Research ArticleImaging

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

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Science Translational Medicine  26 Apr 2017:
Vol. 9, Issue 387, eaah6518
DOI: 10.1126/scitranslmed.aah6518

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Peeking in on osteoprogenitors

The eyes may be the windows to the soul, but a window into the bone—specifically bone marrow—would be useful for studying bone development and disease. Greenbaum et al. developed a method of whole-bone optical clearing, using a series of reagents under continuous flow to delipidate and decalcify bone tissue. This process renders the entire bone transparent but does not affect endogenous fluorescence, making this compatible with reporter mice. Using light sheet fluorescence microscopy, the authors counted and mapped the number of fluorescently labeled osteoprogenitors within cleared mouse tibia, vertebral column, and femur bones treated with sclerostin antibody. With reduced variability compared to standard section analysis, this Bone CLARITY and computational analysis will be a useful tool for bone research.

Abstract

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.

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