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

Greenbaum, Alon and Chan, Ken Y. and Dobreva, Tatyana and Brown, David and Balani, Deepak H. and Boyce, Rogely and Kronenberg, Henry M. and McBride, Helen J. and Gradinaru, Viviana (2017) Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow. Science Translational Medicine, 9 (387). Art. No. eaah6518. ISSN 1946-6234.

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[img] Video (QuickTime) (Visualizing endogenous fluorescence throughout a cleared mouse tibia) - Supplemental Material
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[img] Video (QuickTime) (Visualizing endogenous fluorescence throughout a cleared mouse vertebral body) - Supplemental Material
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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.

Item Type:Article
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URLURL TypeDescription Materials
Greenbaum, Alon0000-0002-2897-876X
Chan, Ken Y.0000-0002-8853-5186
Dobreva, Tatyana0000-0002-2625-8873
Gradinaru, Viviana0000-0001-5868-348X
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 Requests for antibody from a for-profit entity should be submitted to
Group:Heritage Medical Research Institute, Tianqiao and Chrissy Chen Institute for Neuroscience
Funding AgencyGrant Number
Heritage Medical Research InstituteUNSPECIFIED
Curci FoundationUNSPECIFIED
Pew Charitable TrustUNSPECIFIED
Sidney Kimmel Foundation for Cancer ResearchUNSPECIFIED
Caltech–City of Hope Biomedical InitiativeUNSPECIFIED
Issue or Number:387
Record Number:CaltechAUTHORS:20161221-135116035
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Official Citation:A. Greenbaum, K. Y. Chan, T. Dobreva, D. Brown, D. H. Balani, R. Boyce, H. M. Kronenberg, H. J. McBride, V. Gradinaru, Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow. Sci. Transl. Med. 9, eaah6518 (2017)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73099
Deposited By: George Porter
Deposited On:21 Dec 2016 21:54
Last Modified:07 Jul 2020 20:35

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