Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Perspective
  • Published:

Microscopy and its focal switch

Nature Methods volume 6, pages 24–32 (2009)Cite this article

Abstract

Until not very long ago, it was widely accepted that lens-based (far-field) optical microscopes cannot visualize details much finer than about half the wavelength of light. The advent of viable physical concepts for overcoming the limiting role of diffraction in the early 1990s set off a quest that has led to readily applicable and widely accessible fluorescence microscopes with nanoscale spatial resolution. Here I discuss the principles of these methods together with their differences in implementation and operation. Finally, I outline potential developments.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Memorial erected in Ernst Abbe's honor displaying his equation describing the diffraction resolution limit, located in front of the physiology building of the University of Jena, Germany.
Figure 2: Fluorescence switching strategies for super-resolution image assembly.

Similar content being viewed by others

References

  1. Verdet, É. Leçons d'optique physique (Victor Masson et fils, Paris, 1869).

    Google Scholar 

  2. Abbe, E. Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Arch. Mikr. Anat. 9, 413–468 (1873).

    Article  Google Scholar 

  3. Lord Rayleigh. On the theory of optical images, with special reference to the microscope. Philos. Mag. XLII, 167–195 (1896).

    Google Scholar 

  4. Hell, S.W. Double confocal microscope. European Patent 0491289 (1990).

  5. Hell, S.W. & Stelzer, E.H.K. Fundamental improvement of resolution with a 4Pi-confocal fluorescence microscope using two-photon excitation. Opt. Commun. 93, 277–282 (1992).

    Article  Google Scholar 

  6. Schrader, M. & Hell, S.W. 4Pi-confocal images with axial superresolution. J. Microsc. 183, 189–193 (1996).

    Article  Google Scholar 

  7. Hell, S.W., Schrader, M. & van der Voort, H.T.M. Far-field fluorescence microscopy with three-dimensional resolution in the 100 nm range. J. Microsc. 185, 1–5 (1997).

    Article  Google Scholar 

  8. Gustafsson, M.G.L., Agard, D.A. & Sedat, J.W. Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses. Proc. SPIE 2412, 147–156 (1995).

    Article  Google Scholar 

  9. Gustafsson, M.G.L., Agard, D.A. & Sedat, J.W. I5M: 3D widefield light microscopy with better than 100 nm axial resolution. J. Microsc. 195, 10–16 (1999).

    Article  CAS  PubMed  Google Scholar 

  10. Burns, D.H., Callis, G.D., Christian, G.D. & Davidson, E.R. Strategies for attaining superresolution using spectroscopic data as constraints. Appl. Opt. 24, 154–160 (1985).

    Article  CAS  PubMed  Google Scholar 

  11. Hell, S.W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated emission depletion microscopy. Opt. Lett. 19, 780–782 (1994).

    Article  CAS  PubMed  Google Scholar 

  12. Klar, T.A., Jakobs, S., Dyba, M., Egner, A. & Hell, S.W. Fluorescence microscopy with diffraction resolution limit broken by stimulated emission. Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  Google Scholar 

  14. Heintzmann, R., Jovin, T.M. & Cremer, C. Saturated patterned excitation microscopy - a concept for optical resolution improvement. J. Opt. Soc. Am. A 19, 1599–1609 (2002).

    Article  Google Scholar 

  15. Gustafsson, M.G.L. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. USA 102, 13081–13086 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  17. Hell, S.W. Strategy for far-field optical imaging and writing without diffraction limit. Phys. Lett. A 326, 140–145 (2004).

    Article  CAS  Google Scholar 

  18. Hell, S.W., Dyba, M. & Jakobs, S. Concepts for nanoscale resolution in fluorescence microscopy. Curr. Opin. Neurobiol. 14, 599–609 (2004).

    Article  CAS  PubMed  Google Scholar 

  19. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  21. Rust, M.J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–796 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Biteen, J.S. et al. Super-resolution imaging in live Caulobacter crescentus cells using photoswitchable EYFP. Nat. Methods 5, 947–949 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  28. Schwentker, M.A. et al. Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching. Microsc. Res. Tech. 70, 269–280 (2007).

    Article  CAS  PubMed  Google Scholar 

  29. Harke, B. et al. Resolution scaling in STED microscopy. Opt. Express 16, 4154–4162 (2008).

    Article  PubMed  Google Scholar 

  30. Hell, S.W. Increasing the resolution in fluorescence light microscopy by point-spread-function engineering. in Topics in Fluorescence Spectroscopy (ed. Lakowicz, J.R.) 361–422 (Plenum, New York, 1997).

    Google Scholar 

  31. Bretschneider, S., Eggeling, C. & Hell, S.W. Breaking the diffraction barrier in fluorescence microscopy by optical shelving. Phys. Rev. Lett. 98, 218103 (2007).

    Article  PubMed  CAS  Google Scholar 

  32. Hell, S.W. & Schönle, A. Nanoscale resolution in far-field fluorescence microscopy. in Science of Microscopy (eds., Hawkes, P.W. and Spence, J.C.H) 790–834 (Springer, New York, 2007).

