Structured Illumination Microscopy

  • Barry R. MastersEmail author
Part of the Springer Series in Optical Sciences book series (SSOS, volume 227)


Structured illumination microscopy/microscope (SIM) is an optical technique that has the capability of enhancing the lateral and axial resolution of a fluorescence widefield microscope.


  1. Bailey, B., Farkas, D. L., Taylor, D. L., and Lanni, F. (1993). Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation. Nature, 366, 44–48.Google Scholar
  2. Bailey, B., Krishnamurthi, V., Farkas, D. L., Taylor, D. L., and Lanni, F. (1994). Three-dimensional imaging of biological specimens with standing wave fluorescence microscopy. Proceedings of SPIE, 2184, 208–213.Google Scholar
  3. Best, G., Amberger, R., Baddeley, D., Ach, T., Dithmar, S., Heintzmann, R., and Cremer, C. (2011). Structured illumination microscopy of autofluorescent aggregations in human tissue. Micron, 42, 330–335.Google Scholar
  4. Boyd, R. W. (2008). Nonlinear Optics, Third Edition. San Diego: Academic Press.Google Scholar
  5. Bracewell, R. (1999). The Fourier Transform and Its Applications, Third Edition, New York: McGraw-Hill.Google Scholar
  6. Carlson, A. B. (1986). Communications Systems. New York, McGraw-Hill.Google Scholar
  7. Cragg, G. E., and So, P. T. C. (2000). Lateral resolution enhancement with standing evanescent waves. Optics Letters, 25, 46–48.Google Scholar
  8. Creath, K., Schmitt, J., and Wyant, J. C. (2007). Optical Metrology of Diffuse Surfaces, in: Optical Shop Testing, Third Edition, Daniel Malacara, Editor. Hoboken: John Wiley & Sons, pp. 756-807.Google Scholar
  9. Frohn, J. T., Knapp, H. F., and Stemmer, A. (2000). True optical resolution beyond the Rayleigh limit achieved by standing wave illumination. Proceedings of the National Academy of Sciences USA, 97, 7232–7236.Google Scholar
  10. Frohn, J. T., Knapp, H. F., and Stemmer, A. (2001). Three-dimensional resolution enhancement in fluorescence microscopy by harmonic excitation. Optics Letters, 26, 828–830.Google Scholar
  11. Gaskill, J. D. (1978). Linear systems, Fourier Transforms, and Optics. New York: John Wiley and Sons.Google Scholar
  12. Goodman, J. W. (2005). Introduction to Fourier Optics, Third Edition. Greenwood Village, Colorado: Roberts & Company.Google Scholar
  13. Goodman, J. W. (2017). Introduction to Fourier optics (4th ed.). New York: W. H. Freeman and Company.Google Scholar
  14. Grimm, M. A., and Lohmann, A. W. (1966). Super resolution image for one-dimensional objects. Journal of the Optical Society of America, 56, 1151–1156.Google Scholar
  15. Gustafsson, M. G. L., Sedat, J. W., and Agard, D. A. Method and apparatus for three-dimensional microscopy with enhanced depth resolution. US Patent RE38,307, E1. Filed 1995, reissued 11 November 2003.Google Scholar
  16. Gustafsson, M. G. L. (1999). Extended resolution fluorescence microscopy. Current Opinion in Structural Biology, 9, 627–634.Google Scholar
  17. Gustafsson, M. G. L. (2000). Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. Journal of Microscopy, 198, 82–87.Google Scholar
  18. Gustafsson, M. G. L., Agard, D. A., and Sedat, J. W. (1995). Sevenfold improvement of axial resolution in 3D widefield microscopy using two objective lenses. Proceedings of SPIE, 2412, 147–156.Google Scholar
  19. Gustafsson, M. G. L., Agard, D. A., and Sedat, J. W. (1999). I5M: 3D widefield light microscopy with better than 100 nm axial resolution. Journal of Microscopy, 195, 10–16.Google Scholar
