Fluorescence nanoscopy in cell biology

Key Points

  • Fluorescence nanoscopy (also known as super-resolution microscopy) methods have expanded optical imaging to reach the nanometre resolution range, typically 20–50 nm and even down to the 1 nm level.

  • Diffraction-unlimited nanoscopy methods, which neutralize the resolution-limiting role of diffraction, separate fluorophores by transiently transferring them between (at least) two discernible states, typically an 'on' and an 'off' state of fluorescence.

  • The counting of molecules in nanoscale settings such as within organelles is a crucially important development, along with labelling strategies to reliably pinpoint the locations and spatial proximities of all the molecules investigated in an imaging experiment.

  • Dynamic nanoscopy and extensions of nanoscopy imaging to tissue and in vivo contexts are further frontiers.

  • Examples taken from mitochondrial biology and neurobiology illustrate the capabilities and discovery potential of nanoscale molecule-specific imaging with focused light.


Fluorescence nanoscopy uniquely combines minimally invasive optical access to the internal nanoscale structure and dynamics of cells and tissues with molecular detection specificity. While the basic physical principles of 'super-resolution' imaging were discovered in the 1990s, with initial experimental demonstrations following in 2000, the broad application of super-resolution imaging to address cell-biological questions has only more recently emerged. Nanoscopy approaches have begun to facilitate discoveries in cell biology and to add new knowledge. One current direction for method improvement is the ambition to quantitatively account for each molecule under investigation and assess true molecular colocalization patterns via multi-colour analyses. In pursuing this goal, the labelling of individual molecules to enable their visualization has emerged as a central challenge. Extending nanoscale imaging into (sliced) tissue and whole-animal contexts is a further goal. In this Review we describe the successes to date and discuss current obstacles and possibilities for further development.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Dissecting animal cells with fluorescence nanoscopy.
Figure 2: Examples of fluorescence nanoscopy in bacteria and yeast.
Figure 3: The state of the art in fluorescence nanoscopy: basic working principles and comparisons of 3D resolution.
Figure 4: Nanoscopy of neurons.
Figure 5: Nanoscopy of mitochondria.
Figure 6: Sizes of commonly used binding probes: a challenge for nanoscopy.
Figure 7: Super-resolution microscopy in vivo mouse and fruitfly nanoscopy.


  1. 1

    Abbe, E. Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Archiv. Mikrosk. Anat. 9, 413–418 (1873).

  2. 2

    Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994). First viable proposal to utilize elementary molecular state transitions to fundamentally break the diffraction barrier of lens-based fluorescence microscopy. The report describes the concepts and quantitatively outlines the potential of spatial resolution at the nanoscale.

  3. 3

    Hell, S. W. & Kroug, M. Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit. Appl. Phys. B 60, 495–497 (1995).

  4. 4

    Hell, S. W. Toward fluorescence nanoscopy. Nat. Biotechnol. 21, 1347–1355 (2003).

  5. 5

    Hell, S. W. Nanoscopy with focused light (Nobel Lecture). Angew. Chem. Int. Ed. Engl. 54, 8054–8066 (2015).

  6. 6

    Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

  7. 7

    Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).

  8. 8

    Klar, T. A., Jakobs, S., Dyba, M., Egner, A. & Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000). First experimental report of fluorescence nanoscopy in a cellular context. Reports STED nanoscopy in a living cell.

  9. 9

    Donnert, G. et al. Macromolecular-scale resolution in biological fluorescence microscopy. Proc. Natl Acad. Sci. USA 103, 11440–11445 (2006).

  10. 10

    Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R. & Hell, S. W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939 (2006). First application of fluorescence nanoscopy to a biological research question, investigating clustering of the membrane protein synaptotagmin I under different stimulation conditions.

  11. 11

    Göttfert, F. et al. Coaligned dual-channel STED nanoscopy and molecular diffusion analysis at 20 nm resolution. Biophys. J. 105, L01–L03 (2013).

  12. 12

    Hofmann, M., Eggeling, C., Jakobs, S. & Hell, S. W. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc. Natl Acad. Sci. USA 102, 17565–17569 (2005).

  13. 13

    Brakemann, T. et al. A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nat. Biotechnol. 29, 942–947 (2011).

  14. 14

    Grotjohann, T. et al. Diffraction-unlimited all-optical imaging and writing with a photochromic GFP. Nature 478, 204–208 (2011). Refs 13 and 14 provide the first experimental demonstrations of low-light-level RESOLFT nanoscopy in cells.

  15. 15

    Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

  16. 16

    Hess, S. T., Girirajan, T. P. K. & Mason, M. D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

  17. 17

    Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006). Refs 15–17 provide the first demonstrations of the PALM/STORM concept.

  18. 18

    Bates, M., Huang, B., Dempsey, G. T. & Zhuang, X. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749–1753 (2007).

  19. 19

    Bock, H. et al. Two-color far-field fluorescence nanoscopy based on photoswitchable emitters. Appl. Phys. B 88, 161–165 (2007).

  20. 20

    Egner, A. et al. Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys. J. 93, 3285–3290 (2007).

  21. 21

    Fölling, J. et al. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat. Methods 5, 943–945 (2008).

  22. 22

    Heilemann, M. et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew. Chem. Int. Ed. Engl. 47, 6172–6176 (2008).

  23. 23

    Dempsey, G. T., Vaughan, J. C., Chen, K. H., Bates, M. & Zhuang, X. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat. Methods 8, 1027–1036 (2011).

  24. 24

    Sharonov, A. & Hochstrasser, R. M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl Acad. Sci. USA 103, 18911–18916 (2006).

  25. 25

    Dickson, R. M., Cubitt, A. B., Tsien, R. Y. & Moerner, W. E. On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388, 355–358 (1997). First report of single-molecule-level switching of a fluorescent protein with light between active and inactive states.

  26. 26

    Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606–612 (2016). Reports MINFLUX, a concept for nanometre-level molecule localization and nanoscopy with minimal fluxes of emitted (fluorescence) photons.

  27. 27

    Danzl, J. G. et al. Coordinate-targeted fluorescence nanoscopy with multiple off states. Nat. Photonics 10, 122–128 (2016).

