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Surgical implantation of an abdominal imaging window for intravital microscopy

Nature Protocols volume 8, pages 583–594 (2013)Cite this article

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Abstract

High-resolution intravital microscopy through imaging windows has become an indispensable technique for the long-term visualization of dynamic processes in living animals. Easily accessible sites such as the skin, the breast and the skull can be imaged using various different imaging windows; however, long-term imaging studies on cellular processes in abdominal organs are more challenging. These processes include colonization of the liver by metastatic tumor cells and the development of an immune response in the spleen. We have recently developed an abdominal imaging window (AIW) that allows long-term imaging of the liver, the pancreas, the intestine, the kidney and the spleen. Here we describe the detailed protocol for the optimal surgical implantation of the AIW, which takes ∼1 h, and subsequent multiphoton imaging, which takes up to 1 month.

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Figure 1: The specifics of the AIW.
Figure 2: The AIW liver surgery.
Figure 3: Fixation of the mouse in the imaging box.
Figure 4: Tools to retrace areas of interest over multiple days.
Figure 5: Imaging of the liver through the AIW.
Figure 6: Imaging of the spleen through the AIW.
Figure 7: Imaging of the pancreas through the AIW.
Figure 8: Imaging of the small intestine through the AIW.

References

  1. Beerling, E., Ritsma, L., Vrisekoop, N., Derksen, P.W.B. & van Rheenen, J. Intravital microscopy: new insights into metastasis of tumors. J. Cell Sci. 124, 299–310 (2011).

    Article  CAS  Google Scholar 

  2. Ritsma, L., Ponsioen, B. & van Rheenen, J. Intravital imaging of cell signaling in mice. Intravital 1, 2–10 (2012).

    Article  Google Scholar 

  3. Theer, P., Hasan, M.T. & Denk, W. Two-photon imaging to a depth of 1000 μm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003).

    Article  CAS  Google Scholar 

  4. Egeblad, M. et al. Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy. Dis. Model. Mech. 1, 155–167 (2008).

    Article  Google Scholar 

  5. Ritsma, L. et al. Intravital microscopy through an abdominal imaging window reveals a pre-micrometastasis stage during liver metastasis. Sci. Transl. Med. 4, 158ra145 (2012).

    Article  Google Scholar 

  6. Pan, F. & Gan, W.-B. Two-photon imaging of dendritic spine development in the mouse cortex. Dev. Neurobiol. 68, 771–778 (2008).

    Article  Google Scholar 

  7. Yuan, F. et al. Vascular permeability and microcirculation of gliomas and mammary carcinomas transplanted in rat and mouse cranial Windows. Cancer Res. 54, 4564–4568 (1994).

    CAS  PubMed  Google Scholar 

  8. Kienast, Y. et al. Real-time imaging reveals the single steps of brain metastasis formation. Nat. Med. 16, 116–122 (2010).

    Article  CAS  Google Scholar 

  9. Lehr, H., Leunig, M., Menger, M., Nolte, D. & Messmer, K. Dorsal skinfold chamber technique for intravital microscopy in nude mice. Am. J. Pathol. 143, 1055–1062 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Palmer, G.M. et al. In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters. Nat. Protoc. 6, 1355–1366 (2011).

    Article  CAS  Google Scholar 

  11. Perentes, J.Y. et al. In vivo imaging of extracellular matrix remodeling by tumor-associated fibroblasts. Nat. Meth. 6, 143–145 (2009).

    Article  CAS  Google Scholar 

  12. Gligorijevic, B., Kedrin, D., Segall, J., Condeelis, J. & van Rheenen, J. Dendra2 photoswitching through the Mammary Imaging Window. J. Vis. Exp. 28 doi:10.3791/1278 (2009).

  13. Kedrin, D. et al. Intravital imaging of metastatic behavior through a mammary imaging window. Nat. Meth. 5, 1019–1021 (2008).

