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

Greenbaum, Alon; Chan, Ken Y.; Dobreva, Tatyana; Brown, David Alan; Balani, Deepak H.; Boyce, Rogely; Kronenberg, Henry M.; McBride, Helen J.; Gradinaru, Viviana

doi:10.1126/scitranslmed.aah6518

Cited by 158 publications

(162 citation statements)

References 50 publications

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“…In the raw data, inhomogeneity was evident between sections, but there was also marked inhomogeneity between tiles. Cells were preferentially located near the endplates, notably adjacent to the endocortex, which has been described for these cells using bone clearing techniques and 3-dimensional imaging of whole mouse vertebral bodies (Greenbaum et al, 2017). Inhomogeneity or non-uniform distribution of cell populations, which may or may not be sparse, is a common situation in the neurosciences where proportionator sampling can offer significant improvements in efficiency.…”

Section: Discussionmentioning

confidence: 73%

The Automatic Proportionator Estimator Is Highly Efficient for Estimation of Total Number of Sparse Cell Populations

Boyce

Gundersen

2018

Front. Neuroanat.

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Estimation of total number of a population of cells that are sparsely distributed in an organ or anatomically-defined region of interest represents a challenge for conventional stereological methods. In these situations, classic fractionator approaches that rely on systematic uniform random sampling are highly inefficient and, in many cases, impractical due to the intense sampling of the organ and tissue sections that is required to obtain sufficient counts for an acceptable level of precision. The proportionator, an estimator based on non-uniform sampling theory, marries automated image analysis with stereological principles and is the only estimator that provides a highly efficient and precise method to address these challenging quantification problems. In this paper, the practical considerations of the proportionator estimator and its implementation with Proportionator™ software and digital slide imaging are reviewed. The power of the proportionator as a stereological tool is illustrated in its application to the estimation of the total number of a very rare (~50/vertebrae) and sparsely distributed population of osteoprogenitor cells in mouse vertebral body. The proportionator offers a solution to neuroscientists interested in quantifying total cell number of sparse cell populations in the central and peripheral nervous system where systematic uniform random sampling-based stereological estimators are impractical.

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Section: Discussionmentioning

confidence: 73%

The Automatic Proportionator Estimator Is Highly Efficient for Estimation of Total Number of Sparse Cell Populations

Boyce

Gundersen

2018

Front. Neuroanat.

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“…PACT has been modified to expand tissues for better separation of compact structures (ePACT) . It has also been optimized to clear difficult‐to‐image tissues, such as bone through the introduction of EDTA (PACT‐deCAL) or the combination of EDTA with amino alcohol (Bone CLARITY, a bone tissue clearing method based on CLARITY) , and lipid droplet‐rich tissues by lipase assist . Woo et al proposed a faster method by adding α‐thioglycerol to the original PACT clearing solution, named mPACT .…”

Section: Optical Clearing Methodsmentioning

confidence: 99%

“…The clearing agents and protocols mentioned above can potentially be shuffled and combined to develop better protocols or satisfy certain applications based on their chemical compositions and putative functions in the clearing process. Many researchers have explored the combining strategies for specific purposes, such as the combinations of TDE, SeeDB, CUBIC‐R2 or RapiClear with CLARITY for economical refractive index‐matching alternatives ; fructose in SeeDB with urea in Sca l e for low viscosity of clearing solutions (FRUIT ); RapiClear with Sca l e for better transparency ; tert ‐butanol in FluoClearBABB with 3DISCO (uDISCO ) for better fluorescence preservation; SDS in CLARITY with detergents in iDISCO for immunostaining improvement (FASTClear ); and amino alcohol in CUBIC with CLARITY for decolorization of bone tissues (Bone CLARITY ).…”

Section: Optical Clearing Methodsmentioning

confidence: 99%

Optical clearing for multiscale biological tissues

Gong

et al. 2017

Journal of Biophotonics

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Three-dimensional reconstruction of tissue structures is essential for biomedical research. The development of light microscopes and various fluorescent labeling techniques provides powerful tools for this motivation. However, optical imaging depth suffers from strong light scattering due to inherent heterogeneity of biological tissues. Tissue optical clearing technology provides a distinct solution and permits us to image large volumes with high resolution. Until now, various clearing methods have been developed. In this study, from the perspective of the end users, we review in vitro tissue optical clearing techniques based on the sample features in terms of size and age, enumerate the methods suitable for immunostaining and lipophilic dyes and summarize the combinations with various imaging techniques. We hope this review will be helpful for researchers to choose the most suitable clearing method from a variety of protocols to meet their specific needs.

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“…Whereas it is hard to obtain images from deep part of tissue by using confocal microscopy, the light sheet microscopy allows us to visualize the deep portion of bone marrow (Chen et al, 2016; Greenbaum et al, 2017). Intravital deep imaging enabled by multi-photon microscopy has allowed analysis of cellular and oxygen dynamics in murine calvarial bone marrow.…”

Section: Hsc Visualizationmentioning

confidence: 99%

Use of Imaging Techniques to Illuminate Dynamics of Hematopoietic Stem Cells and Their Niches

Morikawa

Takubo

2017

Front. Cell Dev. Biol.

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Continuous generation of blood cells over an organism's lifetime is supported by hematopoietic stem/progenitor cells (HSPCs) capable of producing all hematopoietic cell subtypes. Adult mammalian HSPCs are localized to bone marrow and regulated by their neighboring microenvironment, or “niche.” Because interactions of HSPCs with their niches are highly dynamic and complex, the recent development of imaging technologies provides a powerful new tool to understand stem cell/niche biology. In this review, we discuss recent advances in our understanding of dynamic HSPC/niche interactions during development, homeostasis, disease states or aging with a focus on studies advanced by imaging analysis. We also summarize methods to visualize HSPCs and niche cells in vivo, including use of HSPC reporter mice and chemical probes. Findings emerging from these investigations could suggest novel therapies for diseases and aging.

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

Abstract: Bone clearing and light-sheet microscopy enables visualization and quantification of fluorescent single cells in intact bone.

Cited by 158 publications

References 50 publications

The Automatic Proportionator Estimator Is Highly Efficient for Estimation of Total Number of Sparse Cell Populations

The Automatic Proportionator Estimator Is Highly Efficient for Estimation of Total Number of Sparse Cell Populations

Optical clearing for multiscale biological tissues

Use of Imaging Techniques to Illuminate Dynamics of Hematopoietic Stem Cells and Their Niches

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