  28. 28

    Göttfert, F. et al. Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent. Proc. Natl Acad. Sci. USA 114, 2125–2130 (2017).

  29. 29

    Westphal, V. et al. Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320, 246–249 (2008).

  30. 30

    Minsky, M. Microscopy apparatus. US patent 3013467 A (1961).

  31. 31

    Sheppard, C. J. R. Super-resolution in confocal imaging. Optik 80, 53–54 (1988).

  32. 32

    Müller, C. B. & Enderlein, J. Image scanning microscopy. Phys. Rev. Lett. 104, 198101 (2010).

  33. 33

    Chen, B.-C. et al. Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).

  34. 34

    Hookway, C. et al. Microtubule-dependent transport and dynamics of vimentin intermediate filaments. Mol. Biol. Cell 26, 1675–1686 (2015).

  35. 35

    Hagen, C. et al. Structural basis of vesicle formation at the inner nuclear membrane. Cell 163, 1692–1701 (2015).

  36. 36

    Burnette, D. T. et al. A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells. J. Cell Biol. 205, 83–96 (2014).

  37. 37

    Nixon-Abell, J. et al. Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER. Science 354, aaf3928 (2016).

  38. 38

    Li, D. et al. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349, aab3500 (2015).

  39. 39

    Sahl, S. J. et al. Comment on “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics”. Science 352, 527 (2016).

  40. 40

    Dertinger, T., Colyer, R., Iyer, G., Weiss, S. & Enderlein, J. Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI). Proc. Natl Acad. Sci. USA 106, 22287–22292 (2009).

  41. 41

    Geissbuehler, S. et al. Live-cell multiplane three-dimensional super-resolution optical fluctuation imaging. Nat. Commun. 5, 5830 (2014).

  42. 42

    Chen, F., Tillberg, P. W. & Boyden, E. S. Expansion microscopy. Science 347, 543–548 (2015).

  43. 43

    Harke, B., Ullal, C. K., Keller, J. & Hell, S. W. Three-dimensional nanoscopy of colloidal crystals. Nano Lett. 8, 1309–1313 (2008).

  44. 44

    Hell, S. & Stelzer, E. H. K. Properties of a 4Pi confocal fluorescence microscope. J. Opt. Soc. Am. A 9, 2159–2166 (1992).

  45. 45

    Schmidt, R. et al. Spherical nanosized focal spot unravels the interior of cells. Nat. Methods 5, 539–544 (2008).

  46. 46

    Schmidt, R. et al. Mitochondrial cristae revealed with focused light. Nano Lett. 9, 2508–2510 (2009).

  47. 47

    Curdt, F. et al. isoSTED nanoscopy with intrinsic beam alignment. Opt. Express 23, 30891–30903 (2015).

  48. 48

    Böhm, U., Hell, S. W. & Schmidt, R. 4Pi-RESOLFT nanoscopy. Nat. Commun. 7, 10504 (2016).

  49. 49

    Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl Acad. Sci. USA 106, 3125–3130 (2009).

  50. 50

    Aquino, D. et al. Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores. Nat. Methods 8, 353–359 (2011).

  51. 51

    Huang, F. et al. Ultra-high resolution 3D imaging of whole cells. Cell 166, 1028–1040 (2016). Comprehensive demonstration of 3D nanoscopy at 10–20 nm resolution across a wide range of cellular structures based on the 4Pi approach (Ref. 44) and its combination with PALM/STORM (Refs 49 and 50).

  52. 52

    Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

  53. 53

    Juette, M. F. et al. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat. Methods 5, 527–529 (2008).

  54. 54

    Pavani, S. R. P. et al. Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function. Proc. Natl Acad. Sci. USA 106, 2995–2999 (2009).

  55. 55

    Lee, H.-l. D., Sahl, S. J., Lew, M. D. & Moerner, W. E. The double-helix microscope super-resolves extended biological structures by localizing single blinking molecules in three dimensions with nanoscale precision. Appl. Phys. Lett. 100, 153701 (2012).

  56. 56

    Backlund, M. P. et al. Simultaneous, accurate measurement of the 3D position and orientation of single molecules. Proc. Natl Acad. Sci. USA 109, 19087–19092 (2012).

  57. 57

    Gahlmann, A. et al. Quantitative multicolor subdiffraction imaging of bacterial protein ultrastructures in three dimensions. Nano Lett. 13, 987–993 (2013).

  58. 58

    Jia, S., Vaughan, J. C. & Zhuang, X. Isotropic three-dimensional super-resolution imaging with a self-bending point spread function. Nat. Photonics 8, 302–306 (2014).

  59. 59

    Shechtman, Y., Sahl, S. J., Backer, A. S. & Moerner, W. E. Optimal point spread function design for 3D imaging. Phys. Rev. Lett. 113, 133902 (2014).

  60. 60

    Shechtman, Y., Weiss, L. E., Backer, A. S., Lee, M. Y. & Moerner, W. E. Multicolour localization microscopy by point-spread-function engineering. Nat. Photonics 10, 590–594 (2016).

  61. 61

    Smith, C., Huisman, M., Siemons, M., Grünwald, D. & Stallinga, S. Simultaneous measurement of emission color and 3D position of single molecules. Opt. Express 24, 4996–5013 (2016).

  62. 62

    Vaughan, J. C., Jia, S. & Zhuang, X. Ultrabright photoactivatable fluorophores created by reductive caging. Nat. Methods 9, 1181–1184 (2012).

  63. 63

    Xu, K., Babcock, H. P. & Zhuang, X. Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat. Methods 9, 185–188 (2012).

  64. 64

    Staudt, T. et al. Far-field optical nanoscopy with reduced number of state transition cycles. Opt. Express 19, 5644–5657 (2011).

  65. 65

    Zhu, L., Zhang, W., Elnatan, D. & Huang, B. Faster STORM using compressed sensing. Nat. Methods 9, 721–723 (2012).

  66. 66

    Huang, F. et al. Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms. Nat. Methods 10, 653–658 (2013).

  67. 67

    Schneider, J. et al. Ultrafast, temporally stochastic STED nanoscopy of millisecond dynamics. Nat. Methods 12, 827–830 (2015).