  20. Gustafsson, M. G. L., Agard, D. A., and Sedat, J. W. (2000). Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination. Proceedings of SPIE, 3919, 141–150.Google Scholar
  21. Gustafsson, M. G. L. (2005). Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proceedings of the National Academy of Sciences USA, 102, 13081–13086.Google Scholar
  22. Gustafsson, M. G. L. (2008). Super-resolution light microscopy goes live. Nature Methods, 5, 385–387.Google Scholar
  23. Gustafsson, M. G. L., Shao, L., Carlton, P. M., Wang, C. J., Golubovskaya, I. N., Cande, W. Z., Agard, D. A., and Sedat, J. W. (2008). Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophysical Journal, 94, 4957–4970.Google Scholar
  24. Hausmann, M., Schneider, B., Bradl, J., and Cremer, C. (1997). High-precision distance microscopy of 3D-nanostructures by a spatially modulated excitation fluorescence microscope. Proceedings of SPIE, 3197, 217–222.Google Scholar
  25. Heintzmann, R. (2003). Saturated patterned excitation microscopy with two-dimensional excitation patterns. Micron, 34, 283–291.Google Scholar
  26. Heintzmann, R., and Benedetti, P. A. (2006). High-resolution image reconstruction in fluorescence microscopy with patterned excitation. Applied Optics, 45, 5037–5045.Google Scholar
  27. Heintzmann, R., and Cremer, C. (1999). Laterally modulated excitation microscopy: improvement of resolution by using a diffraction grating. Proceedings of SPIE, 3568, 185–196.Google Scholar
  28. Heintzmann, R., and Cremer, C. (2002). Axial tomographic confocal fluorescence microscopy. Journal of Microscopy, 206, 7–23.Google Scholar
  29. Heintzmann, R., Jovin, T. M., and Cremer, C. (2002). Saturated patterned excitation microscopy-a concept for optical resolution improvement. Journal of the Optical Society of America A, 19, 1599–1609.Google Scholar
  30. Hell, S., and Stelzer, E. H. K. (1992a). Properties of a 4Pi confocal fluorescence microscope. Journal of the Optical Society of America A, 9, 2159–2166.Google Scholar
  31. Hell, S., and Stelzer, E. H. K. (1992b). Fundamental improvement of a 4Pi confocal fluorescence microscope using two-photon excitation. Optics Communications, 93, 277–282.Google Scholar
  32. Hirvonen, L. (2008). Structured illumination microscopy using photoswitchable fluorescent proteins. PhD thesis, King’s College London, UK.Google Scholar
  33. Jost, A., Tolstik, E., Feldmann, P., Wicker, K., Sentenac, A., and Heintzmann, R. (2015). Optical sectioning and high resolution in single-slice structured illumination microscopy by thick slice blind-SIM reconstruction. PLoS ONE, 10(7), e0132174.
  34. Kafri, O., and Glatt, I. (1990). The Physics of Moiré Metrology. New York: John Wiley & Sons.Google Scholar
  35. Kner, P., Chhun, B. B., Griffis, E. R., Winoto, L., and Gustafsson, M. G. (2009). Super-resolution video microscopy of live cells by structured illumination. Nature Methods, 6, 339–342.Google Scholar
  36. Krzewina, L. G., and Kim, M. K. (2006). Single-exposure optical sectioning by color structured illumination microscopy. Optics Letters, 31, 477–479.Google Scholar
  37. Lanni, F., and Bailey, B. (1994). Standing-wave excitation for fluorescence microscopy. Trends in Cell Biology, 4, 262–265.Google Scholar
  38. Lanni, F., Bailey, B., Farkas, D. L., and Taylor, D. L. (1993). Excitation field synthesis as a means for obtaining enhanced axial resolution in fluorescence microscopes. Bioimaging, 1, 187–196.Google Scholar
