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Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain

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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Figure 1: Principle of ultramicroscopy.
Figure 2: Brain imaging.
Figure 3: Imaging of different sources of fluorescence.
Figure 4: Imaging of mouse embryos.
Figure 5: Imaging of D. melanogaster

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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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Correspondence to Hans-Ulrich Dodt.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

whole mouse brain reconstructed from 550 optical sections. (MOV 2686 kb)

Supplementary Video 2

Granule cells with dendrites in the hippocampus of a thy-1 GFP mouse. (MOV 2732 kb)

Supplementary Video 3

Excised whole hippocampus. (MOV 1016 kb)

Supplementary Video 4

Optical sectioning of a whole hippocampus. (MOV 1521 kb)

Supplementary Video 5

3D-reconstruction and animation of a part of a whole hippocampus. (MOV 2262 kb)

Supplementary Video 6

3D reconstruction and animation of axonal bundles in the hippocampal alveus and dendritic spines of CA1 pyramidal neurons. (MOV 2134 kb)

Supplementary Video 7

Primary and secondary barrel field made visible by excitation of autofluorescence in the whole brain of a 10 day old mouse. (MOV 1040 kb)

Supplementary Video 8

Optical sectioning of a mouse brain imaged by detection of scattered light. Note the appearance of fibre tracts during the movement of the optical sectioning plane through the brain. (MOV 2115 kb)

Supplementary Methods (PDF 10 kb)

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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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  • DOI: https://doi.org/10.1038/nmeth1036

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