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

Nature Methods volume 4, pages311–313(2007)Cite this article

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.

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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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Author notes

  1. Peter J Verveer & Jim Swoger

    Present address: Present addresses: Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227, Dortmund, Germany (P.J.V.) and Systems Biology Programme, Centre for Genomic Regulation, C/Dr. Aiguader 88, 08003 Barcelona, Spain (J.S.).,

Affiliations

  1. Cell Biology & Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, 69117, Germany

    Peter J Verveer, Jim Swoger, Francesco Pampaloni, Klaus Greger & Ernst H K Stelzer

  2. Biomedical Structure Group, German Cancer Research Center, Im Neuenheimer Feld 280, Heidelberg, 69120, Germany

    Marco Marcello

Authors
  1. Peter J Verveer
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  2. Jim Swoger
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  3. Francesco Pampaloni
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  4. Klaus Greger
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  5. Marco Marcello
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  6. Ernst H K Stelzer
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Corresponding authors

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

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The authors declare no competing financial interests.

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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