  39. Lanni, F., Taylor, D. L., and Waggoner, A. S. (1986). Standing wave luminescence microscopy. Patent, US 4621911 A, Nov 11, 1986.Google Scholar
  40. Lohmann, A. W. (1978). Three-dimensional properties of wave-fields. Optik, 51, 105–117.Google Scholar
  41. Lukosz, W. (1966). Optical systems with resolving powers exceeding the classical limit, Part 1. Journal of the Optical Society of America, 56, 1463–1471.Google Scholar
  42. Lukosz, W. (1967). Optical systems with resolving powers exceeding the classical limit. II. Journal of the Optical Society of America, 57, 932–941.Google Scholar
  43. Lukosz, W., and Marchand, M. (1963). Optischen Abbildung Unter Überschreitung der Beugungsbedingten Auflösungsgrenze. Optica Acta, 10, 241–255.Google Scholar
  44. Masters, B. R. (1996). Selected Papers on Confocal Microscopy. Bellingham, SPIE Press.Google Scholar
  45. Neil, M. A. A., Juškaitis, R., and Wilson, T. (1997). Method of obtaining optical sectioning by using structured light in a conventional microscopy. Optics Letters, 22, 1905–1907.Google Scholar
  46. Neil, M. A. A., Juškaitis, R., and Wilson, T. (1998a). Real time 3D fluorescence microscopy by two beam interference illumination. Optics Communications, 153, 1–4.Google Scholar
  47. Neil, M. A. A., Wilson, T., and Juškaitis, R. (1998b). A light efficient optically sectioning microscope. Journal of Microscopy, 189, 114–117.Google Scholar
  48. Oster, G., and Nishijima, Y. (1963). Moiré patterns. Scientific American, 208, 54–63.Google Scholar
  49. Rayleigh, L. (1881). On copying diffraction gratings and on some phenomenon connected therewith. Philosophical Magazine, 11, 196–205.Google Scholar
  50. Rego, E. H., Shao, L., Macklin, J. J., Winoto, L., Johansson, G. A., Kamps-Hughes, N., Davidson, M. W., and Gustafsson, M. G. L. (2012). Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution. Proceedings of the National Academy of Sciences USA, 109, E135–E143.Google Scholar
  51. Rowe, S. H., and Welford, W. T. (1967). Surface topography of non-optical surfaces by projected interference fringes. Nature, 216, 786–787.Google Scholar
  52. Schermelleh, L., Carlton, P. M., Haase, S., Shao, L., Winoto, L., Kner, P., Burke, B., Cardoso, M. C., Agard, D. A., Gustafsson, M. G. L., Leonhardt, H., and Sedat, J. W. (2008). Subdiffraction multicolor imaging of the nuclear periphery with 3D structured illumination microscopy. Science, 320, 1332–1336.Google Scholar
  53. Schropp, M., and Uhl, R. (2014). Two-dimensional structured illumination microscopy. Journal of Microscopy, 256, 23–36.Google Scholar
  54. Shao, L., Isaac, B., Uzawa, S., Agard, D. A., Sedat, J. W., and Gustafsson, M. G. L. (2008). I5S: wide-field light microscopy with 100-nm-scale resolution in three dimensions. Biophysical Journal, 94, 4971–4983.Google Scholar
  55. Streibl, N. (1984). Fundamental restrictions for 3-D light distributions. Optik, 66, 341–354.Google Scholar
  56. Streibl, N. (1985). Three-dimensional imaging by a microscope. Journal of the Optical Society of America A, 2, 121–127.Google Scholar
  57. Talbot, H. F. (1836). Facts relating to optical science. Philosophical Magazine, 9, 401–407.Google Scholar
  58. Wicker, K., Mandula, O., Best, G., Fiolka, R., and Heintzmann, R. (2013). Phase optimization for structured illumination microscopy. Optics Express, 21, 2032–2049.Google Scholar
  59. Williams, C. S., and Becklund, O. A. (1989). Introduction to the Optical Transfer Function. New York: Wiley-Interscience.Google Scholar