  68. 68

    Komis, G., Samajova, O., Ovecka, M. & Samaj, J. Super-resolution microscopy in plant cell imaging. Trends Plant Sci. 20, 834–843 (2015).

  69. 69

    Williamson, D. J. et al. Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat. Immunol. 12, 655–662 (2011).

  70. 70

    Dudok, B. et al. Cell-specific STORM super-resolution imaging reveals nanoscale organization of cannabinoid signaling. Nat. Neurosci. 18, 75–86 (2015).

  71. 71

    Chojnacki, J. et al. Maturation-dependent HIV-1 surface protein redistribution revealed by fluorescence nanoscopy. Science 338, 524–528 (2012).

  72. 72

    Van Engelenburg, S. B. et al. Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits. Science 343, 653–656 (2014).

  73. 73

    Bleck, M. et al. Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding. Proc. Natl Acad. Sci. USA 111, 12211–12216 (2014).

  74. 74

    Prescher, J. et al. Super-resolution imaging of ESCRT-proteins at HIV-1 assembly sites. PLOS Pathog. 11, e1004677 (2015).

  75. 75

    Hanne, J. et al. Stimulated emission depletion nanoscopy reveals time-course of human immunodeficiency virus proteolytic maturation. ACS Nano 10, 8215–8222 (2016).

  76. 76

    Gahlmann, A. & Moerner, W. E. Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging. Nat. Rev. Microbiol. 12, 9–22 (2014).

  77. 77

    Chen, C. et al. Imaging and intracellular tracking of cancer-derived exosomes using single-molecule localization-based super-resolution microscope. ACS Appl. Mater. Interfaces 8, 25825–25833 (2016).

  78. 78

    Ilgen, P. et al. STED super-resolution microscopy of clinical paraffin-embedded human rectal cancer tissue. PLoS ONE 9, e101563 (2014).

  79. 79

    Benda, A., Aitken, H., Davies, D. S., Whan, R. & Goldsbury, C. STED imaging of tau filaments in Alzheimer's disease cortical grey matter. J. Struct. Biol. 195, 345–352 (2016).

  80. 80

    Löschberger, A. et al. Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution. J. Cell Sci. 125, 570–575 (2012).

  81. 81

    Szymborska, A. et al. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 341, 655–658 (2013). Refs 80 and 81: pioneering studies of NPC architecture by fluorescence nanoscopy employing image-averaging techniques.

  82. 82

    Broeken, J. et al. Resolution improvement by 3D particle averaging in localization microscopy. Methods Appl. Fluoresc. 3, 014003 (2015).

  83. 83

    Yang, T. T. et al. Superresolution pattern recognition reveals the architectural map of the ciliary transition zone. Sci. Rep. 5, 14096 (2015).

  84. 84

    Laine, R. F. et al. Structural analysis of herpes simplex virus by optical super-resolution imaging. Nat. Commun. 6, 5980 (2015).

  85. 85

    Xu, K., Zhong, G. & Zhuang, X. Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science 339, 452–456 (2013). Nanoscopy-enabled discovery of a periodic lattice of various cytoskeleton proteins in the axons of neuronal cells.

  86. 86

    Zhong, G. et al. Developmental mechanism of the periodic membrane skeleton in axons. eLife 3, e04581 (2014).

  87. 87

    D'Este, E., Kamin, D., Gottfert, F., El-Hady, A. & Hell, S. W. STED nanoscopy reveals the ubiquity of subcortical cytoskeleton periodicity in living neurons. Cell Rep. 10, 1246–1251 (2015).

  88. 88

    Sidenstein, S. C. et al. Multicolour multilevel STED nanoscopy of actin/spectrin organization at synapses. Sci. Rep. 6, 26725 (2016).

  89. 89

    Bär, J., Kobler, O., van Bommel, B. & Mikhaylova, M. Periodic F-actin structures shape the neck of dendritic spines. 6, 37136 (2016).

  90. 90

    Leterrier, C. et al. Nanoscale architecture of the axon initial segment reveals an organized and robust scaffold. Cell Rep. 13, 2781–2793 (2015).

  91. 91

    Leite, S. C. et al. The actin-binding protein α-adducin is required for maintaining axon diameter. Cell Rep. 15, 490–498 (2016).

  92. 92

    Lukinavicius, G. et al. Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat. Methods 11, 731–733 (2014).

  93. 93

    D'Este, E. et al. Subcortical cytoskeleton periodicity throughout the nervous system. Sci. Rep. 6, 22741 (2016).

  94. 94

    He, J. et al. Prevalent presence of periodic actin-spectrin-based membrane skeleton in a broad range of neuronal cell types and animal species. Proc. Natl Acad. Sci. USA 113, 6029–6034 (2016).

  95. 95

    Albrecht, D. et al. Nanoscopic compartmentalization of membrane protein motion at the axon initial segment. J. Cell Biol. 215, 37–46 (2016).

  96. 96

    D'Este, E., Kamin, D., Balzarotti, F. & Hell, S. W. Ultrastructural anatomy of nodes of Ranvier in the peripheral nervous system as revealed by STED microscopy. Proc. Natl Acad. Sci. USA 114, E191–E199 (2017).

  97. 97

    Wilhelm, B. G. et al. Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science 344, 1023–1028 (2014).

  98. 98

    Chazeau, A. & Giannone, G. Organization and dynamics of the actin cytoskeleton during dendritic spine morphological remodeling. Cell. Mol. Life Sci. 73, 3053–3073 (2016).

  99. 99

    Ehmann, N., Sauer, M. & Kittel, R. J. Super-resolution microscopy of the synaptic active zone. Front. Cell. Neurosci. 9, 7 (2015).

  100. 100

    Kittel, R. J. et al. Bruchpilot promotes active zone assembly, Ca2+ channel clustering, and vesicle release. Science 312, 1051–1054 (2006). Pioneering study of the molecular organization of presynaptic active zones using fluorescence nanoscopy.

  101. 101

    Fouquet, W. et al. Maturation of active zone assembly by Drosophila Bruchpilot. J. Cell Biol. 186, 129–145 (2009).

  102. 102

    Owald, D. et al. A Syd-1 homologue regulates pre- and postsynaptic maturation in Drosophila. J. Cell Biol. 188, 565–579 (2010).

