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Real-time intravital imaging of pH variation associated with osteoclast activity

Abstract

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

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Figure 1
Figure 2: Chemical structures and spectroscopic data of pHocas-1–3 and pHocas-AL.
Figure 3: Evaluation of the availability of pHocas-3 for TPEM imaging of osteoclast activity in living mice.
Figure 4: Quantitative analysis of osteoclast activity in vivo based on colocalization analysis of pHocas-3 and tdTomato.
Figure 5: Two-photon time-lapse imaging of bone tissues, acquired at 5-min intervals for 8 h after injection of pHocas-3.
Figure 6: Relationship between the formation of an acidic compartment and cell motility.

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Acknowledgements

This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (grant nos. 25220207, 26102529, and 15K12754 to K.K., 14J00794 to H.M.). K.K. and M.I. thank the Naito Foundation for financial support. K.K. and S.M. thank the Asahi Glass Foundation for financial support. K.K. and M.I. also thank the Uehara Memorial Foundation for financial support.

Author information

Authors and Affiliations

Authors

Contributions

H.M., T.K., S.M., and K.K. contributed to the development of chemical probes, and M.F., M.S., J.K., and M.I. conducted imaging experiments in vivo. H.M. and T.K. synthesized and characterized chemical probes. M.F., M.S., and J.K. performed in vivo studies. H.M. and T.K. co-wrote the initial draft. J.K., M.F., S.M., M.I., and K.K. revised the final draft.

Corresponding authors

Correspondence to Masaru Ishii or Kazuya Kikuchi.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1 and 2 and Supplementary Figures 1–10. (PDF 1095 kb)

Supplementary Note

Synthetic Procedures. (PDF 563 kb)

Real-time fluorescence imaging of osteoclasts in living mice with pHocas-AL.

Long-term intravital two-photon microscopy of mouse calvaria bone tissues was performed. PBS solution of pHocas-AL was injected subcutaneously into TRAP-tdTomato mice daily for 3 d prior to imaging. Images were captured every 5 min for 8 h and processed using spectral unmixing algorithms. The detailed experimental procedure is described in the Online Methods. (MOV 7989 kb)

Real-time fluorescence imaging of osteoclasts in living mice with pHocas-3.

Long-term intravital two-photon microscopy of mouse calvaria bone tissues was performed. PBS solution of pHocas-3 was injected subcutaneously into TRAP-tdTomato mice daily for 3 d prior to imaging. Images were captured every 5 min for 8 h and processed using spectral unmixing algorithms. The detailed experimental procedure is described in the Online Methods. (MOV 7242 kb)

In vivo imaging of osteoclast activation for short intervals.

Short-term intravital two-photon microscopy of mouse calvaria bone tissues was performed. PBS solution of pHocas-3 was injected subcutaneously into TRAP-tdTomato mice daily for 3 d prior to imaging. Images were captured every 1 min for 130 min without spectral unmixing algorithms. The detailed experimental procedure is described in the Online Methods. (MOV 6999 kb)

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Maeda, H., Kowada, T., Kikuta, J. et al. Real-time intravital imaging of pH variation associated with osteoclast activity. Nat Chem Biol 12, 579–585 (2016). https://doi.org/10.1038/nchembio.2096

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