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Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue

Nature Protocols volume 16, pages 1907–1935 (2021)Cite this article

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Abstract

Multiphoton intravital imaging is essential for understanding cellular behavior and function in vivo. The adipose-rich environment of the mammary gland poses a unique challenge to in vivo microscopy due to light scattering that impedes high-resolution imaging. Here we provide a protocol for high-quality, six-color 3D intravital imaging of regions across the entire mouse mammary gland and associated tissues for several hours while maintaining tissue access for microdissection and labeling. An incision at the ventral midline and along the right hind leg creates a skin flap that is then secured to a raised platform skin side down. This allows for fluorescence-guided microdissection of connective tissue to provide unimpeded imaging of mammary ducts. A sealed imaging chamber over the skin flap creates a stable environment while maintaining access to large tissue regions for imaging with an upright microscope. We provide a strategy for imaging single cells and the tissue microenvironment utilizing multicolor Confetti lineage-tracing and additional dyes using custom-designed filters and sequential excitation with dual multiphoton lasers. Furthermore, we describe a strategy for simultaneous imaging and photomanipulation of single cells using the Olympus SIM scanner and provide steps for 3D video processing, visualization and high-dimensional analysis of single-cell behavior. We then provide steps for multiplexing intravital imaging with fixation, immunostaining, tissue clearing and 3D confocal imaging to associate cell behavior with protein expression. The skin-flap surgery and chamber preparation take 1.5 h, followed by up to 12 h of imaging. Applications range from basic filming in 1 d to 5 d for multiplexing and complex analysis.

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Fig. 1: Schematic overview of the intravital imaging protocol.
Fig. 2: Surgical preparation of the mammary skin-flap chamber.
Fig. 3: Six-color intravital imaging to separate Confetti fluorescence and additional dyes.
Fig. 4: Cyan/red stereo 3D visualization.
Fig. 5: 3D intravital imaging of epithelial cells and stromal interactions.
Fig. 6: Dynamics of mammary basal progenitor cells in ducts and terminal end buds.

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Data availability

Data in Fig. 5a,b were published previously12. The source data for Fig. 6e are included in a source data file to this protocol. All other data are too large to include in the paper but are available from the authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank F. Jackling for animal management and S. Devi and R. Yip for imaging assistance. We are grateful to the Walter and Eliza Hall Institute (WEHI) Center for Dynamic Imaging and WEHI Bioservices. This work was supported by the Australian National Health and Medical Research Council (NHMRC) grants #1016701, 1113133; NHMRC IRIISS; the Victorian State Government through VCA funding and Operational Infrastructure Support; and the Australian Cancer Research Foundation. C.A.D was supported by an Australian Government Research Training Program Scholarship; A.C.R. was supported by a National Breast Cancer Foundation (NBCF)/Cure Cancer Australia Fellowship; G.J.L. by NHMRC Fellowship #1078730, #1175960; J.E.V. by NHMRC Fellowships #1037230, 1102742; S.N.M. by NHMRC Fellowship #1136550.

Author information

Author notes

  1. These authors contributed equally: Anne C. Rios, Jane E. Visvader.

Authors and Affiliations

  1. Cancer Biology and Stem Cells Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Caleb A. Dawson, Geoffrey J. Lindeman, Anne C. Rios & Jane E. Visvader

  2. Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia

    Caleb A. Dawson, Anne C. Rios & Jane E. Visvader

  3. Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, Victoria, Australia

    Scott N. Mueller

  4. The Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Melbourne, Victoria, Australia

    Scott N. Mueller

  5. Department of Medicine, The University of Melbourne, Parkville, Victoria, Australia

    Geoffrey J. Lindeman

  6. Parkville Familial Cancer Centre and Department of Medical Oncology, The Royal Melbourne Hospital and Peter MacCallum Cancer Centre, Parkville, Victoria, Australia

    Geoffrey J. Lindeman

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Contributions

C.A.D. designed and performed experiments, analyzed data and wrote the manuscript. S.N.M. designed experiments. G.J.L provided general guidance. A.C.R and J.E.V. designed experiments and provided general guidance.

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Correspondence to Jane E. Visvader.

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Dawson, C. A. et al. Nat. Cell Biol. 546–558 (2020): https://doi.org/10.1038/s41556-020-0505-0

Supplementary information

Supplementary Information

Supplementary Discussion.