  103. 103

    Liu, K. S. et al. RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release. Science 334, 1565–1569 (2011).

  104. 104

    Ehmann, N. et al. Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states. Nat. Commun. 5, 4650 (2014).

  105. 105

    Nishimune, H., Badawi, Y., Mori, S. & Shigemoto, K. Dual-color STED microscopy reveals a sandwich structure of Bassoon and Piccolo in active zones of adult and aged mice. Sci. Rep. 6, 27935 (2016).

  106. 106

    Chamma, I. et al. Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin. Nat. Commun. 7, 10773 (2016).

  107. 107

    Dani, A., Huang, B., Bergan, J., Dulac, C. & Zhuang, X. Superresolution imaging of chemical synapses in the brain. Neuron 68, 843–856 (2010). STORM analysis of a large number of chemical synapses from different brain regions, quantifying variations in synapse morphology and the distribution of synaptic proteins.

  108. 108

    Hoze, N. et al. Heterogeneity of AMPA receptor trafficking and molecular interactions revealed by superresolution analysis of live cell imaging. Proc. Natl Acad. Sci. USA 109, 17052–17057 (2012).

  109. 109

    MacGillavry, H. D., Song, Y., Raghavachari, S. & Blanpied, T. A. Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78, 615–622 (2013).

  110. 110

    Fukata, Y. et al. Local palmitoylation cycles define activity-regulated postsynaptic subdomains. J. Cell Biol. 202, 145–161 (2013).

  111. 111

    Nair, D. et al. Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organized in nanodomains regulated by PSD95. J. Neurosci. 33, 13204–13224 (2013).

  112. 112

    Tang, A.-H. et al. A trans-synaptic nanocolumn aligns neurotransmitter release to receptors. Nature 536, 210–214 (2016).

  113. 113

    Izeddin, I. et al. Super-resolution dynamic imaging of dendritic spines using a low-affinity photoconvertible actin probe. PLoS ONE 6, e15611 (2011).

  114. 114

    Urban, N. T., Willig, K. I., Hell, S. W. & Nagerl, U. V. STED nanoscopy of actin dynamics in synapses deep inside living brain slices. Biophys. J. 101, 1277–1284 (2011).

  115. 115

    Chazeau, A. et al. Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion. EMBO J. 33, 2745–2764 (2014).

  116. 116

    Nägerl, U. V., Willig, K. I., Hein, B., Hell, S. W. & Bonhoeffer, T. Live-cell imaging of dendritic spines by STED microscopy. Proc. Natl Acad. Sci. USA 105, 18982–18987 (2008).

  117. 117

    Takasaki, K. & Sabatini, B. L. Super-resolution 2-photon microscopy reveals that the morphology of each dendritic spine correlates with diffusive but not synaptic properties. Front. Neuroanat 8, 29 (2014).

  118. 118

    Tonnesen, J., Katona, G., Rozsa, B. & Nägerl, U. V. Spine neck plasticity regulates compartmentalization of synapses. Nat. Neurosci. 17, 678–685 (2014). Reports on the link between nanoscale anatomy and compartmentalization in live spines of mouse brain slices by using time-lapse STED imaging in combination with FRAP measurements, glutamate uncaging, electrophysiology and simulations.

  119. 119

    Duim, W. C., Jiang, Y., Shen, K., Frydman, J. & Moerner, W. E. Super-resolution fluorescence of huntingtin reveals growth of globular species into short fibers and coexistence of distinct aggregates. ACS Chem. Biol. 9, 2767–2778 (2014).

  120. 120

    Pinotsi, D. et al. Direct observation of heterogeneous amyloid fibril growth kinetics via two-color super-resolution microscopy. Nano Lett. 14, 339–345 (2014).

  121. 121

    Kaminski Schierle, G. S. et al. In situ measurements of the formation and morphology of intracellular β-amyloid fibrils by super-resolution fluorescence imaging. J. Am. Chem. Soc. 133, 12902–12905 (2011).

  122. 122

    Sahl, S. J., Weiss, L. E., Duim, W. C., Frydman, J. & Moerner, W. E. Cellular inclusion bodies of mutant huntingtin exon 1 obscure small fibrillar aggregate species. Sci. Rep. 2, 895 (2012).

  123. 123

    Roberti, M. J. et al. Imaging nanometer-sized α-synuclein aggregates by superresolution fluorescence localization microscopy. Biophys. J. 102, 1598–1607 (2012).

  124. 124

    Sontag, E. M. et al. Exogenous delivery of chaperonin subunit fragment ApiCCT1 modulates mutant Huntingtin cellular phenotypes. Proc. Natl Acad. Sci. USA 110, 3077–3082 (2013).

  125. 125

    Sahl, S. J. et al. Delayed emergence of subdiffraction-sized mutant huntingtin fibrils following inclusion body formation. Q. Rev. Biophys. 49, e2 (2016).

  126. 126

    Li, L. et al. Real-time imaging of Huntingtin aggregates diverting target search and gene transcription. eLife 5, e17056 (2016).

  127. 127

    Donnert, G. et al. Two-color far-field fluorescence nanoscopy. Biophys. J. 92, L67–L69 (2007).

  128. 128

    Kehrein, K. et al. Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies. Cell Rep. 10, 843–853 (2015).

  129. 129

    Beinlich, F. R., Drees, C., Piehler, J. & Busch, K. B. Shuttling of PINK1 between mitochondrial microcompartments resolved by triple-color superresolution microscopy. ACS Chem. Biol. 10, 1970–1976 (2015).

  130. 130

    Das, A., Nag, S., Mason, A. B. & Barroso, M. M. Endosome-mitochondria interactions are modulated by iron release from transferrin. J. Cell Biol. 214, 831–845 (2016).

  131. 131

    French, J. B. et al. Spatial colocalization and functional link of purinosomes with mitochondria. Science 351, 733–737 (2016).

  132. 132

    Huang, B., Jones, S. A., Brandenburg, B. & Zhuang, X. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat. Methods 5, 1047–1052 (2008).

  133. 133

    Wurm, C. A., Neumann, D., Schmidt, R., Egner, A. & Jakobs, S. Sample Preparation for STED Microscopy. In Live Cell Imaging: Methods and Protocols, Vol. 591 (ed. Papkovsky, D. B.) 185–199 (2010).

