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In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography

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Abstract

We report a technique for fluorescence tomography that operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The method uses multi-projection illumination and photon transport description in opaque tissues. We demonstrate whole-body three-dimensional visualization of the morphogenesis of GFP-expressing salivary glands and wing imaginal discs in living Drosophila melanogaster pupae in vivo and over time.

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Figure 1: Overview of the experimental configuration.
Figure 2: In vivo reconstructions of D. melanogaster salivary glands.
Figure 3: Time-lapse imaging of D. melanogaster wing imaginal discs.

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Acknowledgements

We thank A. Soubret for his help and suggestions, and Polymicro Technologies LLC for providing us the fused silica capillary tubing. This work was supported by the US National Institutes of Health National Institute of Biomedical Imaging and BioEngineering grant RO1000750. C.P. is supported by an European Molecular Biology Organization fellowship. N.P. is a Howard Hughes Medical Institute investigator.

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Author notes

  1. Claudio Vinegoni, Chrysoula Pitsouli and Daniel Razansky: These authors contributed equally to this work.

Authors and Affiliations

  1. Center for Molecular Imaging Research, Massachusetts General Hospital and Harvard Medical School, Building 149, 13th Street, Charlestown, 02129, Massachusetts, USA

    Claudio Vinegoni, Daniel Razansky & Vasilis Ntziachristos

  2. Center for System Biology, Massachusetts General Hospital and Harvard Medical School, Richard B. Simches Research Center, 185 Cambridge Street, Boston, 02114, Massachusetts, USA

    Claudio Vinegoni

  3. Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute, 77 Avenue Louis Pasteur, Boston, 02115, Massachusetts, USA

    Chrysoula Pitsouli & Norbert Perrimon

  4. Institute for Biological and Medical Imaging, Technical University of Munich and Helmholtz Center Munich, Ingolstädter Landstraße 1, D-85764, Neuherberg, Germany

    Daniel Razansky & Vasilis Ntziachristos

Authors
  1. Claudio Vinegoni
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  2. Chrysoula Pitsouli
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  3. Daniel Razansky
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  4. Norbert Perrimon
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  5. Vasilis Ntziachristos
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Contributions

C.V. and C.P. designed the experiment. C.V. developed the system, performed the experiments, developed the image processing and inversion schemes, analyzed data and wrote most of the paper. C.P. generated the GFP-expressing flies and performed histology and confocal microscopy. D.R. developed the inversion schemes, image reconstruction software and contributed to developing the photon propagation model. N.P. and V.N. supervised the project.

Corresponding author

Correspondence to Claudio Vinegoni.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Methods (PDF 1281 kb)

Supplementary Video 1

Time evolution wing imaginal discs (0 degrees projection). Development of a D. melanogaster pupa expressing GFP in the wing imaginal discs at 0 degrees projection during 474 minutes. The red box in the bottom left part of the video indicates the relative time. (MOV 3490 kb)

Supplementary Video 2

Time evolution wing imaginal discs (90 degrees projection). Development of a D. melanogaster pupa expressing GFP in the wing imaginal discs at 90 degrees projection during 474 minutes. The red box in the bottom left part of the video indicates the relative time. (MOV 2828 kb)

Supplementary Video 3

Time evolution wing imaginal discs (180 degrees projection). Development of a D. melanogaster pupa expressing GFP in the wing imaginal discs at 180 degrees projection during 474 minutes. The red box in the bottom left part of the video indicates the relative time. (MOV 3255 kb)

Supplementary Video 4

Wing imaginal discs imaged at different projections. 6.5 hr D. melanogaster pupa expressing GFP in the imaginal discs; the movie corresponds to fluorescent projections detected by the CCD at different rotation angles. (MOV 2640 kb)

Supplementary Video 5

Head eversion. D. melanogaster pupa expressing GFP in the imaginal discs during the head eversion process (∼12 hrs APF). The images are acquired at one fixed projection. The red box in the bottom left part of the video indicates the relative time. (MOV 5892 kb)

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Vinegoni, C., Pitsouli, C., Razansky, D. et al. In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography. Nat Methods 5, 45–47 (2008). https://doi.org/10.1038/nmeth1149

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  • DOI: https://doi.org/10.1038/nmeth1149

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