Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain (original) (raw)

Nature Methods volume 4, pages 331–336 (2007)Cite this article

Abstract

Visualizing entire neuronal networks for analysis in the intact brain has been impossible up to now. Techniques like computer tomography or magnetic resonance imaging (MRI) do not yield cellular resolution, and mechanical slicing procedures are insufficient to achieve high-resolution reconstructions in three dimensions. Here we present an approach that allows imaging of whole fixed mouse brains. We modified 'ultramicroscopy' by combining it with a special procedure to clear tissue. We show that this new technique allows optical sectioning of fixed mouse brains with cellular resolution and can be used to detect single GFP-labeled neurons in excised mouse hippocampi. We obtained three-dimensional (3D) images of dendritic trees and spines of populations of CA1 neurons in isolated hippocampi. Also in fruit flies and in mouse embryos, we were able to visualize details of the anatomy by imaging autofluorescence. Our method is ideally suited for high-throughput phenotype screening of transgenic mice and thus will benefit the investigation of disease models.

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References

  1. Tyszka, J.M., Fraser, S.E. & Jacobs, R.E. Magnetic resonance microscopy: recent advantages and applications. Curr. Opin. Biotechnol. 16, 93–99 (2005).
    Article CAS Google Scholar
  2. Kalender, W.A. CT: the unexpected evolution of an imaging modality. Eur. Radiol. 15 (Suppl. 4), d21–d24 (2005).
    Article Google Scholar
  3. Johnson, G.A., Cofer, G.P., Gewalt, S.L. & Hedlund, L.W. Morphological phenotyping with MR microscopy: The visible mouse. Radiology 222, 789–793 (2002).
    Article Google Scholar
  4. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).
    Article CAS Google Scholar
  5. Weninger, W.J. & Mohun, T. Phenotyping transgenic embryos: a rapid 3-D screening method based on episcopic fluorescence image capturing. Nat. Genet. 30, 59–65 (2002).
    Article CAS Google Scholar
  6. Streicher, J. et al. Computer-based three-dimensional visualization of developmental gene expression. Nat. Genet. 25, 147–152 (2000).
    Article CAS Google Scholar
  7. Sharpe, J. et al. Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 296, 541–545 (2002).
    Article CAS Google Scholar
  8. Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).
    Article CAS Google Scholar
  9. Siedentopf, H. & Zsigmondy, R. Über Sichtbarmachung und Größenbestimmung ultramikroskopischer Teilchen, mit besonderer Anwendung auf Goldrubingläser. Annalen der Physik 10, 1–39 (1903).
    CAS Google Scholar
  10. Spalteholz, W. Über das Durchsichtigmachen von menschlichen und tierischen Präparaten. (S. Hierzel, Leipzig, 1914).
  11. Dent, J.A., Polson, A.G. & Klymkowski, M.W. A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus. Development 105, 61–74 (1989).
    CAS PubMed Google Scholar
  12. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).
    Article CAS Google Scholar
  13. Dickinson, M.E., Bearman, G., Tille, S., Lansford, R. & Fraser, S.E. Multi-spectral imaging and linear unmixing add a whole new dimension to laser scanning fluorescence microscopy. Biotechniques 31, 1272–1278 (2001).
    Article CAS Google Scholar
  14. Jacobsen, H. & Hell, S.W. Effect of the specimen refractive index on the imaging of a confocal fluorescence microscope employing high aperture oil immersion lenses. Bioimaging 3, 39–47 (1995).
    Article Google Scholar
  15. Ikegaya, Y. et al. Synfire chains and cortical songs: temporal modules of cortical activity. Science 304, 559–564 (2004).
    Article CAS Google Scholar
  16. Dalva, M.B. & Katz, L.C. Rearrangements of synaptic connections in visual cortex revealed by laser photostimulation. Science 265, 255–258 (1994).
    Article CAS Google Scholar
  17. Dodt, H.-U., Eder, M., Frick, A. & Zieglgänsberger, W. Precisely localized LTD in the neocortex revealed by infrared-guided laser stimulation. Science 286, 110–113 (1999).
    Article CAS Google Scholar
  18. Melles Griot Optics Guide 4, Gaussian beam optics, 17.15–17.20, (Melles Griot, Irvine, 1988).

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Acknowledgements

We thank G. Ryseck for help with initial experiments and S. Espinoza, L. Luo, E. Kramer and C. Wotjak for specimens. This work was supported by grants of the Hertie foundation and the SFB391.

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Author notes

  1. Hans-Ulrich Dodt, Nina Jährling & Klaus Becker
    Present address: Present address: Department of Bioelectronics, Institute of Solid State Electronics, Vienna University of Technology, Floragasse 7, 1040 Vienna, Austria.,

Authors and Affiliations

  1. Max Planck Institute of Psychiatry, Kraepelinstr. 2, Munich, 80804, Germany
    Hans-Ulrich Dodt, Ulrich Leischner, Anja Schierloh, Nina Jährling, Christoph Peter Mauch, Jan Michael Deussing, Matthias Eder, Walter Zieglgänsberger & Klaus Becker
  2. Department of Molecular Neurobiology, Max Planck Institute of Neurobiology, Am Klopferspitz 18, Martinsried, 82152, Germany
    Katrin Deininger

Authors

  1. Hans-Ulrich Dodt
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  2. Ulrich Leischner
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  3. Anja Schierloh
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  4. Nina Jährling
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  5. Christoph Peter Mauch
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  6. Katrin Deininger
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  7. Jan Michael Deussing
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  8. Matthias Eder
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  9. Walter Zieglgänsberger
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  10. Klaus Becker
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Correspondence toHans-Ulrich Dodt.

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Dodt, HU., Leischner, U., Schierloh, A. et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain.Nat Methods 4, 331–336 (2007). https://doi.org/10.1038/nmeth1036

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