Advanced CLARITY for rapid and high-resolution imaging of intact tissues
Open accessAbstract
CLARITY is a method for chemical transformation of intact biological tissues into a hydrogel-tissue hybrid, which becomes amenable to interrogation with light and macromolecular labels while retaining fine structure and native biological molecules. This emerging accessibility of information from large intact samples has created both new opportunities and new challenges. Here we describe protocols spanning multiple dimensions of the CLARITY workflow, ranging from simple, reliable and efficient lipid removal without electrophoretic instrumentation (passive CLARITY) to optimized objectives and integration with light-sheet optics (CLARITY-optimized light-sheet microscopy (COLM)) for accelerating data collection from clarified samples by several orders of magnitude while maintaining or increasing quality and resolution. The entire protocol takes from 7-28 d to complete for an adult mouse brain, including hydrogel embedding, full lipid removal, whole-brain antibody staining (which, if needed, accounts for 7-10 of the days), and whole-brain high-resolution imaging; timing within this window depends on the choice of lipid removal options, on the size of the tissue, and on the number and type of immunostaining rounds performed. This protocol has been successfully applied to the study of adult mouse, adult zebrafish and adult human brains, and it may find many other applications in the structural and molecular analysis of large assembled biological systems.
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When coupled with two-photon or light-sheet laser microscopy, whole mouse brains can be imaged entirely intact with sub-cellular resolution.
“When coupled with two-photon or light-sheet laser microscopy, whole mouse brains can be imaged entirely intact with sub-cellular resolution (Tomer et al., 2014).”
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 Tomer et al., 2014
The Overview of procedure and Anticipated Results (page unavailable) report high-resolution imaging through an intact mouse brain and whole-brain imaging with CLARITY-optimized light-sheet microscopy. The paper does not demonstrate whole-brain imaging with two-photon microscopy or establish sub-cellular resolution throughout the whole brain. (LLM, conf=0.86)
Experimental — accuracy not yet measuredVirus-mediated neuronal tract-tracing combined with CLARITY-optimized light-sheet microscopy revealed complete interruption of corticospinal tract fibres and pronounced depletion of glutamatergic reticulospinal fibres below the injury.
“Virus-mediated neuronal tract-tracing combined with CLARITY-optimized light-sheet microscopy23 revealed the complete interruption of corticospinal tract fibres and pronounced depletion of glutamatergic reticulospinal fibres below the injury (Fig. 1e and Extended Data Fig. 2a).”
From The neurons that restore walking after paralysis (2022) · cites this paper as [23]
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- Authors
- Tomer, R., Ye, L., Hsueh, B., & Deisseroth, K.
- Affiliations
- Howard Hughes Medical Institute (US)
- Stanford University (US)
- Corresponding author
- Deisseroth, Karl
- Venue
- Nature Protocols
- Published
- 2014-06-19
- Volume
- 9
- Issue
- 7
- Pages
- 1682-1697
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- PMC
- PMC4096681
- OpenAlex
- W2010114799
- Cited by
- 842
- References
- 42
- Citations by year
- 2026: 25 · 2025: 51 · 2024: 54 · 2023: 38 · 2022: 45 · 2021: 78 · 2020: 99 · 2019: 96 · 2018: 94 · 2017: 82 · 2016: 93 · 2015: 73 · 2014: 11
- FWCI
- 51.24
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institute of Mental Health (T32MH020016)
- National Institute on Drug Abuse (R37DA035377)
- 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, lipid removal, light-sheet microscopy, whole-brain imaging, antibody staining, mouse brain, zebrafish brain, human brain
- MeSH terms
- Animals, Brain, Microscopy, Tissue Embedding, Hydrogel, Polyethylene Glycol Dimethacrylate, Mice, Optics and Photonics
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