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In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography

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.

References

  1. Helmchen, F. & Denk, W. Nat. Methods 2, 932–940 (2005).

    CAS  Article  Google Scholar 

  2. Yuste, R. Nat. Methods 2, 902–904 (2005).

    CAS  Article  Google Scholar 

  3. Minsky, M. US patent 3,013,467 (1961).

  4. Huang, D. et al. Science 254, 1178–1181 (1991).

    CAS  Article  Google Scholar 

  5. Weissleder, R., Tung, C.H., Mahmood, U. & Bogdanov, A. Nat. Biotechnol. 17, 375–378 (1999).

    CAS  Article  Google Scholar 

  6. Ntziachristos, V., Ripoll, J., Wang, L.H.V. & Weissleder, R. Nat. Biotechnol. 23, 313–320 (2005).

    CAS  Article  Google Scholar 

  7. Arridge, S.R. Inverse Probl. 15, R41–R93 (1999).

    Article  Google Scholar 

  8. Sharpe, J. et al. Science 296, 541–545 (2002).

    CAS  Article  Google Scholar 

  9. Dodt, H.U. et al. Nat. Methods 4, 331–336 (2007).

    CAS  Article  Google Scholar 

  10. Pomraning, G.C. Nucl. Sci. Eng. 127, 182–198 (1997).

    CAS  Article  Google Scholar 

  11. Ward, R.E., Reid, P., Bashirullah, A., D'Avino, P.P. & Thummel, C.S. Dev. Biol. 256, 389–402 (2003).

    CAS  Article  Google Scholar 

  12. Sun, B., Xu, P. & Salvaterra, P.M. Proc. Natl. Acad. Sci. USA 96, 10438–10443 (1999).

    CAS  Article  Google Scholar 

  13. Ninov, N., Chiarelli, D.A. & Martin-Blanco, E. Development 134, 367–379 (2007).

    CAS  Article  Google Scholar 

  14. Zeitlinger, J. & Bohmann, D. Development 126, 3947–3956 (1999).

    CAS  PubMed  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

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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