High-resolution three-dimensional imaging of large specimens with light sheet–based microscopy

Abstract

We report that single (or selective) plane illumination microscopy (SPIM), combined with a new deconvolution algorithm, provides a three-dimensional spatial resolution exceeding that of confocal fluorescence microscopy in large samples. We demonstrate this by imaging large living multicellular specimens obtained in a three-dimensional cell culture. The ability to rapidly image large samples at high resolution with minimal photodamage provides new opportunities especially for the study of subcellular processes in large living specimens.

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Figure 1: Comparison of SPIM and confocal fluorescence microscopy by imaging the autofluorescence of grains of paper mulberry pollen.
Figure 2: Comparison of SPIM and confocal fluorescence microscopy of MDCK cysts.
Figure 3: Images recorded using SPIM of a large cellular spheroid of BxPC3 human pancreatic cancer cells labeled with DRAQ5.

References

  1. 1

    Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H.K. Science 305, 1007–1009 (2004).

    CAS  Article  Google Scholar 

  2. 2

    Voie, A.H., Burns, D.H. & Spelman, F.A. J. Microsc. 170, 229–236 (1993).

    CAS  Article  Google Scholar 

  3. 3

    Keller, P.J., Pampaloni, F. & Stelzer, E.H.K. Curr. Opin. Cell Biol. 18, 117–124 (2006).

    CAS  Article  Google Scholar 

  4. 4

    Greger, K., Swoger, J. & Stelzer, E.H.K. Rev. Sci. Instrum. (in the press).

  5. 5

    Swoger, J., Huisken, J. & Stelzer, E.H.K. Opt. Lett. 28, 1654–1656 (2003).

    Article  Google Scholar 

  6. 6

    Agard, D.A. & Sedat, J.W. Nature 302, 676–681 (1983).

    CAS  Article  Google Scholar 

  7. 7

    Carrington, W.A. et al. Science 268, 1483–1487 (1995).

    CAS  Article  Google Scholar 

  8. 8

    Verveer, P.J. & Jovin, T.M. Appl. Opt. 37, 6240–6246 (1998).

    CAS  Article  Google Scholar 

  9. 9

    Timmins, N.E., Dietmair, S. & Nielsen, L.K. Angiogenesis 7, 97–103 (2004).

    Article  Google Scholar 

  10. 10

    Kunz-Schughart, L.A., Freyer, J.P., Hofstaedter, F. & Ebner, R. J. Biomol. Screen. 9, 273–285 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Kam, Z., Hanser, B., Gustafsson, M.G., Agard, D.A. & Sedat, J.W. Proc. Natl. Acad. Sci. USA 98, 3790–3795 (2001).

    CAS  Article  Google Scholar 

  12. 12

    Engelbrecht, C.J. & Stelzer, E.H.K. Opt. Lett. 31, 1477–1479 (2006).

    Article  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank D. Holzer (European Molecular Biology Laboratory, Heidelberg) for help with MDCK cell culture and transfection, and A. Schrödel and M. Löhr (German Cancer Research Center, Heidelberg) for providing the BxPC3 spheroids. E.H.K.S. and F.P. acknowledge the Forschungsprogramm Optische Technologien der Landesstiftung Baden-Württemberg for financial support. M.M. was supported by a joint collaboration between Hamamatsu and the German Cancer Research Center (Project PA 11631).

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Correspondence to Peter J Verveer or Jim Swoger or Ernst H K Stelzer.

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Supplementary information

Supplementary Fig. 1

SPIM and confocal microscopy of CHO cells. (PDF 141 kb)

Supplementary Fig. 2

SPIM deconvolution of a Medaka embryo. (PDF 148 kb)

Supplementary Fig. 3

Confocal microscopy of pancreatic tumor cells. (PDF 91 kb)

Supplementary Methods (PDF 114 kb)

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Verveer, P., Swoger, J., Pampaloni, F. et al. High-resolution three-dimensional imaging of large specimens with light sheet–based microscopy. Nat Methods 4, 311–313 (2007). https://doi.org/10.1038/nmeth1017

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