    Chapter  Google Scholar 

  33. Heisenberg, W. The Physical Principles of the Quantum Theory (Chicago Univ. Press, Chicago, 1930).

    Google Scholar 

  34. Bobroff, N. Position measurement with a resolution and noise-limited instrument. Rev. Sci. Instrum. 57, 1152–1157 (1986).

    Article  Google Scholar 

  35. Betzig, E. Proposed method for molecular optical imaging. Opt. Lett. 20, 237–239 (1995).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Fölling, J. et al. Photochromic rhodamines provide nanoscopy with optical sectioning. Angew. Chem. Int. Ed. 46, 6266–6270 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Geisler, C. et al. Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching. Appl. Phys. A Mater. Sci. Process. 88, 223–226 (2007).

    Article  CAS  Google Scholar 

  40. Steinhauer, C., Forthmann, C., Vogelsang, J. & Tinnefeld, P. Superresolution microscopy on the basis of engineered dark states. J. Am. Chem. Soc. 10.1021/ja806590m (2008).

  41. Shroff, H., Galbraith, C.G., Galbraith, J.A. & Betzig, E. Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat. Methods 5, 417–423 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Westphal, V., Lauterbach, M.A., Di Nicola, A. & Hell, S.W. Dynamic far-field fluorescence nanoscopy. New J. Phys. 9, 435 (2007).

    Article  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  45. Moerner, W.E. & Kador, L. Optical detection and spectroscopy of single molecules in a solid. Phys. Rev. Lett. 62, 2535–2538 (1989).

    Article  CAS  PubMed  Google Scholar 

  46. Orrit, M. & Bernard, J. Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal. Phys. Rev. Lett. 65, 2716–2719 (1990).

    Article  CAS  PubMed  Google Scholar 

  47. Moerner, W.E. Single-molecule mountains yield nanoscale cell images. Nat. Methods 3, 781–782 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Schönle, A. & Hell, S.W. Fluorescence nanoscopy goes multicolor. Nat. Biotechnol. 25, 1234–1235 (2007).

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Nägerl, V.U., 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).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Kastrup, L., Blom, H., Eggeling, C. & Hell, S.W. Fluorescence fluctuation spectroscopy in subdiffraction focal volumes. Phys. Rev. Lett. 94, 178104 (2005).

    Article  PubMed  CAS  Google Scholar 

  52. Dedecker, P. et al. Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: numerical analysis and application to the fluorescent protein Dronpa. J. Am. Chem. Soc. 129, 16132–16141 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. Hell, S.W., Soukka, J. & Hänninen, P.E. Two- and multiphoton detection as an imaging mode and means of increasing the resolution in far-field light microscopy. Bioimaging 3, 64–69 (1995).

    Article  Google Scholar 

  54. Lidke, K.A., Rieger, B., Jovin, T.M. & Heintzmann, R. Superresolution by localization of quantum dots using blinking statistics. Opt. Express 13, 7052–7062 (2005).

    Article  PubMed  Google Scholar 

  55. Heintzmann, R. & Ficz, G. Breaking the resolution limit in light microscopy. Brief. Funct. Genomic. Proteomic 5, 289–301 (2006).

    Article  PubMed  Google Scholar 

  56. Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    Article  CAS  PubMed  Google Scholar 

  57. Zumbusch, A., Holtom, G.R. & Xie, X.S. Three-dimensional vibrational imaging by coherent anti-stokes Raman scattering. Phys. Rev. Lett. 82, 4142–4145 (1999).

    Article  CAS  Google Scholar 

  58. Schönle, A., Hänninen, P.E. & Hell, S.W. Nonlinear fluorescence through intermolecular energy transfer and resolution increase in fluorescence microscopy. Ann. Phys. 8, 115–133 (1999).

    Article  Google Scholar 

  59. Schönle, A. & Hell, S.W. Far-field fluorescence microscopy with repetetive excitation. Eur. Phys. J. D 6, 283–290 (1999).

    Article  Google Scholar 

  60. Weiss, S. Fluorescence spectroscopy of single biomolecules. Science 283, 1676–1683 (1999).

    Article  CAS  PubMed  Google Scholar 

  61. Bornfleth, H., Satzler, K., Eils, R. & Cremer, C. High-precision distance measurements and volume-conserving segmentation of objects near and below the resolution limit in three-dimensional confocal fluorescence microscopy. J. Microsc. 189, 118–136 (1998).

    Article  Google Scholar 

  62. Hildenbrand, G. et al. Nano-sizing of specific gene domains in intact human cell nuclei by Spatially Modulated Illumination (SMI) light microscopy. Biophys. J. 88, 4312–4318 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. van Oijen, A.M., Köhler, J., Schmidt, J., Müller, M. & Brakenhoff, G.J. Far-field fluorescence microscopy beyond the diffraction limit. J. Opt. Soc. Am. A 16, 909–915 (1999).

    Article  CAS  Google Scholar 

  64. Qu, X., Wu, D., Mets, L. & Scherer, N.F. Nanometer-localized multiple single-molecule fluorescence microscopy. Proc. Natl. Acad. Sci. USA 101, 11298–11303 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Pendry, J.B. Negative refraction makes a perfect lens. Phys. Rev. Lett. 85, 3966–3969 (2000).

