Structural and molecular interrogation of intact biological systems

Journal name:
Nature
Volume:
497,
Pages:
332–337
Date published:
DOI:
doi:10.1038/nature12107
Received
Accepted
Published online

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.

At a glance

Figures

  1. CLARITY.
    Figure 1: CLARITY.

    Tissue is crosslinked with formaldehyde (red) in the presence of hydrogel monomers (blue), covalently linking tissue elements to monomers that are then polymerized into a hydrogel mesh (followed by a day-4 wash step; Methods). Electric fields applied across the sample in ionic detergent actively transport micelles into, and lipids out of, the tissue, leaving fine-structure and crosslinked biomolecules in place. The ETC chamber is depicted in the boxed region (Supplementary Fig. 2).

  2. Intact adult mouse brain imaging.
    Figure 2: Intact adult mouse brain imaging.

    Imaging was performed in adult mouse brains (3 months old). a, Cajal quote before CLARITY. b, Cajal quote after CLARITY: Thy1–eYFP line-H mouse brain after hydrogel–tissue hybridization, ETC and refractive-index matching (Methods). c, Fluorescence image of brain depicted in b. d, Dorsal aspect is imaged (single-photon (1p) microscopy), then brain is inverted and ventral aspect imaged. e, Three-dimensional rendering of clarified brain imaged (×10 water-immersion objective; numerical aperture, 0.3; working distance, 3.6mm). Left, dorsal half (stack size, 3,100μm; step size, 20μm). Right, ventral half (stack size, 3,400μm; step size, 20μm). Scale bar, 1mm (Supplementary Videos 3, 4, 5). f, Non-sectioned mouse brain tissue showing cortex, hippocampus and thalamus (×10 objective; stack size, 3,400μm; step size, 2μm). Scale bar, 400μm (Supplementary Video 2). gl, Optical sections from f showing negligible resolution loss even at ~3,400-μm deep: z = 446μm (g, h), z = 1,683μm (i, j) and z = 3,384μm (k, l). h, j and l, boxed regions in g, i and k, respectively. Scale bars, 100μm. m, Cross-section of axons in clarified Thy1–channelrhodopsin2 (ChR2)–eYFP striatum: membrane-localized ChR2–eYFP (1-mm-thick coronal block; ×63 glycerol-immersion objective; numerical aperture, 1.3; working distance, 280μm). Scale bar, 5μm. n, Dendrites and spines of neurons in clarified Thy1–eYFP line-H cortex (1-mm-thick coronal block; ×63 glycerol objective). Scale bar, 5μm.

  3. Molecular phenotyping in intact tissue.
    Figure 3: Molecular phenotyping in intact tissue.

    a, Protein loss in clarified mouse brain compared to conventional methods (see Supplementary Information for more details); error bars denote s.e.m.; n = 4 for each condition. b, Rendering of a 1-mm-thick non-sectioned coronal block of Thy1–eYFP mouse brain immunostained for GFP. The tissue was ETC-cleared (1 day), immunostained (3 days) and imaged (×10 water-immersion objective; single-photon excitation). Left, eYFP (green); middle, anti-GFP (red); right, overlay. Scale bar, 500μm (Supplementary Video 6). c, Three-dimensional rendering of the boxed region in the cortex in b shows eYFP fluorescence (left) and anti-GFP staining (right). d, Left, co-localization: Manders overlap coefficient plotted versus depth43. Right, optical sections at different depths in three-dimensional rendering. Scale bar, 100μm. e, f, 500-μm-thick block of line-H mouse brain (2 months old) clarified for 1day and immunostained for synapsin I (red) and PSD-95 (blue) for 3days (Methods) (×63 glycerol objective; single-photon excitation). e, Left, optical sections (z = 20μm, z = 200μm). Right, enlarged images of boxed regions on left. Individual synaptic puncta resolved throughout depth. White depicts eYFP staining. f, Average immunofluorescence cross-section of PSD-95 puncta at z = 20μm (top) and z = 200μm (bottom). g, Full width at half maximum (FWHM) of average immunofluorescence cross-section of PSD-95 puncta versus depth. Insets, average puncta at z = 20μm and z = 200μm. h, Hippocampal staining. Left, GABA; middle, parvalbumin (PV); right, overlay. 500-μm-thick block of wild-type mouse brain (3 months) clarified (1 day) and immunostained (3 days) (×25 water-immersion objective; numerical aperture, 0.95; working distance,2.5mm; single-photon excitation). Scale bar, 20μm. i, in situ hybridization. Clarified 500-μm mouse brain block showing dopamine receptor D2 (Drd2) mRNA in the striatum. LV, lateral ventricle. Blue, DAPI. 50-base-pair RNA probes for Drd2 visualized with FastRed (×25 water-immersion objective; single-photon excitation (555nm) for FastRed, two-photon excitation (720nm) for DAPI). Scale bars: left, 100μm; right, 20μm. j, k, Axonal fibres of tyrosine hydroxylase (TH)-positive neurons in the nucleus accumbens (NAc) and caudate–putamen (CPu). j, Three-dimensional rendering of 1-mm-thick clarified mouse brain block stained for tyrosine hydroxylase (red) and DAPI (green). aca, anterior commissure. Scale bar, 500μm. k, Maximum projection, NAc/aca volume in j. Scale bar, 50μm.

  4. Multi-round molecular phenotyping of intact tissue.
    Figure 4: Multi-round molecular phenotyping of intact tissue.

    a, First round. Rendering of 1-mm-thick Thy1–eYFP block immunostained for tyrosine hydroxylase in non-sectioned form. ETC-cleared (1 day) and immunostained (6 days). Scale bar, 500μm (Supplementary Video 10). b, Antibodies eluted from block in a (4% SDS, 60 °C for 0.5days). Tyrosine hydroxylase signal was removed and eYFP fluorescence retained (Supplementary Video 11). c, Second round. Three-dimensional rendering of same block now immunostained for parvalbumin (red), glial fribrillary acidic protein (GFAP) (blue) and DAPI (white) (Supplementary Video 12). df, Maximum projections of 100μm volume of yellow-boxed regions in a, b and c, respectively. eYFP-positive neurons preserved. cp, cerebral peduncle; SNR, substantia nigra. Scale bar, 100μm. g, Optical section of white/dotted-box region in c showing DAPI. CA, cornu ammonis; DG, dentate gyrus. Scale bar, 100μm. h, i, Tyrosine hydroxylase channel of white box regions in a (h) and j (i). Tyrosine hydroxylase antigenicity preserved through multiple elutions. Scale bar, 100μm. j, Third round. Block in ac immunostained for tyrosine hydroxylase (red) and choline acetyltransferase (ChAT) (blue) (Supplementary Video 13). k, Three-dimensional view of hippocampus in c showing eYFP-expressing neurons (green), parvalbumin-positive neurons (red) and GFAP (blue). Alv, alveus. Scale bar, 200μm (Supplementary Video 14).

  5. Human brain structural/molecular phenotyping.
    Figure 5: Human brain structural/molecular phenotyping.

    Human BA10 500-μm-thick intact blocks clarified (1day) and immunostained (3days) (×25 water-immersion objective). a, Optical section: myelin basic protein (MBP) and parvalbumin staining. White arrowheads indicate membrane-localized myelin basic protein around parvalbumin-positive projections. Scale bar, 10μm. b, Tyrosine hydroxylase and parvalbumin staining (maximum projection; 120μm volume; step size, 0.5μm). Scale bar, 50μm. c, Optical section: neurofilament (NP) and GFAP. Scale bar, 20 μm. d, Somatostatin and parvalbumin staining (maximum projection; 63μm volume; step size, 0.5μm). Scale bar, 20μm. e, Rendering of neurofilament-positive axonal fibres. Red, traced axon across volume. Scale bar, 500μm. Inset: boxed region. Scale bar, 20μm (Supplementary Video 15). f, Visualization of parvalbumin-positive neurons in the neocortex of autism case; layers identified as described in ref. 44. Scale bar, 500μm (Supplementary Video 16). g, Yellow-boxed region in f showing parvalbumin-positive cell bodies and fibres in layers 4, 5 and 6. Three representative parvalbumin-positive interneurons in layer 6 with ladder-shaped hetero- or iso-neuronal connections were traced (green, purple, blue). Scale bar, 100μm (Supplementary Video 17). h, Three-dimensional rendering of abnormal neurons in g; yellow arrowheads (1, 2) indicate ladder-shaped structures shown below in i and k. Scale bar, 80μm. i, Zoomed-in maximum projection of 8μm volume showing morphology of ladder-shaped structure formed by neurites from a single neuron. Scale bar, 10μm. j, Tracing of structure in i. k, Maximum projection of 18μm volume showing ladder-shaped structure formed by neurites from two different neurons. Scale bar, 10μm. l, Tracing of structure in k. m, Iso- and hetero-neuronal dendritic bridges per neuron. Neurons selected randomly and traced in software (Methods); dendritic bridges were manually counted. **P<0.05; error bars denote s.e.m. n = 6 neurons for both superficial and deep layers of autism case and n = 4 neurons for both superficial and deep layers of control case. n, Three-dimensional reconstruction of a neuron in layer 2 (superficial) of the autism case. Typical avoidance of iso-dendritic contact was observed.

