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Structural and molecular interrogation of intact biological systems

Chung, Kwanghun and Wallace, Jenelle and Kim, Sung-Yon and Kalyanasundaram, Sandhiya and Andalman, Aaron S. and Davidson, Thomas J. and Mirzabekov, Julie J. and Zalocusky, Kelly A. and Mattis, Joanna and Denisin, Aleksandra K. and Pak, Sally and Bernstein, Hannah and Ramakrishnan, Charu and Grosenick, Logan and Gradinaru, Viviana and Deisseroth, Karl (2013) Structural and molecular interrogation of intact biological systems. Nature, 497 (7449). pp. 332-337. ISSN 0028-0836. PMCID PMC4092167. http://resolver.caltech.edu/CaltechAUTHORS:20130513-151857819

[img] PDF ( Supplementary Information. This file contains Supplementary Figures 1-14 and Supplementary Tables 1-3.) - Supplemental Material
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[img] Video (QuickTime) ( Video 1: Maximum projection of 300μm-volume of the eGFP-expressing neuronal circuit elements in the hippocampus in the 1mm-thick block of Thy-1:eGFP mouse brain shown in Supplementary Fig. 1a) - Supplemental Material
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[img] Video (QuickTime) (Video 2: 3D visualization of the YFP-expressing neuronal circuit elements from pial surface to the thalamus in the intact Thy-1:eYFP mouse brain (16 weeks old) shown in Fig. 2.) - Supplemental Material
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[img] Video (QuickTime) (Video 3: 3D visualization of YFP-expressing neuronal circuit elements in the ventral half of the intact Thy-1:eYFP mouse brain (16 weeks old).) - Supplemental Material
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[img] Video (QuickTime) ( Video 4: Raw data from Supplementary Video 1) - Supplemental Material
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[img] Video (QuickTime) ( Video 5: Brainstem region of the raw data from Supplementary Video 2) - Supplemental Material
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[img] Video (QuickTime) (Video 6: 3D visualization of a 1mm-thick coronal block of Thy1-eYFP mouse (12 weeks old) immunostained for GFP in intact nonsectioned form highlighting uniform immunostaining (6,604 Å~ 6,164 Å~ 918 μm volume; step-size=4.99 μm).) - Supplemental Material
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[img] Video (QuickTime) ( Video 7: TH staining of the intact mouse brain.) - Supplemental Material
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[img] Video (QuickTime) (Video 8: TH-positive fibers in the amygdala of the mouse brain.) - Supplemental Material
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[img] Video (QuickTime) ( Video 9: MAP2-positive processes and neuronal cell bodies in the DG of the mouse brain.) - Supplemental Material
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[img] Video (QuickTime) (Video 10: 3D visualization of a 1mm-thick coronal block of H line mouse brain (12 weeks old) immunostained for tyrosine hydroxylase (TH) (green, eYFP; red, TH).) - Supplemental Material
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[img] Video (QuickTime) (Video 11: 3D visualization of the 1mm-thick coronal block shown in Video 9 after the antibody elution process.) - Supplemental Material
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[img] Video (QuickTime) ( Video 12: 3D visualization of 2nd round immunostaining on the same 1mm-thick coronal block shown in Videos 9 and 10.) - Supplemental Material
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[img] Video (QuickTime) (Video 13: 3D visualization of 3rd round immunostaining on the same tissue shown in Videos 9-11.) - Supplemental Material
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[img] Video (QuickTime) (Video 14: Hippocampus region showing networks of YFP-expressing neurons (green), distribution of PV-positive neurons (red) and astrocytes (blue).) - Supplemental Material
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[img] Video (QuickTime) (Video 15: Animation illustrating single axonal tracing in intact postmortem human brain tissue shown in Fig. 5.) - Supplemental Material
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[img] Video (QuickTime) (Video 16: 3D Visualization of PV-positive neurons in the neocortex of the autism case shown in Fig. 5.) - Supplemental Material
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[img] Video (QuickTime) (Video 17: Animation illustrating tracing of the PV-positive neurons in layer 6 with abnormal dendritic bridging shown in Fig. 5.) - Supplemental Material
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[img] PDF - Accepted Version
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Abstract

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1038/nature12107DOIArticle
http://www.nature.com/nature/journal/v497/n7449/full/nature12107.htmlPublisherArticle
http://www.nature.com/nature/journal/v497/n7449/full/nature12107.html#supplementary-informationPublisherSupporting information
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092167/PubMed CentralArticle
http://rdcu.be/mSyTPublisherFree ReadCube access
ORCID:
AuthorORCID
Gradinaru, Viviana0000-0001-5868-348X
Additional Information:©2013 Macmillan Publishers Limited. Received 01 September 2012. Accepted 20 March 2013. Published online 10 April 2013. This work was funded by a National Institutes of Health (NIH) Director’s Transformative Research Award (TR01) to K.D. from NIMH, as well as by NSF, the Simons Foundation, and the President and Provost of Stanford University. K.D. is also funded by NIDA, the DARPA REPAIR program, and the Wiegers, Snyder, Reeves, Gatsby, and Yu Foundations. K.C. is supported by the Burroughs Wellcome Fund Career Award at the Scientific Interface. S.-Y.K. is supported by a Samsung Scholarship, A.S.A. by the Helen Hay Whitney Foundation, K.A.Z. and A.K.D. by an NSF Graduate Research Fellowship and J.M. by the NIH MSTP. We acknowledge H. Vogel, L. Luo, L. Schwarz, M. Monje, S. Hestrin and D. Castaneda for advice, and the Autism Tissue Program for providing human brain tissue, as well as J. J. Perrino, J. Mulholland and the Cell Sciences Imaging Facility at Stanford for electron microscopy imaging and advice. We would also like to thank the entire Deisseroth laboratory for discussions and support. CLARITY resources and protocols are freely supported online (http://CLARITYresourcecenter.org).
Funders:
Funding AgencyGrant Number
NIHTR01
National Institute of Mental Health (NIMH)UNSPECIFIED
NSFUNSPECIFIED
Simons FoundationUNSPECIFIED
National Institute on Drug AbuseUNSPECIFIED
Defense Advanced Research Projects Agency (DARPA)UNSPECIFIED
Wiegers FoundationUNSPECIFIED
H. L. Snyder Medical FoundationUNSPECIFIED
Reeves FoundationUNSPECIFIED
Gatsby FoundationUNSPECIFIED
Albert Yu and Mary Bechmann FoundationUNSPECIFIED
Burroughs Wellcome FundUNSPECIFIED
Samsung ScholarshipUNSPECIFIED
Helen Hay Whitney FoundationUNSPECIFIED
NSF Graduate Research FellowshipUNSPECIFIED
Stanford UniversityUNSPECIFIED
Subject Keywords:Molecular neuroscience
PubMed Central ID:PMC4092167
Record Number:CaltechAUTHORS:20130513-151857819
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20130513-151857819
Official Citation:Chung, K., Wallace, J., Kim, S.-Y., Kalyanasundaram, S., Andalman, A. S., Davidson, T. J., … Deisseroth, K. (2013). Structural and molecular interrogation of intact biological systems. Nature, 497(7449), 332–337. doi:10.1038/nature12107
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:38457
Collection:CaltechAUTHORS
Deposited By: Katherine Johnson
Deposited On:23 Mar 2015 18:53
Last Modified:22 Nov 2016 23:03

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