    Article  CAS  PubMed  Google Scholar 

  66. Synge, E.H. A suggested method for extending microscopic resolution into the ultra-microscopic region. Philos. Mag. 6, 356 (1928).

    Article  CAS  Google Scholar 

  67. Pohl, D.W., Denk, W. & Lanz, M. Optical stethoscopy: image recording with resolution λ/20. Appl. Phys. Lett. 44, 651–653 (1984).

    Article  Google Scholar 

  68. Lewis, A., Isaacson, M., Harootunian, A. & Murray, A. Development of a 500 Å resolution light microscope. Ultramicroscopy 13, 227–231 (1984).

    Article  Google Scholar 

  69. Betzig, E., Chichester, R.J., Lanni, F. & Taylor, D.L. Near-field fluorescence imaging of cytoskeletal actin. Bioimaging 1, 129–136 (1993).

    Article  CAS  Google Scholar 

  70. Toraldo di Francia, G. Supergain antennas and optical resolving power. Nuovo Cimento 9 Suppl., 426–435 (1952).

    Article  Google Scholar 

  71. Lukosz, W. Optical systems with resolving powers exceeding the classical limit. J. Opt. Soc. Am. 56, 1463–1472 (1966).

    Article  CAS  Google Scholar 

  72. Minsky, M. Microscopy apparatus. US patent 3,013,467 (1961).

  73. Sheppard, C.J.R. & Wilson, T. The theory of scanning microscopes with Gaussian pupil functions. J. Microsc. 114, 179–197 (1978).

    Article  Google Scholar 

  74. Cremer, C. & Cremer, T. Considerations on a laser-scanning-microscope with high resolution and depth of field. Microsc. Acta 81, 31–44 (1978).

    CAS  PubMed  Google Scholar 

  75. Carrington, W.A. et al. Superresolution in three-dimensional images of fluorescence in cells with minimal light exposure. Science 268, 1483–1487 (1995).

    Article  CAS  PubMed  Google Scholar 

  76. Hell, S.W. Improvement of lateral resolution in far-field light microscopy using two-photon excitation with offset beams. Opt. Commun. 106, 19–24 (1994).

    Article  Google Scholar 

  77. Dyba, M. & Hell, S.W. Focal spots of size λ/23 open up far-field fluorescence microscopy at 33 nm axial resolution. Phys. Rev. Lett. 88, 163901 (2002).

    Article  PubMed  CAS  Google Scholar 

  78. Fölling, J. et al. Fluorescence nanoscopy with optical sectioning by two-photon induced molecular switching using continuous-wave lasers. ChemPhysChem 9, 321–326 (2008).

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  80. Dyba, M., Jakobs, S. & Hell, S.W. Immunofluorescence stimulated emission depletion microscopy. Nat. Biotechnol. 21, 1303–1304 (2003).

    Article  CAS  PubMed  Google Scholar 

  81. Schmidt, R. et al. Spherical nanosized spot unravel the interior of cells. Nat. Methods 4, 81–86 (2008).

    Article  CAS  Google Scholar 

  82. v Middendorff, C., Egner, A., Geisler, C., Hell, S.W. & Schönle, A. Isotropic 3D Nanoscopy based on single emitter switching. Opt. Express 16, 20774–20788 (2008).

    Article  Google Scholar 

  83. Nagorni, M. & Hell, S.W. 4Pi-confocal microscopy provides three-dimensional images of the microtubule network with 100- to 150-nm resolution. J. Struct. Biol. 123, 236–247 (1998).

    Article  CAS  PubMed  Google Scholar 

  84. Egner, A. & Hell, S.W. Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol. 15, 207–215 (2005).

    Article  CAS  PubMed  Google Scholar 

  85. Shao, L. et al. I5S: wide-field light microscopy with 100-nm-scale resolution in three dimensions. Biophys. J. 94, 4971–4983 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. 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).

    Article  CAS  PubMed  Google Scholar 

  87. Hell, S.W. et al. Nanoscale resolution with focused light: stimulated emission depletion and other reversible saturable optical fluorescence transitions microscopy concepts. in Handbook of Biological Confocal Microscopy (ed. Pawley, J.) 571–579 (Springer, New York, 2006).

    Chapter  Google Scholar 

  88. Hell, S.W., Jakobs, S. & Kastrup, L. Imaging and writing at the nanoscale with focused visible light through saturable optical transitions. Appl. Phys. A 77, 859–860 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

I thank E. Rittweger and B. Rankin for help in preparing the figures and various suggestions for improving the presentation. Critical reading by A. Schönle, and also by S. Jakobs, C. Eggeling and J. Jethwa, is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, 37070, Germany

    Stefan W Hell

  2. German Cancer Research Center (DKFZ), Heidelberg, 69120, Germany

    Stefan W Hell

Authors
  1. Stefan W Hell
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stefan W Hell.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hell, S. Microscopy and its focal switch. Nat Methods 6, 24–32 (2009). https://doi.org/10.1038/nmeth.1291

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1291

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing