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.
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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).
K.C. and K.D. have disclosed these findings to the Stanford Office of Technology Licensing, which has filed a patent to ensure broad use of the methods in microscopy systems and for studying disease mechanisms and treatments. All protocols and methods remain freely available for academic and non-profit research in perpetuity, and supported by the authors, through the CLARITY website (http://CLARITYresourcecenter.org)
This file contains Supplementary Figures 1-14 and Supplementary Tables 1-3. (PDF 7475 kb)
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.
The video illustrates well-preserved eGFP signals from the fine axonal and dendritic branches after the CLARITY process. 1p excitation (488nm) and the 25Å~ objective (NA 0.95, WD 2.4 mm) were used for imaging. (MOV 7953 kb)
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.
Fly-through animation of the 3D volume data (2,037 Å~ 1,694 Å~ 3,405 μm; step-size=1.976 μm) illustrates visualization of all layers of cortex, the hippocampus, and the thalamus without degradation of resolution at depth. 1p excitation (514nm) and a 10Å~ objective (NA 0.3, WD 3.6 mm) were used. (MOV 11385 kb)
3D visualization of YFP-expressing neuronal circuit elements in the ventral half of the intact Thy-1:eYFP mouse brain (16 weeks old).
The clarified intact brain was mounted as described and the ventral half (18,100 Å~ 13,900 Å~ 3,400 μm; step-size=20 μm) was imaged using 1p excitation (514nm) and a 10Å~ water immersion objective (NA 0.3, WD 3.6 mm). Fly-through animation shows the YFP expressing neuronal networks in all regions of the brain. Note that cellular resolution is achieved even in highly scattering regions, such as the brainstem and the thalamus (see Video 3 for optical slices of the whole brain and Video 4 for optical slices of the brainstem region). (MOV 12465 kb)
Raw data from Supplementary Video 1
The video shows a series of xy-plane images through the 3D reconstruction data (18,100 Å~ 13,900 Å~ 3,400 μm volume; step-size=20 μm) from the ventral surface to center of the brain. Z position is indicated on the left. (MOV 9258 kb)
Brainstem region of the raw data from Supplementary Video 2
The video shows a series of xy-plane images through the 3D reconstruction data (5,482 Å~ 5,825 Å~ 3,400 μm volume, step-size=20 μm) from the ventral surface to center of the brain. Z-position is indicated on the left. (MOV 5396 kb)
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).
Left, eYFP (green). Middle, anti-GFP staining (red). Right, overlay. The intact block was ETC-treated for one day and immunostained for three days (two days in GFP antibody conjugated with alexa 594 and one day wash) at 37°C. 1p excitation (514nm and 594nm) and the 10Å~ objective (NA 0.3, WD 3.6 mm) were used for imaging. (MOV 3763 kb)
TH staining of the intact mouse brain.
A series of xy-plane images (4,492 Å~ 6,237 Å~ 2,504 μm volume; step-size=3 μm) from the ventral surface to center of the intact brain shown in Supplementary Fig. 4. The video shows immunohistologically labeled TH-positive neurons and their projections throughout the imaging depth and demonstrates that CLARITY enables whole mouse brain molecular phenotyping. The intact mouse brain was ETC-cleared for three days and stained for six weeks: primary (2 weeks) – wash (1 week) – secondary (2 weeks) – wash (1 week). The stained intact brain was then imaged 2500 μm from ventral side using the 10x water immersion objective (2p excitation, 780nm). Zposition is indicated on the left. (MOV 1583 kb)
TH-positive fibers in the amygdala of the mouse brain.
A series of xy-plane images through the 3D reconstruction image shown in Supplementary Figure 7 (2,078 Å~ 2,075 Å~ 915 μm volume; step mm) were used for imaging. The video shows extensive innervation of neurons in BLA and CeA by TH+ fibers. (MOV 14220 kb)
MAP2-positive processes and neuronal cell bodies in the DG of the mouse brain.
A series of xyplane images (18,100 Å~ 13,900 Å~ 300 μm volume; step-size=0.1 μm) showing that weakly-labeled cell bodies can be clearly identified, and among densely packed fibers that individual neurites are traceable. The video first shows a series of xy-plane images through the 3D reconstruction data (from bottom to top), and then shows 3D rendering while in reverse showing the individual xy-plane images. 2p excitation (780nm) and the 63Å~ glycerol immersion objective (NA 1.3, WD 280μm) were used. (MOV 7059 kb)
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).
The video first shows a series of xy-plane images through the 3D reconstruction data (6,695 Å~ 5,364 Å~ 968 μm volume; step-size=10 μm; anterior to posterior), and then shows 3D rendering. 1p excitation (514nm and 594nm) and the 10Å~ objective (NA 0.3, WD 3.6 mm) were used for imaging. Note that non-specific TH signals on pial surface are caused by using whole antiserum TH antibody (ab113, Abcam, Cambridge, MA). When immunogen affinity-purified TH antibody (ab51191, Abcam, Cambridge, MA) was used on the same tissue, no nonspecific signals were seen. (MOV 5701 kb)
3D visualization of the 1mm-thick coronal block shown in Video 9 after the antibody elution process.
The video first shows a series of xy-plane images through the 3D reconstruction data (6,679 Å~5,361 Å~ 1,018 μm volume; step-size=10 μm; from anterior to posterior), and then shows 3D rendering. The imaged antibodies were eluted from the block by incubating in 4% SDS solution at 60°C for 0.5 day. Note that TH signal is completely removed while the fluorescence signal of eYFP is retained. 1p excitation (514nm and 594nm) and the 10Å~ objective (NA 0.3, WD 3.6 mm) were used for imaging. (MOV 5619 kb)
3D visualization of 2nd round immunostaining on the same 1mm-thick coronal block shown in Videos 9 and 10.
The video first shows a series of xy-plane images through the 3D reconstruction data (6,662 Å~ 6,000 Å~ 948 μm volume; step-size=10 μm; from anterior to posterior), and then shows 3D rendering. The block of tissue was immunostained for PV (red) and GFAP (blue). DAPI (white) was used to counterstain nuclei. Note that DAPI staining is possible after the elution process. 1p excitation (514nm and 647nm) were used for imaging eYFP and PV. 2p excitation (780nm) was used for imaging DAPI and GFAP. (MOV 5983 kb)
3D visualization of 3rd round immunostaining on the same tissue shown in Videos 9-11.
The video first shows a series of xy-plane images through the 3D reconstruction data (6,690 Å~ 5,354 Å~ 1028 μm volume; step-size=10 μm; from anterior to posterior), and then shows 3D rendering. The block of tissue was immunostained for TH (red) and ChAT (blue). The pattern and signal intensity of TH staining is very similar between the 1st round and 3rd round (see Fig. 4). This demonstrates that antigens and antigenicity are well-preserved after two sequential antibody elution processes. 1p excitation (514nm, 594nm, and 647nm) and the 10Å~ objective (NA 0.3, WD 3.6 mm) were used for imaging. (MOV 5860 kb)
Hippocampus region showing networks of YFP-expressing neurons (green), distribution of PV-positive neurons (red) and astrocytes (blue).
The video first shows a series of xy-plane images through the 3D reconstruction data (2,962 Å~ 2,737 Å~ 940 μm volume, step-size=10 μm, from anterior to posterior), and then shows 3D rendering. (MOV 9061 kb)
Animation illustrating single axonal tracing in intact postmortem human brain tissue shown in Fig. 5.
A 500 μm-thick intact block of the frontal lobe (BA 10) of postmortem human brain (autism case, #AN13961; age, 7 years; sex, male; storage, 82 months in 10% formalin at room temperature) was clarified and immunostained for neurofilament protein. The stained block was imaged using 2p-excitation (780nm) and a 25x water immersion objective (NA = 0.95, working distance = 2.4 mm). A single axon crossing the volume (1,083 Å~ 1,079 Å~ 470 μm; step-size=0.49μm) from left bottom corner to top right corner was traced and highlighted by red line. (MOV 13157 kb)
3D Visualization of PV-positive neurons in the neocortex of the autism case shown in Fig. 5.
A 500 μm-thick intact block of the postmortem human brain tissue was clarified and immunostained for PV.The stained block was imaged using 2p-excitation (780nm) and the 10x water immersion objective (NA 0.3, WD 3.6 mm). The video (6,708 Å~ 4,713 Å~ 509 μm; step size=9.98 μm) shows overall distribution of PV-positive neurons in all layers of cortex (from the pial surface to white matter) and zooms into layer 6. (MOV 8892 kb)
Animation illustrating tracing of the PV-positive neurons in layer 6 with abnormal dendritic bridging shown in Fig. 5.
A subregion (1,083 Å~ 1,079 Å~ 470 μm; step-size=0.47μm) of the tissue shown in Video 14 was imaged using the 25x objective. The video shows a series of xy-optical slices (side view) as well as the traced neurons. The neurons were manually traced using Imaris software (Bitplane). (MOV 12175 kb)
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Chung, K., Wallace, J., Kim, SY. et al. Structural and molecular interrogation of intact biological systems. Nature 497, 332–337 (2013). https://doi.org/10.1038/nature12107
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