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.

  • Review Article
  • Published:

Optical sectioning microscopy with planar or structured illumination

Nature Methods volume 8, pages 811–819 (2011)Cite this article

Subjects

Abstract

A key requirement for performing three-dimensional (3D) imaging using optical microscopes is that they be capable of optical sectioning by distinguishing in-focus signal from out-of-focus background. Common techniques for fluorescence optical sectioning are confocal laser scanning microscopy and two-photon microscopy. But there is increasing interest in alternative optical sectioning techniques, particularly for applications involving high speeds, large fields of view or long-term imaging. In this Review, I examine two such techniques, based on planar illumination or structured illumination. The goal is to describe the advantages and disadvantages of these techniques.

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: PIM configurations.
Figure 2: Demonstration of high-speed volumetric imaging with 2P-PIM.
Figure 3: Demonstrations of long-term developmental imaging with PIM.
Figure 4: Demonstration of anatomical imaging with 1P-PIM operated in bidirectional widefield illumination mode.
Figure 5: Schematics of different optical sectioning configurations with structured illumination.
Figure 6: Molecular Probes fluorescently labeled mouse intestine slide imaged with grid-illumination DSD microscopy.
Figure 7: Confocal versus HiLo microscopy.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Pawley, J., ed. Handbook of Biological Confocal Microscopy, 3rd edn. (Springer, 2006).

    Book  Google Scholar 

  3. Conchello, J.-A. & Lichtman, J.W. Optical sectioning microscopy. Nat. Methods 2, 920–931 (2005).

    Article  CAS  Google Scholar 

  4. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

    Article  CAS  Google Scholar 

  5. Svoboda, K. & Yasuda, R. Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 50, 823–839 (2006).

    Article  CAS  Google Scholar 

  6. Siedentopf, H. & Zsigmondy, R. Uber sichtbarmachung und groessenbestimmung ultramikroskopischer teilchen, mit besonder anwendung auf goldrubinglaesern. Ann. Phys. 10, 1–39 (1903).

    CAS  Google Scholar 

  7. Voie, A.H., Burns, D.H. & Spelman, F.A. Orthogonal-plane fluorescence optical sectioning: three dimensional imaging of macroscopic biological specimens. J. Microsc. 170, 229–236 (1993).

    Article  CAS  Google Scholar 

  8. Fuchs, E., Jaffe, J., Long, R. & Azam, F. Thin laser light sheet microscope for microbial oceanography. Opt. Express 10, 145–154 (2002).

    Article  Google Scholar 

  9. Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H.K. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004). The first paper to highlight the advantages of planar illumination for developmental imaging, essentially launching the renaissance of PIM.

    Article  CAS  Google Scholar 

  10. Dodt, H.-U. et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat. Methods 4, 331–336 (2007). Optical clearing and PIM are combined for the first time, demonstrating high resolution, anatomical imaging over wide fields of view.

    Article  CAS  Google Scholar 

  11. Buytaert, J.A.N. & Dirckx, J.J.J. Design and quantitative resolution measurements of an optical virtual sectioning three-dimensional imaging technique for biomedical specimens, featuring two-micrometer slicing resolution. J. Biomed. Opt. 12, 014039 (2007).

    Article  Google Scholar 

  12. Keller, P.J., Schmidt, A.D., Wittbrodt, J. & Stelzer, E.H.K. Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322, 1065–1069 (2008).

    Article  CAS  Google Scholar 

  13. Holekamp, T.F., Turaga, D. & Holy, T.E. Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy. Neuron 57, 661–672 (2008).

    Article  CAS  Google Scholar 

  14. Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–162 (2008).

    Article  CAS  Google Scholar 

  15. Santi, P.A. et al. Thin-sheet laser imaging microscopy for optical sectioning of thick tissues. Biotechniques 46, 287–294 (2009).

    Article  CAS  Google Scholar 

  16. Palero, J., Santos, S.I.C.O., Artigas, D. & Loza-Alvarez, P. A simple scanless two-photon fluorescence microscope using selective plane illumination. Opt. Express 18, 8491–8498 (2010).

    Article  CAS  Google Scholar 

  17. Truong, T.V., Supatto, W., Koos, D.S., Choi, J.M. & Fraser, S.E. Deep and fast live imaging with two-photon scanned light-sheet microscopy. Nat. Methods 8, 757–760 (2011). Advantages of 2P-PIM compared to 1P-PIM for imaging thick samples are highlighted.

    Article  CAS  Google Scholar 

  18. Planchon, T.A. et al. Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat. Methods 8, 417–423 (2011). The combination of PIM with high-numerical-aperture optics provides ultrafast volumetric imaging with isotropic sub-micrometer resolution.