    Article  CAS  Google Scholar 

  14. Shan, S., Sorg, B. & Dewhirst, M.W. A novel rodent mammary window of orthotopic breast cancer for intravital microscopy. Microvasc. Res. 65, 109–117 (2003).

    Article  Google Scholar 

  15. Farrar, M.J. et al. Chronic in vivo imaging in the mouse spinal cord using an implanted chamber. Nat. Meth. 9, 297–302 (2012).

    Article  CAS  Google Scholar 

  16. Holtmaat, A. et al. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat. Protoc. 4, 1128–1144 (2009).

    Article  CAS  Google Scholar 

  17. Niinomi, M. Recent research and development in titanium alloys for biomedical applications and healthcare goods. Sci. Technol. Adv. Mater. 4, 445–454 (2003).

    Article  CAS  Google Scholar 

  18. Zomer, A. et al. Intravital imaging of cancer stem cell plasticity in mammary tumors. Stem Cells. doi:10.1002/stem.1296 (7 December 2012).

  19. van Rheenen, J. et al. EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells. J. Cell Biol. 179, 1247–1259 (2007).

    Article  CAS  Google Scholar 

  20. Stock, P. et al. The generation of hepatocytes from mesenchymal stem cells and engraftment into murine liver. Nat. Protoc. 5, 617–627 (2010).

    Article  CAS  Google Scholar 

  21. Drixler, T.A. et al. Continuous administration of angiostatin inhibits accelerated growth of colorectal liver metastases after partial hepatectomy. Cancer Res. 60, 1761–1765 (2000).

    CAS  PubMed  Google Scholar 

  22. Bajénoff, M. et al. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25, 989–1001 (2006).

    Article  Google Scholar 

  23. Snippert, H.J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank A. de Graaff and the Hubrecht Imaging Centre for imaging support and T. Faase for help with the replaceable window. This work was supported by a Vidi fellowship (91710330, to J.v.R.) from the Dutch Organization of Scientific Research (NWO), by grants from the Dutch Cancer Society (KWF, HUBR 2009-4621 to J.v.R. and 2009-4367 to E.J.A.S.) and the PON foundation (to E.J.A.S.), and by equipment grants (175.010.2007.00 and 834.11.002) from the NWO.

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Author notes

  1. Laila Ritsma, Ernst J A Steller, Inne H M Borel Rinkes and Jacco van Rheenen: These authors contributed equally to this work.

Authors and Affiliations

  1. Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW)—University Medical Centre Utrecht, Utrecht, The Netherlands

    Laila Ritsma, Saskia I J Ellenbroek & Jacco van Rheenen

  2. Department of Surgery, University Medical Centre Utrecht, Utrecht, The Netherlands

    Ernst J A Steller, Onno Kranenburg & Inne H M Borel Rinkes

  3. Cancer Genomics Netherlands,

    Jacco van Rheenen

Authors
  1. Laila Ritsma
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  2. Ernst J A Steller
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  3. Saskia I J Ellenbroek
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  4. Onno Kranenburg
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  5. Inne H M Borel Rinkes
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  6. Jacco van Rheenen
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Contributions

L.R. and E.J.A.S. wrote the manuscript, collected the data presented and optimized the surgical procedure and window design. L.R. prepared the figures. J.v.R. conceived and supervised the study and helped write the manuscript. O.K. helped design and supervise the study. I.H.M.B.R. and S.I.J.E. helped to optimize the surgical procedure and window design, respectively, and helped to write the manuscript. All authors had the opportunity to discuss the results and comment on the manuscript.

Corresponding authors

Correspondence to Inne H M Borel Rinkes or Jacco van Rheenen.

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The authors declare no competing financial interests.

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Ritsma, L., Steller, E., Ellenbroek, S. et al. Surgical implantation of an abdominal imaging window for intravital microscopy. Nat Protoc 8, 583–594 (2013). https://doi.org/10.1038/nprot.2013.026

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