Optimization of CLARITY for clearing whole-brain and other intact organs
Open accessAbstract
The development, refinement, and use of techniques that allow high-throughput imaging of whole brains with cellular resolution will help us understand the complex functions of the brain. Such techniques are crucial for the analysis of complete neuronal morphology—anatomical and functional—connectivity, and repeated molecular phenotyping. CLARITY is a recently introduced technique that produces structurally intact, yet optically transparent tissue, which may be labeled and imaged without sectioning. However, the utility of this technique depends on several procedural variables during the process in which the light-scattering lipids in a tissue are replaced by a transparent hydrogel matrix. Here, we systematically varied a number of factors (including temperature, hydrogel composition, and polymerization conditions) to provide an optimized, highly replicable CLARITY procedure for clearing mouse brains. We found that for these preparations optimal tissue clearing requires electrophoresis (and cannot be achieved with passive clearing alone) for 5 d with a combination of 37 and 55°C temperature. Although this protocol is optimized for brains, we also show that it can be used to clear and analyze a variety of organs. Brain or other tissue prepared using this protocol is suitable for high-throughput imaging with confocal or single-plane illumination microscopy.
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While it appears that we have labeled the major myelinated WM tracts of the mouse brain, there may be areas that are not fully captured due to incomplete antibody penetration or uneven labeling.
“First, while it appears that we have labeled the major myelinated WM tracts of the mouse brain, there may be areas that are not fully captured due to incomplete antibody penetration or uneven labeling, which have been reported by other groups using intact brain approaches (Epp et al., 2015; Li et al., 2015).”
From The role of myelination in measures of white matter integrity: Combination of diffusion tensor imaging and two-photon microscopy of CLARITY intact brains (2017) · cites this paper as Epp et al., 2015
The citing authors’ statement about which tracts they labeled is their own; the point attributed to Epp et al. is the reported risk of incomplete antibody penetration or uneven labeling in intact-brain approaches. The Materials and Methods section describes antibody incubation, but neither it nor the passages found by searching for penetration and uneven labeling report that problem (page unavailable). (LLM, conf=0.72)
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Research works printed but not linked: 12
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- Authors
- Epp, J. R., Niibori, Y., (Liz) Hsiang, H.-L., Mercaldo, V., Deisseroth, K., Josselyn, S. A., & Frankland, P. W.
- Affiliations
- University of Toronto (CA)
- Stanford University (US)
- Corresponding author
- Josselyn, Sheena A.
- Frankland, Paul W.
- Venue
- eneuro
- Publisher
- Society for Neuroscience
- Published
- 2015-05
- Volume
- 2
- Issue
- 3
- Pages
- ENEURO.0022-15.2015
- ISSN
- 2373-2822 (ISSN-L)
- PMC
- PMC4586927
- OpenAlex
- W2180979457
- Cited by
- 133
- References
- 0
- Citations by year
- 2026: 4 · 2025: 8 · 2024: 8 · 2023: 4 · 2022: 9 · 2021: 13 · 2020: 15 · 2019: 20 · 2018: 19 · 2017: 17 · 2016: 13 · 2015: 2
- FWCI
- 10.33
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by
- Locations
- eNeuro (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- Canadian Institutes of Health Research (MOP86762, MOP-74650)
- NARSAD young investigator grant
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Cell Image Analysis Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Keywords
- clarity, tissue clearing, whole-brain imaging, hydrogel matrix, temperature optimization, polymerization conditions, mouse brain, single plane illumination microscopy, confocal microscopy, high-throughput imaging
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