Reporting Summary

Supplementary Video 1

Six-color IVM of immune cell interactions with mammary terminal end bud cap cells during morphogenesis in puberty. IVM of a TEB in a 5 week-old K5/TetCre/Confetti mouse treated with doxycycline for 3 d at 4 weeks of age and labeled with CD45 APC antibody placed in the imaging chamber (n = 2 mice). CFP, cyan; GFP, pink; YFP, yellow; RFP, magenta; SHG, gray; CD45, orange. Related to Fig. 3c. All experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee.

Supplementary Video 2

IVM of resident ductal macrophage behavior in mammary ducts. Animated IVM of mammary ducts in an Elf5-GFP mouse with immunolabeling by MHCII Alexa Fluor 647 antibody in the imaging chamber12. The movie cycles over a 6 h time span with images acquired every 10 min. GFP, magenta; Duct-adjacent MHCII, yellow; stromal MHCII, cyan; collagen SHG, pink. Duct-adjacent MHCII was isolated in Imaris by creating a low resolution GFP surface and masking the MHCII signal. The duct structure is 300 µm across (n = 6 mice). Related to Figure 5a. All experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee.

Supplementary Video 3

IVM of the resident macrophage response to damage of the mammary epithelium. An animation of IVM of a mammary duct in a Cx3cr1GFP/+ mouse12. The movie cycles through time points prior to damage showing the arrangement of GFP-high ductal macrophages (yellow) around a duct, then views an optical section through ductal macrophages before and after precise photoablation at 4 h (bolt symbol). Images were acquired every 3 min (n = 3 mice). Related to Figure 5b. All experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee.

Supplementary Video 4

IVM of single photoactivated mammary terminal end bud cells. IVM of a TEB in a 4-week-old Kaede mouse after photoconversion of single cells using the Olympus SIM scanner (n = 2 mice). Related to Figure 5c. All experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee.

Supplementary Video 5

IVM of mammary duct basal cell division. IVM of a duct in an 8-week-old K5/TetCre/Confetti mouse after treatment with doxycycline at 4 weeks of age showing a myoepithelial cell dividing longitudinally (n = 5 mice). The duct was imaged every 10 min for 8 h and 40 min. Related to Fig. 6a. All experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee.

Supplementary Video 6

IVM of mammary duct basal cells, example 2. IVM of a duct in a 6-week-old K5/TetCre/Confetti mouse after treatment with doxycycline at 4 weeks of age (n = 5 mice). The duct was imaged every 10 min for 4 h. All experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee.

Supplementary Video 7

IVM of mammary terminal end bud cap cell dynamics. IVM of a TEB in a 5-week-old K5/TetCre/Confetti mouse treated with doxycycline for 3 d at 4 weeks of age. An optical slice through the outer cap layer is shown to reveal cap cell dynamics (n = 4 mice). Related to Fig. 6c. The TEB is pointing upward. All experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee.

Supplementary Video 8

IVM of mammary terminal end bud cap cells in stereo 3D. IVM of a TEB in a 5-week-old K5/TetCre/Confetti mouse treated with doxycycline for 3 d at 4 weeks of age. The full image volume is displayed for viewing with cyan/red 3D glasses. The TEB is pointing to the left (n = 4 mice). Related to Fig. 6c-d. All experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee.

Supplementary Video 9

IVM of mammary cap cell migration into the terminal end bud body. IVM of a TEB in a 5 week-old K5/TetCre/Confetti mouse treated with doxycycline for 3 d at 4 weeks of age. An optical slice through the center of the TEB (which is pointing to the left), showing cap cells that migrate from the cap into the TEB body. RFP, magenta (n = 4 mice). Related to Fig. 6d. All experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee.

Supplementary Video 10

IVM of mammary terminal end bud cap cells, example 2. IVM of a TEB in a 5 week-old K5/TetCre/Confetti mouse treated with doxycycline for 3 d at 4 weeks of age (n = 4 mice). The TEB is pointing to the right. All experiments were approved by the Walter and Eliza Hall Institute Animal Ethics Committee.

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Dawson, C.A., Mueller, S.N., Lindeman, G.J. et al. Intravital microscopy of dynamic single-cell behavior in mouse mammary tissue. Nat Protoc 16, 1907–1935 (2021). https://doi.org/10.1038/s41596-020-00473-2

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