Videos

  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.
    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.
    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.
  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.
    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.
    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.
  3. 3D visualization of YFP-expressing neuronal circuit elements in the ventral half of the intact Thy-1:eYFP mouse brain (16 weeks old).
    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).
    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).
  4. Raw data from Supplementary Video 1
    Video 4: 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.
  5. Brainstem region of the raw data from Supplementary Video 2
    Video 5: 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.
  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).
    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).
    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.
  7. TH staining of the intact mouse brain.
    Video 7: 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.
  8. TH-positive fibers in the amygdala of the mouse brain.
    Video 8: 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.
  9. MAP2-positive processes and neuronal cell bodies in the DG of the mouse brain.
    Video 9: 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.
  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).
    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).
    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.
  11. 3D visualization of the 1mm-thick coronal block shown in Video 9 after the antibody elution process.
    Video 11: 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.
  12. 3D visualization of 2nd round immunostaining on the same 1mm-thick coronal block shown in Videos 9 and 10.
    Video 12: 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.
  13. 3D visualization of 3rd round immunostaining on the same tissue shown in Videos 9-11.
    Video 13: 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.
  14. Hippocampus region showing networks of YFP-expressing neurons (green), distribution of PV-positive neurons (red) and astrocytes (blue).
    Video 14: 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.
  15. Animation illustrating single axonal tracing in intact postmortem human brain tissue shown in Fig. 5.
    Video 15: 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.
  16. 3D Visualization of PV-positive neurons in the neocortex of the autism case shown in Fig. 5.
    Video 16: 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.
  17. Animation illustrating tracing of the PV-positive neurons in layer 6 with abnormal dendritic bridging shown in Fig. 5.
    Video 17: 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).