  134. 134

    Wurm, C. A. et al. Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient. Proc. Natl Acad. Sci. USA 108, 13546–13551 (2011).

  135. 135

    Palade, G. E. The fine structure of mitochondria. Anat. Rec. 114, 427–451 (1952).

  136. 136

    Shim, S. H. et al. Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proc. Natl Acad. Sci. USA 109, 13978–13983 (2012).

  137. 137

    Jans, D. C. et al. STED super-resolution microscopy reveals an array of MINOS clusters along human mitochondria. Proc. Natl Acad. Sci. USA 110, 8936–8941 (2013).

  138. 138

    Perkins, G. A. et al. The micro-architecture of mitochondria at active zones: electron tomography reveals novel anchoring scaffolds and cristae structured for high-rate metabolism. J. Neurosci. 30, 1015–1026 (2010).

  139. 139

    Stoldt, S. et al. The inner-mitochondrial distribution of Oxa1 depends on the growth conditions and on the availability of substrates. Mol. Biol. Cell 23, 2292–2301 (2012).

  140. 140

    Sukhorukov, V. M. et al. Determination of protein mobility in mitochondrial membranes of living cells. Biochim. Biophys. Acta 1798, 2022–2032 (2010).

  141. 141

    Dieteren, C. E. J. et al. Solute diffusion is hindered in the mitochondrial matrix. Proc. Natl Acad. Sci. USA 108, 8657–8662 (2011).

  142. 142

    Appelhans, T. et al. Nanoscale organization of mitochondrial microcompartments revealed by combining tracking and localization microscopy. Nano Lett. 12, 610–616 (2012).

  143. 143

    Tait, S. W. & Green, D. R. Mitochondria and cell death: outer membrane permeabilization and beyond. Nat. Rev. Mol. Cell Biol. 11, 621–632 (2010).

  144. 144

    Nechushtan, A., Smith, C. L., Lamensdorf, I., Yoon, S. H. & Youle, R. J. Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J. Cell Biol. 153, 1265–1276 (2001).

  145. 145

    Grosse, L. et al. Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis. EMBO J. 35, 402–413 (2016).

  146. 146

    Salvador-Gallego, R. et al. Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores. EMBO J. 35, 389–401 (2016). Refs 145 and 146 are two independent studies reporting on the assembly of Bax in the mitochondrial outer membrane to mediate membrane rupture.

  147. 147

    Kuwana, T., Olson, N. H., Kiosses, W. B., Peters, B. & Newmeyer, D. D. Pro-apoptotic Bax molecules densely populate the edges of membrane pores. Sci. Rep. 6, 27299 (2016).

  148. 148

    Kukat, C. et al. Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA. Proc. Natl Acad. Sci. USA 108, 13534–13539 (2011).

  149. 149

    Brown, T. A. et al. Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction. Mol. Cell. Biol. 31, 4994–5010 (2011).

  150. 150

    Iborra, F. J., Kimura, H. & Cook, P. R. The functional organization of mitochondrial genomes in human cells. BMC Biol. 2, 9 (2004).

  151. 151

    Legros, F., Malka, F., Frachon, P., Lombes, A. & Rojo, M. Organization and dynamics of human mitochondrial DNA. J. Cell Sci. 117, 2653–2662 (2004).

  152. 152

    Kukat, C. et al. Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid. Proc. Natl Acad. Sci. USA 112, 11288–11293 (2015).

  153. 153

    Gustafsson, C. M., Falkenberg, M. & Larsson, N. G. Maintenance and expression of mammalian mitochondrial DNA. Annu. Rev. Biochem. 85, 133–160 (2016).

  154. 154

    Lau, L., Lee, Y. L., Sahl, S. J., Stearns, T. & Moerner, W. E. STED microscopy with optimized labeling density reveals 9-fold arrangement of a centriole protein. Biophys. J. 102, 2926–2935 (2012).

  155. 155

    Pleiner, T. et al. Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation. eLife 4, e11349 (2015).

  156. 156

    Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Methods 9, 582–584 (2012).

  157. 157

    Mikhaylova, M. et al. Resolving bundled microtubules using anti-tubulin nanobodies. Nat. Commun. 6, 7933 (2015).

  158. 158

    Bradbury, A. & Plückthun, A. Reproducibility: standardize antibodies used in research. Nature 518, 27–29 (2015).

  159. 159

    Butkevich, A. N. et al. Fluorescent rhodamines and fluorogenic carbopyronines for super-resolution STED microscopy in living cells. Angew. Chem. Int. Ed. Engl. 55, 3290–3294 (2016).

  160. 160

    Bottanelli, F. et al. Two-colour live-cell nanoscale imaging of intracellular targets. Nat. Commun. 7, 10778 (2016).

  161. 161

    Winter, F. R. et al. Multicolour nanoscopy of fixed and living cells with a single STED beam and hyperspectral detection. Sci. Rep. 7, 46492 (2017).

  162. 162

    Gibson, T. J., Seiler, M. & Veitia, R. A. The transience of transient overexpression. Nat. Methods 10, 715–721 (2013).

  163. 163

    Ratz, M., Testa, I., Hell, S. W. & Jakobs, S. CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells. Sci. Rep. 5, 9592 (2015).

  164. 164

    Nikic, I. & Lemke, E. A. Genetic code expansion enabled site-specific dual-color protein labeling: superresolution microscopy and beyond. Curr. Opin. Chem. Biol. 28, 164–173 (2015).

  165. 165

    Uttamapinant, C. et al. Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins. J. Am. Chem. Soc. 137, 4602–4605 (2015).

  166. 166

    Lee, S.-H., Shin, J. Y., Lee, A. & Bustamante, C. Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM). Proc. Natl Acad. Sci. USA 109, 17436–17441 (2012).

  167. 167

    Finan, K., Raulf, A. & Heilemann, M. A set of homo-oligomeric standards allows accurate protein counting. Angew. Chem. Int. Ed. Engl. 54, 12049–12052 (2015).

  168. 168

    Puchner, E. M., Walter, J. M., Kasper, R., Huang, B. & Lim, W. A. Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory. Proc. Natl Acad. Sci. USA 110, 16015–16020 (2013).

