Light Sheet Microscopy in Cell Biology

  • Raju Tomer
  • Khaled Khairy
  • Philipp J. KellerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 931)


Light sheet-based fluorescence microscopy (LSFM) is emerging as a powerful imaging technique for the life sciences. LSFM provides an exceptionally high imaging speed, high signal-to-noise ratio, low level of photo-bleaching, and good optical penetration depth. This unique combination of capabilities makes light sheet-based microscopes highly suitable for live imaging applications.

Here, we provide an overview of light sheet-based microscopy assays for in vitro and in vivo imaging of biological samples, including cell extracts, soft gels, and large multicellular organisms. We furthermore describe computational tools for basic image processing and data inspection.

Key words

Light sheet microscopy In vivo imaging Cell extracts Quantitative developmental biology Embryonic development Image processing 



This work was supported by the Howard Hughes Medical Institute.


  1. 1.
    Keller PJ, Pampaloni F, Stelzer EHK (2006) Life sciences require the third dimension. Curr Opin Cell Biol 18:117–124PubMedCrossRefGoogle Scholar
  2. 2.
    Keller PJ, Stelzer EH (2008) Quantitative in vivo imaging of entire embryos with digital scanned laser light sheet fluorescence microscopy. Curr Opin Neurobiol 18:624–632PubMedCrossRefGoogle Scholar
  3. 3.
    Khairy K, Keller PJ (2011) Reconstructing embryonic development. Genesis 49(7):488–513PubMedCrossRefGoogle Scholar
  4. 4.
    Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer EHK (2004) Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305:1007–1009PubMedCrossRefGoogle Scholar
  5. 5.
    Keller PJ, Schmidt AD, Wittbrodt J, Stelzer EHK (2008) Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Science 322:1065–1069PubMedCrossRefGoogle Scholar
  6. 6.
    Keller PJ, Pampaloni F, Stelzer EH (2007) Three-dimensional preparation and imaging reveal intrinsic microtubule properties. Nat Methods 4:843–846PubMedCrossRefGoogle Scholar
  7. 7.
    Keller PJ, Schmidt AD, Santella A, Khairy K, Bao Z, Wittbrodt J, Stelzer EH (2010) Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy. Nat Methods 7:637–642PubMedCrossRefGoogle Scholar
  8. 8.
    Fuchs E, Jaffe J, Long R, Azam F (2002) Thin laser light sheet microscope for microbial oceanography. Opt Express 10:145–154PubMedCrossRefGoogle Scholar
  9. 9.
    Voie AH, Burns DH, Spelman FA (1993) Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens. J Microsc 170:229–236PubMedCrossRefGoogle Scholar
  10. 10.
    Engelbrecht CJ, Voigt F, Helmchen F (2010) Miniaturized selective plane illumination microscopy for high-contrast in vivo fluorescence imaging. Opt Lett 35:1413–1415PubMedCrossRefGoogle Scholar
  11. 11.
    Turaga D, Holy TE (2008) Miniaturization and defocus correction for objective-coupled planar illumination microscopy. Opt Lett 33:2302–2304PubMedCrossRefGoogle Scholar
  12. 12.
    Scherz PJ, Huisken J, Sahai-Hernandez P, Stainier DY (2008) High-speed imaging of developing heart valves reveals interplay of morphogenesis and function. Development 135:1179–1187PubMedCrossRefGoogle Scholar
  13. 13.
    Fahrbach FO, Simon P, Rohrbach A (2010) Microscopy with self-reconstructing beams. Nat Photonics 4:780–785CrossRefGoogle Scholar
  14. 14.
    Rohrbach A (2009) Artifacts resulting from imaging in scattering media: a theoretical prediction. Opt Lett 34:3041–3043PubMedCrossRefGoogle Scholar
  15. 15.
    Planchon TA, Gao L, Milkie DE, Davidson MW, Galbraith JA, Galbraith CG, Betzig E (2011) Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat Methods 8:417–423PubMedCrossRefGoogle Scholar
  16. 16.
    Fahrbach FO, Rohrbach A (2010) A line scanned light-sheet microscope with phase shaped self-reconstructing beams. Opt Express 18:24229–24244PubMedCrossRefGoogle Scholar
  17. 17.
    Truong TV, Supatto W, Koos DS, Choi JM, Fraser SE (2011) Deep and fast live imaging with two-photon scanned light-sheet microscopy. Nat Methods 8(9):757–760PubMedCrossRefGoogle Scholar
  18. 18.
    Swoger J, Verveer P, Greger K, Huisken J, Stelzer EH (2007) Multi-view image fusion improves resolution in three-dimensional microscopy. Opt Express 15:8029–8042PubMedCrossRefGoogle Scholar
  19. 19.
    Keller PJ, Pampaloni F, Lattanzi G, Stelzer EHK (2008) Three-dimensional microtubule behavior in Xenopus egg extracts reveals four dynamic states and state-dependent elastic properties. Biophys J 95:1474–1486PubMedCrossRefGoogle Scholar
  20. 20.
    Keller PJ, Stelzer EH (2010) Digital scanned laser light sheet fluorescence microscopy. Cold Spring Harb Protoc 2010:pdb.top78Google Scholar
  21. 21.
    Lucy LB (1974) An iterative technique for the rectification of observed distributions. Astron J 79:745–754CrossRefGoogle Scholar
  22. 22.
    Preibisch S, Saalfeld S, Schindelin J, Tomancak P (2010) Software for bead-based registration of selective plane illumination microscopy data. Nat Methods 7:418–419PubMedCrossRefGoogle Scholar
  23. 23.
    Hom EFY, Marchis F, Lee TK, Haase S, Agard DA, Sedat JW (2007) AIDA: an adaptive image deconvolution algorithm with application to multi-frame and three-dimensional data. J Opt Soc Am A 24:1580–1600CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Janelia Farm Research Campus, Howard Hughes Medical InstituteAshburnUSA

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