    Article  CAS  Google Scholar 

  19. Hoebe, R.A. et al. Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging. Nat. Biotechnol. 25, 249–253 (2007).

    Article  CAS  Google Scholar 

  20. Chu, K.K., Lim, D. & Mertz, J. Enhanced weak-signal sensitivity in two-photon microscopy by adaptive illumination. Opt. Lett. 32, 2846–2848 (2007).

    Article  Google Scholar 

  21. Chu, K.K., Lim, D. & Mertz, J. Practical implementation of log-scale active illumination microscopy. Biomed. Opt. Express 1, 236–245 (2010).

    Article  Google Scholar 

  22. Mertz, J.C. Introduction to Optical Microscopy. (Roberts and Company Publishers, 2009).

    Google Scholar 

  23. Durnin, J., Miceli, J.J. & Eberly, J.H. Diffraction-free beams. Phys. Rev. Lett. 58, 1499–1501 (1987).

    Article  CAS  Google Scholar 

  24. Fahrbach, F.O., Simon, P. & Rohrbach, A. Microscopy with self-reconstructing beams. Nat. Photonics 4, 780–785 (2010).

    Article  CAS  Google Scholar 

  25. Keller, P.J. & Stelzer, E.H.K. Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy. Curr. Opin. Neurobiol. 18, 624–632 (2008).

    Article  CAS  Google Scholar 

  26. Reynaud, E.G., Kržič, U., Greger, K. & Stelzer, E.H.K. Light sheet-based fluorescence microscopy: more dimensions, more photons, and less photodamage. HFSP J. 2, 266–275 (2008).

    Article  Google Scholar 

  27. Hopt, A. & Neher, E. Highly nonlinear photodamage in two-photon fluorescence microscopy. Biophys. J. 80, 2029–2036 (2001).

    Article  CAS  Google Scholar 

  28. Dunsby, C. Optically sectioned imaging by oblique plane microscopy. Opt. Express 16, 20306–20316 (2008).

    Article  CAS  Google Scholar 

  29. Turaga, D. & Holy, T.E. Miniaturization and defocus correction for objective-coupled planar illumination microscopy. Opt. Lett. 33, 2302–2304 (2008).

    Article  Google Scholar 

  30. Engelbrecht, C.J., Voigt, F. & Helmchen, F. Miniaturized selective plane illumination microscopy for high-contrast in vivo fluorescence imaging. Opt. Lett. 35, 1413–1415 (2010).

    Article  Google Scholar 

  31. Olivier, N. et al. Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy. Science 329, 967–971 (2010).

    Article  CAS  Google Scholar 

  32. Huisken, J. & Stainier, D.Y.R. Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM). Opt. Lett. 32, 2608–2610 (2007).

    Article  Google Scholar 

  33. Huisken, J. & Stainier, D.Y.R. Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM). Opt. Lett. 32, 2608–2610 (2007).

    Article  Google Scholar 

  34. Becker, K., Jährling, N., Kramer, E.R., Schnorrer, F. & Dodt, H.-U. Ultramicroscopy: 3D reconstruction of large microscopical specimens. J. Biophotonics 42, 36–42 (2008).

    Article  Google Scholar 

  35. Oron, D., Tal, E. & Silberberg, Y. Scanningless depth resolved microscopy. Opt. Express 13, 1468–1476 (2005).

    Article  Google Scholar 

  36. Zhu, G., Howe, J.V., Durst, M., Zipfel, W. & Xu, C. Simultaneous spatial and temporal focusing of femtosecond pulses. Opt. Express 13, 2153–2159 (2005).

    Article  Google Scholar 

  37. Heintzmann, R. & Benedetti, P.A. High-resolution image reconstruction in fluorescence microscopy with patterned excitation. Appl. Opt. 45, 5037–5045 (2006).

    Article  Google Scholar 

  38. Heintzmann, R. Structured illumination methods. in Handbook of Biological Confocal Microscopy, 3rd edn. (Pawley, J., ed.) 265–279 (Springer, 2006).

    Chapter  Google Scholar 

  39. Petran, M., Hadravsky, M., Egger, M.D. & Galambos, R. Tandem-scanning reflected-light microscope. J. Opt. Soc. Am. 58, 661–664 (1968).

    Article  Google Scholar 

  40. Sheppard, C.J.R. & Mao, X.Q. Confocal microscopes with slit apertures. J. Mod. Opt. 35, 1169–1185 (1988).

    Article  Google Scholar 

  41. Heintzmann, R. et al. Resolution enhancement by subtraction of confocal signals taken at different pinhole sizes. Micron 34, 293–300 (2003).