  169. 169

    Rollins, G. C., Shin, J. Y., Bustamante, C. & Pressé, S. Stochastic approach to the molecular counting problem in superresolution microscopy. Proc. Natl Acad. Sci. USA 112, E110–E118 (2015).

  170. 170

    Hummer, G., Fricke, F. & Heilemann, M. Model-independent counting of molecules in single-molecule localization microscopy. Mol. Biol. Cell 27, 3637–3644 (2016).

  171. 171

    Jungmann, R. et al. Quantitative super-resolution imaging with qPAINT. Nat. Methods 13, 439–442 (2016). Demonstrates qPAINT, a method for quantitative nanoscopy with low counting error.

  172. 172

    Ta, H. et al. Mapping molecules in scanning far-field fluorescence nanoscopy. Nat. Commun. 6, 7977 (2015).

  173. 173

    Annibale, P., Vanni, S., Scarselli, M., Rothlisberger, U. & Radenovic, A. Identification of clustering artifacts in photoactivated localization microscopy. Nat. Methods 8, 527–528 (2011).

  174. 174

    Truan, Z. et al. Quantitative morphological analysis of arrestin2 clustering upon G protein-coupled receptor stimulation by super-resolution microscopy. J. Struct. Biol. 184, 329–334 (2013).

  175. 175

    Sengupta, P. et al. Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat. Methods 8, 969–975 (2011). Method to analyse complex patterns of protein distributions across the plasma membrane.

  176. 176

    Baumgart, F. et al. Varying label density allows artifact-free analysis of membrane-protein nanoclusters. Nat. Methods 13, 661–664 (2016).

  177. 177

    Shroff, H. et al. Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc. Natl Acad. Sci. USA 104, 20308–20313 (2007). Early report on the application of two-colour PALM to study pairs of different proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells.

  178. 178

    Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313–318 (2014).

  179. 179

    Xu, L. et al. Resolution, target density and labeling effects in colocalization studies — suppression of false positives by nanoscopy and modified algorithms. FEBS J. 283, 882–898 (2016).

  180. 180

    Malkusch, S. et al. Coordinate-based colocalization analysis of single-molecule localization microscopy data. Histochem. Cell Biol. 137, 1–10 (2012).

  181. 181

    Pageon, S. V., Nicovich, P. R., Mollazade, M., Tabarin, T. & Gaus, K. Clus-DoC: a combined cluster detection and colocalization analysis for single-molecule localization microscopy data. Mol. Biol. Cell 27, 3627–3636 (2016).

  182. 182

    Eggeling, C. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2009).

  183. 183

    Sahl, S. J., Leutenegger, M., Hilbert, M., Hell, S. W. & Eggeling, C. Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids. Proc. Natl Acad. Sci. USA 107, 6829–6834 (2010). Ref. 182 reports differential diffusion behaviours of phospholipids and sphingolipids in the plasma membranes of living cells, establishing STED-FCS as a powerful tool for live-cell dynamics studies at millisecond timescales, complementary to the fast single-molecule tracking demonstrated in Ref. 183.

  184. 184

    Mueller, V. et al. STED nanoscopy reveals molecular details of cholesterol- and cytoskeleton-modulated lipid interactions in living cells. Biophys. J. 101, 1651–1660 (2011).

  185. 185

    Andrade, D. M. et al. Cortical actin networks induce spatio-temporal confinement of phospholipids in the plasma membrane — a minimally invasive investigation by STED-FCS. Sci. Rep. 5, 11454 (2015).

  186. 186

    Saka, S. K. et al. Multi-protein assemblies underlie the mesoscale organization of the plasma membrane. Nat. Commun. 5, 4509 (2014).

  187. 187

    Honigmann, A. et al. Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment. Nat. Struct. Mol. Biol. 20, 679–686 (2013).

  188. 188

    Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155–157 (2008).

  189. 189

    Das, S. et al. Single-molecule tracking of small GTPase Rac1 uncovers spatial regulation of membrane translocation and mechanism for polarized signaling. Proc. Natl Acad. Sci. USA 112, E267–E276 (2015).

  190. 190

    Jones, S. A., Shim, S. H., He, J. & Zhuang, X. Fast, three-dimensional super-resolution imaging of live cells. Nat. Methods 8, 499–505 (2011).

  191. 191

    Bingen, P., Reuss, M., Engelhardt, J. & Hell, S. W. Parallelized STED fluorescence nanoscopy. Opt. Express 19, 23716–23726 (2011).

  192. 192

    Chmyrov, A. et al. Nanoscopy with more than 100,000 'doughnuts'. Nat. Methods 10, 737–740 (2013).

  193. 193

    Yang, B., Przybilla, F., Mestre, M., Trebbia, J.-B. & Lounis, B. Large parallelization of STED nanoscopy using optical lattices. Opt. Express 22, 5581–5589 (2014).

  194. 194

    Bergermann, F., Alber, L., Sahl, S. J., Engelhardt, J. & Hell, S. W. 2000-fold parallelized dual-color STED fluorescence nanoscopy. Opt. Express 23, 211–223 (2015).

  195. 195

    Chmyrov, A. et al. Achromatic light patterning and improved image reconstruction for parallelized RESOLFT nanoscopy. Sci. Rep. 7, 44619 (2017).

  196. 196

    Hoyer, P. et al. Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT. Proc. Natl Acad. Sci. USA 113, 3442–3446 (2016).

  197. 197

    Shao, L., Kner, P., Rego, E. H. & Gustafsson, M. G. Super-resolution 3D microscopy of live whole cells using structured illumination. Nat. Methods 8, 1044–1046 (2011).

  198. 198

    York, A. G. et al. Instant super-resolution imaging in live cells and embryos via analog image processing. Nat. Methods 10, 1122–1126 (2013).

  199. 199

    Wäldchen, S., Lehmann, J., Klein, T., van de Linde, S. & Sauer, M. Light-induced cell damage in live-cell super-resolution microscopy. Sci. Rep. 5, 15348 (2015).

  200. 200

    Patton, B. R. et al. Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics. Opt. Express 24, 8862–8876 (2016).