Further Reading

  1. Alexandrov, S. A., Hillman, T. R., Gutzler, T., and Sampson, D. D. (2006). Synthetic aperture fourier holographic optical microscopy. Physical Review Letters, 97, 168102-1 to -4.Google Scholar
  2. Ball, G., Demmerle, J., Kaufmann, R., Davis, I., Dobbie, I. M., and Schermelleh, L. (2015). SIMcheck: A toolbox for successful super-resolution structured illumination microscopy. Scientific Reports, 5, 15915.
  3. Betzig, E. (2005). Excitation strategies for optical lattice microscopy. Optics Express, 13, 3021–3036.Google Scholar
  4. Bewersdorf, J., Schmidt, R., and Hell, S. W. (2006). Comparison of I5M and 4Pi-microscopy. Journal of Microscopy, 222, 105–117.Google Scholar
  5. Blanca, C. M., and Hell, S. W. (2002). Axial superresolution with ultrahigh aperture lenses. Optics Express, 10, 893–898.Google Scholar
  6. Buscher, D. F. (2015). Practical Optical Interferometry. Imaging at Visible and Infrared Wavelengths. Cambridge, UK: Cambridge University Press.Google Scholar
  7. Chung, E., Kim, D., Cui, Y., Kim, Y-H., and So, P. T. C. (2007). Two-dimensional standing wave total internal reflection fluorescence microscopy: Superresolution imaging of single molecular and biological specimens. Biophysical Journal, 93, 1747–1757.Google Scholar
  8. Cremer, C., and Masters, B. R. (2013). Resolution enhancement techniques in microscopy. The European Physical Journal H, 38, 281–344. (Open Access article).Google Scholar
  9. Débarre, D., Botcherby, E. J., Booth, M. J., and Wilson, T. (2008). Adaptive optics for structured illumination microscopy. Optics Express, 16, 9290–9305.Google Scholar
  10. Dubois, A., Vabre, L., Boccara, A.-C., and Beaurepaire, E. (2002). High-resolution full-field optical coherence tomography with a Linnik microscope. Applied Optics, 41, 805–812.Google Scholar
  11. Egner, A., and Hell, S. W. (2005). Fluorescence microscopy with super-resolved optical sections. Trends in Cell Biology, 15, 207–215.Google Scholar
  12. Fiolka, R., Shao, L., Rego, E. H., Davidson, M. W., and Gustafsson, M. G. L. (2012). Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination. Proceedings of the National Academy of Sciences USA, 109, 5311–5315.Google Scholar
  13. Heintzmann, R., and Ficz, G. (2007). Breaking the resolution limit in light microscopy. Methods in Cell Biology, 81, 561–580.Google Scholar
  14. Jost, A., and Heintzmann, R. (2013). Superresolution multidimensional imaging with structured illumination microscopy. Annual Review of Materials Research, 43, 261–82.Google Scholar
  15. Kam, Z., Hanser, B., Gustafsson, M. G. L., Agard, D. A., and Sedat, J. W. (2001). Computational adaptive optics for live three-dimensional biological imaging. Proceedings of the National Academy of Sciences USA, 98, 3790–3795.Google Scholar
  16. Lim, D., Chu, K. K., and Mertz, J. (2008). Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy. Optics Letters, 33, 1819–1821.Google Scholar
  17. Lim, D., Ford, T. N., Chu, K. K., and Mertz, J. (2011). Optically sectioned in vivo imaging with speckle illumination HiLo microscopy. Journal of Biomedical Optics, 16, 016014-1 to 016014-8.Google Scholar
  18. Lukyanov, K. A., Fradkov, A. F., Gurskaya, N. G., Matz, M. V., Labas, Y. A., Savitsky, A. P., Markelov, M. L., Zaraisky, A. G., Zhao, X., Tan, W., and Lukyanov, S. A. (2000). Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. Journal of Biological Chemistry, 275, 25879–25882.Google Scholar
