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Published April 26, 2017 | Supplemental Material
Journal Article Open

Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow


Bone tissue harbors unique and essential physiological processes, such as hematopoiesis, bone growth, and bone remodeling. To enable visualization of these processes at the cellular level in an intact environment, we developed "Bone CLARITY," a bone tissue clearing method. We used Bone CLARITY and a custom-built light-sheet fluorescence microscope to detect the endogenous fluorescence of Sox9-tdTomato+ osteoprogenitor cells in the tibia, femur, and vertebral column of adult transgenic mice. To obtain a complete distribution map of these osteoprogenitor cells, we developed a computational pipeline that semiautomatically detects individual Sox9-tdTomato+ cells in their native three-dimensional environment. Our computational method counted all labeled osteoprogenitor cells without relying on sampling techniques and displayed increased precision when compared with traditional stereology techniques for estimating the total number of these rare cells. We demonstrate the value of the clearing-imaging pipeline by quantifying changes in the population of Sox9-tdTomato–labeled osteoprogenitor cells after sclerostin antibody treatment. Bone tissue clearing is able to provide fast and comprehensive visualization of biological processes in intact bone tissue.

Additional Information

© 2017 American Association for the Advancement of Science. Submitted 27 July 2016; Accepted 23 March 2017; Published 26 April 2017. We thank A. Lignell and L. Cai for technical help in building the LSFM and the Amgen Scientific Team (F. Asuncion, D. Hill, M. Ominsky, and E. Pacheco). A.G. is a Good Ventures Fellow of the Life Sciences Research Foundation. Funding: This work was supported by NIH Director's New Innovator Award (IDP20D017782) and Presidential Early Career Award for Scientists and Engineers; Heritage Medical Foundation; Curci Foundation; Amgen Chem-Bio-Engineering Award; Pew Charitable Trust; Kimmel Foundation; and Caltech–City of Hope. Author contributions: H.J.M. and V.G. conceived the project; A.G. and K.Y.C. performed all experiments, data acquisition, and analysis; T.D. and D.B. contributed computational tools and data analysis with input from R.B.; D.H.B. and H.M.K. contributed samples for clearing. A.G., K.Y.C., T.D., D.B., and V.G. generated the figures and wrote the manuscript with input from all authors. V.G. supervised all aspects of the work. Competing interests: V.G., K.Y.C., and A.G. are inventors on patent application 62/447,781 submitted by the California Institute of Technology that covers methods and devices for soft and osseous tissue clearing and fluorescence imaging. H.J.M. and R.B. are employees and shareholders of Amgen. All other authors declare that they have no competing interests. Data and materials availability: The r13c7 antibody can be provided by and at Amgen's sole discretion, with pending scientific review and a completed material transfer agreement with Amgen. Requests for antibody from an academic or nonprofit institution should be submitted to www.ext.amgen.com/partners/academic-collaborations/new-requests/. Requests for antibody from a for-profit entity should be submitted to BDopportunities@amgen.com.

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