  201. 201

    Antonello, J., Kromann, E. B., Burke, D., Bewersdorf, J. & Booth, M. J. Coma aberrations in combined two- and three-dimensional STED nanoscopy. Opt. Lett. 41, 3631–3634 (2016).

  202. 202

    Burke, D., Patton, B., Huang, F., Bewersdorf, J. & Booth, M. J. Adaptive optics correction of specimen-induced aberrations in single-molecule switching microscopy. Optica 2, 177–185 (2015).

  203. 203

    Willig, K. I. et al. Nanoscopy of filamentous actin in cortical dendrites of a living mouse. Biophys. J. 106, L01–L03 (2014).

  204. 204

    Mo, G. C. H. et al. Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution. Nat. Methods 14, 427–434 (2017).

  205. 205

    Testa, I. et al. Nanoscopy of living brain slices with low light levels. Neuron 75, 992–1000 (2012).

  206. 206

    Berning, S., Willig, K. I., Steffens, H., Dibaj, P. & Hell, S. W. Nanoscopy in a living mouse brain. Science 335, 551 (2012).

  207. 207

    Schnorrenberg, S. et al. In vivo super-resolution RESOLFT microscopy of Drosophila melanogaster. eLife 5, e15567 (2016).

  208. 208

    Beliveau, B. J. et al. Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nat. Commun. 6, 7147 (2015).

  209. 209

    Boettiger, A. N. et al. Super-resolution imaging reveals distinct chromatin folding for different epigenetic states. Nature 529, 418–422 (2016). Refs 208 and 209 are pioneering nanoscopy reports using STORM and Oligopaint fluorescence in situ hybridization probes on the spatial organization of DNA, including the classification of genomic domains.

  210. 210

    Viero, G. et al. Three distinct ribosome assemblies modulated by translation are the building blocks of polysomes. J. Cell Biol. 208, 581–596 (2015).

  211. 211

    Galiani, S. et al. Super-resolution microscopy reveals compartmentalization of peroxisomal membrane proteins. J. Biol. Chem. 291, 16948–16962 (2016).

  212. 212

    Kanchanawong, P. et al. Nanoscale architecture of integrin-based cell adhesions. Nature 468, 580–584 (2010). Early 3D nanoscopy study of the complex nanoscale protein organization within focal adhesions, which are involved in force transmission, cytoskeletal regulation and signalling.

  213. 213

    Erdmann, R. S. et al. Super-resolution imaging of the Golgi in live cells with a bioorthogonal ceramide probe. Angew. Chem. Int. Ed. Engl. 53, 10242–10246 (2014).

  214. 214

    Hell, S. W. Microscopy and its focal switch. Nat. Methods 6, 24–32 (2009).

  215. 215

    Olivier, N., Keller, D., Gonczy, P. & Manley, S. Resolution doubling in 3D-STORM imaging through improved buffers. PLoS ONE 8, e69004 (2013).

  216. 216

    Lee, Y. L. et al. Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole-cilium interface and facilitates proper cilium formation and function. Mol. Biol. Cell 25, 2919–2933 (2014).

  217. 217

    Wang, W., Li, G. W., Chen, C., Xie, X. S. & Zhuang, X. Chromosome organization by a nucleoid-associated protein in live bacteria. Science 333, 1445–1449 (2011).

  218. 218

    Ptacin, J. L. et al. A spindle-like apparatus guides bacterial chromosome segregation. Nat. Cell Biol. 12, 791–798 (2010). Nanoscopy visualizes components of a dedicated chromosome segregation apparatus in bacterial cells, which features surprising similarities to eukaryotic spindles.

  219. 219

    Fu, G. et al. In vivo structure of the E. coli FtsZ-ring revealed by photoactivated localization microscopy (PALM). PLoS ONE 5, e12682 (2010).

  220. 220

    Lee, M. K., Rai, P., Williams, J., Twieg, R. J. & Moerner, W. E. Small-molecule labeling of live cell surfaces for three-dimensional super-resolution microscopy. J. Am. Chem. Soc. 136, 14003–14006 (2014).

  221. 221

    Raulf, A. et al. Click chemistry facilitates direct labelling and super-resolution imaging of nucleic acids and proteins. RSC Adv. 4, 30462–30466 (2014).

  222. 222

    Laplante, C., Huang, F., Tebbs, I. R., Bewersdorf, J. & Pollard, T. D. Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast. Proc. Natl Acad. Sci. USA 113, E5876–E5885 (2016).

  223. 223

    Brown, M. S., Grubb, J., Zhang, A., Rust, M. J. & Bishop, D. K. Small Rad51 and Dmc1 complexes often co-occupy both ends of a meiotic DNA double strand break. PLoS Genet. 11, e1005653 (2015).

  224. 224

    Kaplan, C. et al. Absolute arrangement of subunits in cytoskeletal septin filaments in cells measured by fluorescence microscopy. Nano Lett. 15, 3859–3864 (2015).

  225. 225

    Wilkens, V., Kohl, W. & Busch, K. Restricted diffusion of OXPHOS complexes in dynamic mitochondria delays their exchange between cristae and engenders a transitory mosaic distribution. J. Cell Sci. 126, 103–116 (2013).

Download references


S.J. and S.W.H. acknowledge funding through the Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB).

Author information

All three authors contributed equally to all four aspects of preparing the article (researching data for the article, substantial contributions to the discussion of the content, writing, and reviewing and editing of the manuscript before submission).

Correspondence to Steffen J. Sahl or Stefan W. Hell or Stefan Jakobs.

Ethics declarations

Competing interests

S.W.H. is a co-founder of Abberior Instruments GmbH and Abberior GmbH, companies commercializing super-resolution microscopy systems and fluorophores for super-resolution applications, respectively.

Supplementary information

Supplementary information S1 (box)

The diffraction limit of optical microscopy (schematic). (PDF 278 kb)

PowerPoint slides


Numerical aperture

(NA). Measure of the opening angle under which light is collected by an objective lens. The NA (n· sinα, with n being the refractive index and α the semi-aperture angle) determines the tightest focusing possible and thus establishes the resolution of diffraction-limited microscopy.

Fluorophore states

States with defined properties. In the context of nanoscopy, useful pairs of states are pairs for which one of them gives a signal ('on'), whereas the other one does not ('off'), as this allows fluorophores to be distinguished even when they are located in closer proximity to each other than the diffraction limit.