References

  1. Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 7376 (1990)
  2. Carmeliet, P. & Tessier-Lavigne, M. Common mechanisms of nerve and blood vessel wiring. Nature 436, 193200 (2005)
  3. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nature Methods 2, 932940 (2005)
  4. Denk, W. & Horstmann, H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2, e329 (2004)
  5. Micheva, K. D. & Smith, S. J. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55, 2536 (2007)
  6. Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 5662 (2007)
  7. Li, A. et al. Micro-optical sectioning tomography to obtain a high-resolution atlas of the mouse brain. Science 330, 14041408 (2010)
  8. Botcherby, E. J. et al. Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates. Proc. Natl Acad. Sci. USA 109, 29192924 (2012)
  9. DeFelipe, J. From the connectome to the synaptome: an epic love story. Science 330, 11981201 (2010)
  10. Petersen, C. C. H. The functional organization of the barrel cortex. Neuron 56, 339355 (2007)
  11. Kasthuri, N. & Lichtman, J. W. The rise of the “projectome”. Nature Methods 4, 307308 (2007)
  12. Lichtman, J. W., Livet, J. & Sanes, J. R. A technicolour approach to the connectome. Nature Rev. Neurosci. 9, 417422 (2008)
  13. Mombaerts, P. et al. Visualizing an olfactory sensory map. Cell 87, 675686 (1996)
  14. Insel, T. R. & Young, L. J. The neurobiology of attachment. Nature Rev. Neurosci. 2, 129136 (2001)
  15. Micheva, K. D., Busse, B., Weiler, N. C., O’Rourke, N. & Smith, S. J. Single-synapse analysis of a diverse synapse population: proteomic imaging methods and markers. Neuron 68, 639653 (2010)
  16. Bock, D. D. et al. Network anatomy and in vivo physiology of visual cortical neurons. Nature 471, 177182 (2011)
  17. Briggman, K. L., Helmstaedter, M. & Denk, W. Wiring specificity in the direction-selectivity circuit of the retina. Nature 471, 183188 (2011)
  18. Ragan, T. et al. Serial two-photon tomography for automated ex vivo mouse brain imaging. Nature Methods 9, 255258 (2012)
  19. Dodt, H. U., Leischner, U. & Schierloh, A. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nature Methods 4, 331336 (2007)
  20. Ertürk, A., Mauch, C., Hellal, F. & Förstner, F. Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Nature Med. 18, 166171 (2012)
  21. Hama, H. et al. Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nature Neurosci. 14, 14811488 (2011)
  22. Cheong, W., Prahl, S. & Welch, A. A review of the optical properties of biological tissues. IEEE J. Quant. Electron. 26, 21662185 (1990)
  23. Sykova, E. & Nicholson, C. Diffusion in brain extracellular space. Physiol Rev. 88, 12771340 (2008)
  24. Becker, K., Jährling, N., Saghafi, S., Weiler, R. & Dodt, H.-U. Chemical clearing and dehydration of GFP expressing mouse brains. PLoS ONE 7, e33916 (2012)
  25. Porrero, C., Rubio-Garrido, P., Avendaño, C. & Clascá, F. Mapping of fluorescent protein-expressing neurons and axon pathways in adult and developing Thy1-eYFP-H transgenic mice. Brain Res. 1345, 5972 (2010)
  26. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 4151 (2000)
  27. Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nature Methods 2, 905909 (2005)
  28. Drobizhev, M., Makarov, N. S., Tillo, S. E., Hughes, T. E. & Rebane, A. Two-photon absorption properties of fluorescent proteins. Nature Methods 8, 393399 (2011)
  29. Goldenthal, K. L., Hedman, K., Chen, J. W., August, J. T. & Willingham, M. C. Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins. J. Histochem. Cytochem. 33, 813820 (1985)
  30. Tramu, G. An efficient method of antibody elution for the successive or simultaneous localization of two antigens by immunocytochemistry. J. Histochem. Cytochem. 26, 322324 (1978)
  31. Wählby, C., Erlandsson, F., Begntsson, E. & Zetterberg, A. Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei. Cytometry 71, 3241 (2002)
  32. Kolodziejczyk, E. & Baertschi, A. J. Multiple immunolabeling in histology: a new method using thermo-inactivation of immunoglobulins. J. Histochem. Cytochem. 34, 17251729 (1986)
  33. Zhou, Q., Homma, K. J. & Poo, M. Shrinkage of dendritic spines associated with long-term depression of hippocampal synapses. Neuron 44, 749757 (2004)
  34. Harlow, M. L., Ress, D., Stoschek, A., Marshall, R. M. & McMahan, U. J. The architecture of active zone material at the frog’s neuromuscular junction. Nature 409, 479484 (2001)
  35. Chen, X. et al. Organization of the core structure of the postsynaptic density. Proc. Natl Acad. Sci. USA 105, 44534458 (2008)
  36. Macdonald, R. Zebrafish immunohistochemistry. Methods Mol. Biol. 127, 7788 (1999)
  37. Sporns, O., Tononi, G. & Kötter, R. The human connectome: a structural description of the human brain. PLOS Comput. Biol. 1, e42 (2005)
  38. Wickersham, I. R., Finke, S., Conzelmann, K.-K. & Callaway, E. M. Retrograde neuronal tracing with a deletion-mutant rabies virus. Nature Methods 4, 4749 (2007)
  39. Miyamichi, K. et al. Cortical representations of olfactory input by trans-synaptic tracing. Nature 472, 191196 (2011)
  40. Matthews, B. J. et al. Dendrite self-avoidance is controlled by Dscam. Cell 129, 593604 (2007)
  41. Lefebvre, J. L., Kostadinov, D., Chen, W. V., Maniatis, T. & Sanes, J. R. Protocadherins mediate dendritic self-avoidance in the mammalian nervous system. Nature 488, 517521 (2012)
  42. Morrow, E. M. et al. Identifying autism loci and genes by tracing recent shared ancestry. Science 321, 218223 (2008)
  43. Dunn, K. W., Kamocka, M. M. & McDonald, J. H. A practical guide to evaluating colocalization in biological microscopy. Am. J. Physiol. Cell Physiol. 300, C723C742 (2011)
  44. Condé, F., Lund, J. S., Jacobowitz, D. M., Baimbridge, K. G. & Lewis, D. A. Local circuit neurons immunoreactive for calretinin, calbindin D-28k or parvalbumin in monkey prefrontal cortex: distribution and morphology. J. Comp. Neurol. 341, 95116 (1994)
  45. Cheng, C. M. et al. Biochemical and morphometric analyses show that myelination in the insulin-like growth factor 1 null brain is proportionate to its neuronal composition. J. Neurosci. 18, 56735681 (1998)
  46. Grossfeld, R. M. & Shooter, E. M. A study of the changes in protein composition of mouse brain during ontogenetic development. J. Neurochem. 18, 22652277 (1971)

