (2011) Live‐cell 3D super‐resolution imaging in thick biological samples. Nature Methods 8 (12): 1047–1049.

Zurek‐Biesiada D, Szczurek AT, Prakash K, et al. (2016) Quantitative super‐resolution localization microscopy of DNA in situ using Vybrant(R) DyeCycle Violet fluorescent probe. Data in Brief 7: 157–171.

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.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Zimmermann, Timo(Jul 2017) Superresolution Microscopy. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0025317]