Stimulated emission depletion

(STED). The stimulated emission process transfers the excited fluorophore to its ground state. The stimulating photon induces the generation of a stimulated identical photon, which is not detected. The STED light thus exits the specimen, providing a clean fluorophore off-switch. The near-infrared light used in STED is hardly absorbed by the cell.

Reversible saturable/switchable optical linear (fluorescence) transitions

(RESOLFT). The general conceptual framework for coordinate-targeted nanoscopy. The term is mostly used in reference to approaches using reversibly switchable fluorescent proteins (RSFPs, see below) or photochromic organic compounds.

Photo-activated localization microscopy/stochastic optical reconstruction microscopy

(PALM/STORM). Coordinate-stochastic nanoscopy concepts based on the switching and localization of single molecules. Conceptually similar techniques include fluorescent PALM (fPALM) and ground state depletion with individual molecule return (GSDIM).

Points accumulation for imaging in nanoscale topography

(PAINT). A coordinate-stochastic nanoscopy concept based on separating fluorophores by registering only the bound ones ('on'), with the diffusing fluorophores remaining undetected ('off').

Nanoscopy with minimal photon fluxes

(MINFLUX). A concept that allows precise localization of fluorophores with minimal fluxes of emitted photons. MINFLUX nanoscopy combines coordinate-targeted and coordinate-stochastic aspects.

Multiple off-state transitions for nanoscopy

(MOST). A concept that synergistically combines two or more state-transfer mechanisms to, for example, protect the fluorophore from pathways related to photobleaching and improve signal-to-background in coordinate-targeted nanoscopy.


A method for increasing the signal (photobleaching reduction) in coordinate-targeted nanoscopy. Using scan fields below the diffraction limit around an intensity minimum (for example, at the centre of a doughnut shape) avoids subjecting the fluorophores to the excess intensities of switching light at the maxima of the off-switching pattern.

Optical sectioning

Used to obtain an image with sufficient contrast that is not compromised by fluorescence originating in other axial planes of the specimen. For example, a confocal pinhole can act to reject the out-of-plane background. Other sectioning strategies include selective excitation or photoactivation by multi-photon absorption or light sheets.


An algorithm to reverse the effects of convolution in the image formation process. By removing the optical blur, a sharper image is computed based on the (ideally) exact knowledge of the blurring (formalized by the so-called point spread function (PSF)). Because knowledge of this PSF is in practice imperfect, and registered images are compromised by noise, artefacts can easily arise in the deconvolution process. Deconvolution is not equivalent to methods that actually improve the spatial resolution by a (on-off) state transition.

Structured illumination microscopy

(SIM). A diffraction-limited method that produces up to 2-fold improved resolution and requires the acquisition of several images of a specimen with shifted illumination patterns and computation of a reconstructed image. Further improvements in resolution can be realized if on-off transitions (as in reversible saturable/switchable optical linear (fluorescence) transitions) are incorporated.


A diffraction-limited method that combines conventional confocal laser scanning microscopy with fast widefield detection or other detector designs to achieve close to a doubling of resolution after mathematical processing. Also known as image scanning microscopy (ISM).

Lattice light-sheet microscopy

A diffraction-limited method that uses a structured light sheet to excite fluorescence in successive planes of a specimen, generating a time series of 3D images that can provide information about dynamic biological processes.

Super-resolution optical fluctuation imaging

(SOFI). A method that analyses on-off fluctuations of fluorescence signals (but not strictly at the single-molecule level as in photo-activated localization microscopy and stochastic optical reconstruction microscopy) by examining correlations in time to improve resolution typically 2- to 3-fold in comparison with epifluorescence.


Optical arrangement for coherent excitation and/or collection of fluorescence emissions featuring two juxtaposed lenses of high numerical aperture to expand the solid angle as much as possible, which enables very high axial resolution in nanoscopy (<10 nm).

Single-particle averaging

Computational methods that infer a structure by sorting and averaging data from a large dataset of images showing the same object.


Parts of a protein that are detected by an antibody or other binding probe.

Bio-orthogonal labelling

Chemical labelling reactions that can occur inside living cells without interfering with endogenous biochemical processes.

Genetic code expansion

A process that enables the site-specific incorporation of an amino acid that is not among the 20 common proteinogenic amino acids into a protein.

Click chemistry

A term that encompasses several chemical reactions that facilitate the fast, specific and irreversible attachment of a probe such as a fluorophore to a specific biomolecule.

Labelling coverage

The fraction of epitopes decorated by a binding probe such as an antibody out of all epitopes potentially available for decoration by this binding probe.

Fluorescence fluctuation spectroscopy

A set of methods, in particular fluorescence correlation spectroscopy (FCS), which allow the determination of timescales of dynamic processes. By analysing the (self-) similarity (so-called correlations) of the signal from an observed spot over time, information on, for example, molecular diffusion can be obtained.

Dwell time

Duration for which a scanning nanoscope collects signal at a given position (pixel or voxel).

Reversibly switchable fluorescent proteins

(RSFPs). Fluorescent proteins that can be reversibly switched by light irradiation between long-lived non-fluorescent 'off' and fluorescent 'on' states. RSFPs can be efficiently transferred between the two states at even a low light dose. Because the established state difference remains in place for milliseconds to hours, in RSFP-based reversible saturable/switchable optical linear (fluorescence) transitions nanoscopy, much lower light intensities are needed to break the diffraction barrier than in stimulated emission depletion nanoscopy.

Adaptive optics

Optical strategies to compensate for the effects of aberration and ensure more optimal focusing by deliberately modifying the phase across the light wavefront, often in response to a measurement to characterize the presence of aberrations, which is used as feedback.

Refractive index

A dimensionless number expressing the factor by which light is slowed down when travelling through a material compared with in vacuum. The refractive index of the immersion medium of an objective lens co-determines its numerical aperture.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Sahl, S., Hell, S. & Jakobs, S. Fluorescence nanoscopy in cell biology. Nat Rev Mol Cell Biol 18, 685–701 (2017). https://doi.org/10.1038/nrm.2017.71

Download citation

Further reading