    Article  Google Scholar 

  42. Wilson, T., Juškaitis, R., Neil, M. & Kozubek, M. Confocal microscopy by aperture correlation. Opt. Lett. 21, 1879–1881 (1996). A key precursor to the development of DSD microscopy.

    Article  CAS  Google Scholar 

  43. Neil, M., Wilson, T. & Juskaitis, R. A light efficient optically sectioning microscope. J. Microsc. 189, 114–117 (1998).

    Article  Google Scholar 

  44. Verveer, P., Hanley, Q., Verbeek, P., Van Vliet, L. & Jovin, W. Theory of confocal fluorescence imaging in the programmable array microscope (PAM). J. Microsc. 189, 192–198 (1998). A key precursor to the development of DSD microscopy.

    Article  Google Scholar 

  45. Hanley, Q., Verveer, P., Gemkow, M., Arndt-Jovin, D. & Jovin, T. An optical sectioning programmable array microscope implemented with a digital micromirror device. J. Microsc. 196, 317–331 (1999).

    Article  CAS  Google Scholar 

  46. Heintzmann, R., Hanley, Q.S., Arndt-Jovin, D. & Jovin, T.M. A dual path programmable array microscope (PAM): simultaneous acquisition of conjugate and non-conjugate images. J. Microsc. 204, 119–135 (2001).

    Article  CAS  Google Scholar 

  47. Wilson, T., Neil, M.A.A. & Juskaitis, R. Confocal microscopy apparatus and method. US patent 6,687052 B1 (2004).

  48. Neil, M., Juskaitis, R. & Wilson, T. Method of obtaining optical sectioning by using structured light in a conventional microscope. Opt. Lett. 22, 1905–1907 (1997). The first demonstration of how structured illumination can provide optical sectioning, initiating the technique of SIM in general.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  50. Gustafsson, M.G.L. et al. Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination. Biophys. J. 94, 4957–4970 (2008).

    Article  CAS  Google Scholar 

  51. Shroff, S.A., Fienup, J.R. & Williams, D.R. Phase-shift estimation in sinusoidally illuminated images for lateral superresolution. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 26, 413–424 (2009).

    Article  Google Scholar 

  52. Kner, P., Chhun, B.B., Griffis, E.R., Winoto, L. & Gustafsson, M.G.L. Super-resolution video microscopy of live cells by structured illumination. Nat. Methods 6, 339–342 (2009).

    Article  CAS  Google Scholar 

  53. Lim, D., Chu, K.K. & Mertz, J. Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy. Opt. Lett. 33, 1819–1821 (2008).

    Article  Google Scholar 

  54. Lim, D., Ford, T.N., Chu, K.K. & Mertz, J. Optically sectioned in vivo imaging with speckle illumination HiLo microscopy. J. Biomed. Opt. 16, 016014 (2011). Advantages of HiLo microscopy for fast, in vivo imaging over wide fields of view are highlighted.

    Article  Google Scholar 

  55. Karadaglic, D., Juskaitis, R. & Wilson, T. Confocal endoscopy via structured illumination. Scanning 24, 301–304 (2002).

    Article  CAS  Google Scholar 

  56. Bozinovic, N., Ventalon, C., Ford, T. & Mertz, J. Fluorescence endomicroscopy with structured illumination. Opt. Express 16, 4603–4610 (2008).

    Article  Google Scholar 

  57. Santos, S. et al. Optically sectioned fluorescence endomicroscopy with imaging through a flexible fiber bundle. J. Biomed. Opt. 14, 030502 (2009).

    Article  Google Scholar 

  58. Breuninger, T., Greger, K. & Stelzer, E.H.K. Lateral modulation boosts image quality in single plane illumination fluorescence microscopy. Opt. Lett. 32, 1938–1940 (2007).

    Article  Google Scholar 

  59. Kalchmair, S., Jährling, N., Becker, K. & Dodt, H.-U. Image contrast enhancement in confocal ultramicroscopy. Opt. Lett. 35, 79–81 (2010).

    Article  Google Scholar 

  60. Keller, P.J. et al. Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat. Methods 7, 637–642 (2010).

    Article  CAS  Google Scholar 

  61. Mertz, J. & Kim, J. Scanning light-sheet microscopy in the whole mouse brain with HiLo background rejection. J. Biomed. Opt. 15, 016027 (2010).

    Article  Google Scholar 

Download references

Acknowledgements

I acknowledge the help of W. Supatto, K. Chu, D. Lim and T. Ford for critically reading this manuscript.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA

    Jerome Mertz

Authors
  1. Jerome Mertz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jerome Mertz.

Ethics declarations

Competing interests

The author declares no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mertz, J. Optical sectioning microscopy with planar or structured illumination. Nat Methods 8, 811–819 (2011). https://doi.org/10.1038/nmeth.1709

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1709

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