  19. Martínez-Corral, M., and Saavedra, G. (2009). The Resolution Challenge in 3D Optical Microscopy, in: Progress in Optics, Emil Wolf, Editor, 53, 1–67.Google Scholar
  20. Mertz, J. (2011). Optical sectioning microscopy with planar or structured illumination. Nature Methods, 8, 911–819.Google Scholar
  21. Neil, M. A. A., Juškaitis, R., Wilson, T., Laczik, Z. J., and Sarafis, V. (2000a). Optimized pupil-plane filters for confocal microscope point-spread function engineering. Optics Letters, 25, 245–247.Google Scholar
  22. Neil, M. A. A., Squire, A., Juškaitis, R., Bastiaens, P. I., and Wilson, T. (2000b). Wide-field optically sectioning fluorescence microscopy with laser illumination. Journal of Microscopy, 197, 1–4.Google Scholar
  23. Porter, A. B. (1906). On the diffraction theory of microscope vision. Philosophical Magazine, 6, 154–156.Google Scholar
  24. Rego, E. H., and Shao, L. (2015). Practical structured illumination microscopy. In: Advanced fluorescence Microscopy: Methods and Protocols in Molecular Biology. Peter J. Verveer (ed.), vol. 1251, 175–192.Google Scholar
  25. Reasenberg, R. D. (Ed.) (1998). Astronomical interferometry. Proceedings of SPIE, 3350, entire volume. Bellingham: SPIE.Google Scholar
  26. Schermelleh, L., Heintzmann, R., and Leonhardt, H. (2010). A guide to super-resolution fluorescence microscopy. The Journal of Cell Biology, 190, 165–175.Google Scholar
  27. Schwentker, A., Bock, H., Hofmann, M., Jakobs, S., Bewersdorf, J., Eggeling, C., and Hell, S. W. (2007). Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching. Microscopy Research and Technique, 70, 269–280.Google Scholar
  28. Shabtay, G., Mendlovic, D., Zalevsky, Z., and Lipson, L. (2001). The optimal system for sub-wavelength point source localization. Optics Communications, 198, 311–315.Google Scholar
  29. Shao, L., Kner, P., Rego, E. H., and Gustafsson, M. G. L. (2011). Super-resolution 3D microscopy of live whole cells using structured illumination. Nature Methods, 8, 1044–1046.Google Scholar
  30. Shao, L., Winoto, L., Agard, D. A., Gustafsson, M. G. L., and Sedat, J. W. (2012). Interferometer-based structured-illumination microscopy utilizing complementary phase relationship through constructive and destructive image detection by two cameras. Journal of Microscopy, 246, 229–236.Google Scholar
  31. Sheppard, C. J. R. (2007). Fundamentals of superresolution. Micron, 38, 165–169.Google Scholar
  32. Wicker, K. (2013). Non-iterative determination of pattern phase in structured illumination microscopy using auto-correlations in Fourier space. Optics Express, 21, 24692–24701.Google Scholar
  33. Wicker, K., and Heintzmann, R. (2014). Resolving a misconception about structured illumination. Nature Photonics, 8, 341–344.Google Scholar
  34. Wilson, T., Neil, M. A. A., and Juškaitis, R. (1998). Real-time three-dimensional imaging of microscopic structures. Journal of Microscopy, 191, 116–118.Google Scholar
  35. Zalevsky, Z., and Mendlovic, D. (2004). Optical Superresolution. Springer Series in Optical Sciences. New York: Springer. Google Scholar

Copyright information

© Springer Nature Switzerland AG 2020

Authors and Affiliations

  1. 1.Previously, Visiting Scientist Department of Biological EngineeringMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Previously, Visiting Scholar Department of the History of ScienceHarvard UniversityCambridgeUSA

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