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A permanent window for the murine lung enables high-resolution imaging of cancer metastasis

Nature Methods volume 15, pages 7380 (2018) | Download Citation


Stable, high-resolution intravital imaging of the lung has become possible through the utilization of vacuum-stabilized imaging windows. However, this technique is extremely invasive and limited to only hours in duration. Here we describe a minimally invasive, permanently implantable window for high-resolution intravital imaging of the murine lung that allows the mouse to survive surgery, recover from anesthesia, and breathe independently. Compared to vacuum-stabilized windows, this window produces the same high-quality images without vacuum-induced artifacts; it is also less invasive, which allows imaging of the same lung tissue over a period of weeks. We further adapt the technique of microcartography for reliable relocalization of the same cells longitudinally. Using commonly employed experimental, as well as more clinically relevant, spontaneous metastasis models, we visualize all stages of metastatic seeding, including: tumor cell arrival; extravasation; growth and progression to micrometastases; as well as tumor microenvironment of metastasis function, the hallmark of hematogenous dissemination of tumor cells.

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This technology was developed in the Gruss-Lipper Biophotonics Center and the Integrated Imaging Program at Albert Einstein College of Medicine. We acknowledge the support of these Centers in this work: Einstein's Integrated Imaging Program, Montefiore's Ruth L. Kirschstein T32 Training Grant of Surgeons for the Study of the Tumor Microenvironment (CA200561), NIH grants CA100324, CA216248, P30CA013330, and SIG #1S10OD019961-01. We thank M. Rottenkolber, R. Ibagon and A. Leggiadro of the Einstein Machine Shop for their skilled craftsmanship and design insight; and we thank C. Rodriguez-Tirado, B. Canella and U. Steidl's lab for help with evaluating blood counts.

Author information

Author notes

    • David Entenberg
    •  & Sonia Voiculescu

    These authors contributed equally to this work.


  1. Anatomy and Structural Biology, Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, USA.

    • David Entenberg
    • , Sonia Voiculescu
    • , Peng Guo
    • , Lucia Borriello
    • , Yarong Wang
    • , George S Karagiannis
    • , Joan Jones
    • , Maja Oktay
    •  & John Condeelis
  2. Gruss-Lipper Biophotonics Center, Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, USA.

    • David Entenberg
    • , Peng Guo
    • , Lucia Borriello
    • , Yarong Wang
    • , George S Karagiannis
    • , Joan Jones
    • , Maja Oktay
    •  & John Condeelis
  3. Integrated Imaging Program, Einstein College of Medicine, Montefiore Medical Center, Bronx,New York, USA.

    • David Entenberg
    • , Yarong Wang
    • , George S Karagiannis
    • , Joan Jones
    • , Maja Oktay
    •  & John Condeelis
  4. Department of Surgery, Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, USA.

    • Sonia Voiculescu
    •  & Francis Baccay
  5. Analytical Imaging Facility, Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, USA.

    • Peng Guo
  6. Department of Pathology, Einstein College of Medicine, Montefiore Medical Center, Bronx, New York, USA.

    • Joan Jones
    •  & Maja Oktay


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D.E. and J.C. conceived of the technique; J.C. conceived of the biological goals for performance; D.E. designed the window and stage plate; D.E., S.V. and F.B. developed the surgical protocol; S.V., D.E., G.S.K., M.O. and J.C. designed the validation experiments; D.E. and S.V. performed the imaging; S.V. and L.B. performed the validation experiments; Y.W. bred the mice; P.G. performed 3D reconstructions; D.E., L.B. and P.G. performed the blood-flow measurements; S.V., D.E., G.S.K., M.O. and J.J. evaluated the validation results; D.E. and G.S.K. performed statistical analyses; M.O. and J.J. evaluated HE, IBA1 IHC and TMEM IHC in fixed lung sections; and D.E., S.V. and J.C. wrote the paper.

Competing interests

D.E., S.V., and J.C. have submitted a provisional patent application on the window (US provisional patent application no. 62/548,455).

Corresponding authors

Correspondence to David Entenberg or John Condeelis.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8

  2. 2.

    Life Sciences Reporting Summary

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Protocol

    A Protocol for the Implantation of a Permanent Window for High-Resolution Imaging of the Murine Lung

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  1. 1.

    Supplementary Data 1

    Microcartography software setup file.

Excel files

  1. 1.

    Supplementary Data 2

    Raw data for supplementary figures.


  1. 1.

    The WHRIL is well tolerated.

    WHRIL bearing mice are able to perform tasks of daily living (grooming, feeding, nesting, stretching, etc.) without impediment.

  2. 2.

    Time-lapse video of healthy lung vasculature shown in Supplemental Figure 1.

    Left: raw, unprocessed video showing single cell resolution imaging. Right: Same video with red (blood) channel averaged over time to improve definition of the vascular boundaries. Time between frames = 1 sec, FOV = 170 μm. Cyan = CFP labeled myeloid cells, Red = vasculature labeled by 155kD TMR-dextran.

  3. 3.

    Time lapse video of raw data shown in Figure 4A showing arrival of a tumor cell to join other cells trapped in the lung vasculature.

    Time between frames = 1.35 min. Time stamp = hh:mm:ss. FOV = 117x67 μm. Green = tumor cell, red = vasculature, cyan = macrophages.

  4. 4.

    Time lapse video of blood averaged data shown in Figure 4B showing extravasation of an experimentally metastasized tumor cell from inside the lung vasculature into an alveolus.

    Left: Maximum intensity projection of 3 slices (9 μm) within the lung vasculature. Right: 3D reconstruction of the entire data set (8 slices, 24 μm) FOV = 60 μm. Time between frames = 64 sec. Time stamp = hh:mm:ss.

  5. 5.

    Visualization of tumor cell extravasation in an experimental metastasis model.

    Raw unprocessed data corresponding to Figure 4B showing a single optical section of the lung vasculature. Time between frames = 64 sec. Green = GFP tumor cells, Cyan = CFP macrophages, Red = Ve-Cad labeled endothelia and 155kD TMR dextran. Time stamp = hh:mm:ss.

  6. 6.

    Video of data presented in Figure 5A showing one cell of a group of spontaneously metastasized tumor cells appearing to undergo division in the lung.

    Time between frames = 1.42 min. FOV = 75 μm. Green = GFP labeled tumor cells, Red = 155kD TMR dextran, Blue = SHG from collagen I fibers. Time stamp =mm:ss

  7. 7.

    Two frames from Supplemental Figure 3B showing co-registration of vasculature and an extravasated tumor cell.

  8. 8.

    Video of functional TMEM in the lung showing transient vasculature leakage (permeability).

    Yellow arrow indicates location of transient vascular leakage seen as red dextran appearing in the extravascular space.

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