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Minimally invasive longitudinal intravital imaging of cellular dynamics in intact long bone

Nature Protocols volume 18, pages 3856–3880 (2023)Cite this article

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Abstract

Intravital two-photon microscopy enables deep-tissue imaging at high temporospatial resolution in live animals. However, the endosteal bone compartment and underlying bone marrow pose unique challenges to optical imaging as light is absorbed, scattered and dispersed by thick mineralized bone matrix and the adipose-rich bone marrow. Early bone intravital imaging methods exploited gaps in the cranial sutures to bypass the need to penetrate through cortical bone. More recently, investigators have developed invasive methods to thin the cortical bone or implant imaging windows to image cellular dynamics in weight-bearing long bones. Here, we provide a step-by-step procedure for the preparation of animals for minimally invasive, nondestructive, longitudinal intravital imaging of the murine tibia. This method involves the use of mixed bone marrow radiation chimeras to unambiguously double-label osteoclasts and osteomorphs. The tibia is exposed by a simple skin incision and an imaging chamber constructed using thermoconductive T-putty. Imaging sessions up to 12 h long can be repeated over multiple timepoints to provide a longitudinal time window into the endosteal and marrow niches. The approach can be used to investigate cellular dynamics in bone remodeling, cancer cell life cycle and hematopoiesis, as well as long-lived humoral and cellular immunity. The procedure requires an hour to complete and is suitable for users with minimal prior expertise in small animal surgery.

Key points

  • An imaging approach for tracking osteoclasts in vivo using bone marrow chimeras obtained from two different lineage reporter mice. The labeled cells can be detected in the tibia with minimally invasive surgery and the creation of an imaging chamber.

  • The nondestructive approach enables the longitudinal monitoring of the cells in a weight-bearing bone, over a period of days to weeks, without requiring thinning of the bone.

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Fig. 1: Overview of the protocol for intravital imaging in the intact tibia.
Fig. 2: Preparation of mice for intravital imaging of the tibia.
Fig. 3: Osteoclastic recycling during the steady state.
Fig. 4: Regional differences in osteoclastic recycling rates in the tibia.
Fig. 5: Mapping osteoclastic activity to different regions in the tibia.

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Data availability

All source imaging data are too large to be included but are available from the corresponding author upon request. Scripts used are available at https://github.com/theimagelab/bone_imaging.

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Acknowledgements

We thank the staff of the Australian BioResource, Biological Testing Facility and Australian Cancer Research Foundation INCITe Centre for Intravital Imaging of Niches for Cancer Immune Therapy. This work is supported by the Australian NHMRC grants ID1155678 and ID2009010, the Mrs Janice Gibb and Ernest Heine Family Foundation, Peter and Val Duncan, Kedje Foundation, Cancer Institute New South Wales and the Australian Cancer Research Foundation.

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  1. These authors contributed equally: Nayan Deger Bhattacharyya, Wunna Kyaw.

Authors and Affiliations

  1. Garvan Institute of Medical Research, Sydney, New South Wales, Australia

    Nayan Deger Bhattacharyya, Wunna Kyaw, Michelle M. McDonald, Rama Dhenni, Abigail K. Grootveld, Ya Xiao, Ryan Chai, Weng Hua Khoo, Linda C. Danserau, C. Marcelo Sergio, Paul Timpson, Peter I. Croucher & Tri Giang Phan

  2. St Vincent’s Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, New South Wales, Australia

    Nayan Deger Bhattacharyya, Wunna Kyaw, Michelle M. McDonald, Rama Dhenni, Abigail K. Grootveld, Ya Xiao, Ryan Chai, Weng Hua Khoo, Linda C. Danserau, Paul Timpson, Peter I. Croucher & Tri Giang Phan

  3. School of Medical Sciences, Faculty of Medicine and Health, The University of Sydney, Sydney, New South Wales, Australia

    Michelle M. McDonald

  4. Charles Perkins Centre, The University of Sydney, Sydney, New South Wales, Australia

    Michelle M. McDonald

  5. ACRF INCITe Centre for Intravital Imaging of Niches for Cancer Immune Therapy, Sydney, New South Wales, Australia

    Linda C. Danserau, Paul Timpson, Woei Ming Lee, Peter I. Croucher & Tri Giang Phan

  6. John Curtin School of Medical Research, Australian National University, Canberra, New South Wales, Australia

    Woei Ming Lee

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Contributions

N.D.B., W.K., P.I.C. and T.G.P. conceived and designed experiments. T.G.P. developed the method. N.D.B. refined and improved the method. N.D.B., W.K., M.M.M., R.D., Y.X., R.C., A.K.G. and W.H.K. performed the experiments. L.C.D. and M.S. provided technical support. P.T., W.M.L., P.I.C. and T.G.P. supervized image processing and analysis. N.D.B., W.K., P.I.C. and T.G.P. wrote the manuscript. All authors have reviewed the manuscript.

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Correspondence to Tri Giang Phan.

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Key references using this protocol

McDonald, M. M. et al. Cell 184, 1330–1347.e13 (2021): https://doi.org/10.1016/j.cell.2021.02.002

Lawson, M. A. et al. Nat. Commun. 6, 8983 (2015): https://doi.org/10.1038/ncomms9983

Khoo, W. H. et al. Blood 134, 30–43 (2019): https://doi.org/10.1182/blood.2018880930

Supplementary information

Reporting Summary

Supplementary Video 1

Intravital imaging of steady-state osteoclast recycling related to Fig. 3. a, 3D rotation of the tibial bone (SHG, blue) surface. b, Video depicting osteoclast fission and fusion. Arrow denotes a fission and fusion event in an GFP+ (CSF1R, green) TOM+ (LysM, red) osteoclasts. c, Video depicting the cell fate mapping of segmented osteoclast surfaces, as denoted by the underground plots (UG) in Fig. 3b

Supplementary Video 2

Intravital imaging of steady-state osteoclast recycling in the proximal site of the tibia, related to the top of Fig. 4a,b. a, 3D rotation of the tibial bone (SHG, blue) surface and fused GFP+ (CSF1R, green), TOM+ (LysM, red) osteoclasts. b, Video depicting slower rates of osteoclast fission and fusion. c, Video depicting the cell fate mapping of segmented osteoclast surfaces, as denoted by the underground plots in

Supplementary Video 3

Intravital imaging of steady-state osteoclast recycling in the distal site of the tibia, related to the bottom of Fig. 4 a,b. a, 3D rotation of the tibial bone (SHG, blue) surface and fused GFP+ (CSF1R, green), TOM+ (LysM, red) osteoclasts. b, Video depicting accelerated rates of osteoclast fission and fusion. c, Video depicting the cell fate mapping of segmented osteoclast surfaces, as denoted by the underground plots in

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Bhattacharyya, N.D., Kyaw, W., McDonald, M.M. et al. Minimally invasive longitudinal intravital imaging of cellular dynamics in intact long bone. Nat Protoc 18, 3856–3880 (2023). https://doi.org/10.1038/s41596-023-00894-9

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