Download references

Author information

Affiliations

  1. Department of Bioengineering, Stanford University, Stanford, California 94305, USA

    • Kwanghun Chung,
    • Jenelle Wallace,
    • Sung-Yon Kim,
    • Aaron S. Andalman,
    • Thomas J. Davidson,
    • Julie J. Mirzabekov,
    • Kelly A. Zalocusky,
    • Joanna Mattis,
    • Aleksandra K. Denisin,
    • Sally Pak,
    • Hannah Bernstein,
    • Charu Ramakrishnan,
    • Logan Grosenick &
    • Karl Deisseroth
  2. CNC Program, Stanford University, Stanford, California 94305, USA

    • Kwanghun Chung,
    • Sandhiya Kalyanasundaram,
    • Aaron S. Andalman,
    • Thomas J. Davidson,
    • Kelly A. Zalocusky,
    • Viviana Gradinaru &
    • Karl Deisseroth
  3. Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California 94305, USA

    • Karl Deisseroth
  4. Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA

    • Karl Deisseroth

Contributions

K.C. and K.D. conceived and designed the experiments and wrote the paper. K.C. led development of the CLARITY technology and its implementation. K.C. and S.K. clarified samples. K.C. imaged samples. K.C., S.-Y.K., S.K., J.W., K.A.Z., S.P., J.J.M., J.M., V.G. and H.B. prepared animals. J.W. performed tracing. T.J.D. and A.S.A. performed image processing. K.C., J.W., J.J.M. and A.K.D. wrote the CLARITY protocol. A.K.D. created AutoCAD drawings. K.C. and L.G. created Supplementary Videos. A.S.A. contributed to in situ and zebrafish data. C.R., L.G. and V.G. contributed to set-up of the relevant laboratory infrastructure. K.D. supervised all aspects of the work.

Competing financial interests

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)

Corresponding author

Correspondence to:

Author details

Supplementary information

Video

  1. 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. (7.76 MB, Download)
    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.
  2. 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. (11.11 MB, Download)
    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.
  3. 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). (12.17 MB, Download)
    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).
  4. Video 4: Raw data from Supplementary Video 1 (9.04 MB, Download)
    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.
  5. Video 5: Brainstem region of the raw data from Supplementary Video 2 (5.26 MB, Download)
    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.
  6. 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). (3.67 MB, Download)
    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.
  7. Video 7: TH staining of the intact mouse brain. (1.54 MB, Download)
    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.
  8. Video 8: TH-positive fibers in the amygdala of the mouse brain. (13.88 MB, Download)
    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.
  9. Video 9: MAP2-positive processes and neuronal cell bodies in the DG of the mouse brain. (6.89 MB, Download)
    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.
  10. 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). (5.56 MB, Download)
    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.
  11. Video 11: 3D visualization of the 1mm-thick coronal block shown in Video 9 after the antibody elution process. (5.48 MB, Download)
    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.
  12. Video 12: 3D visualization of 2nd round immunostaining on the same 1mm-thick coronal block shown in Videos 9 and 10. (5.84 MB, Download)
    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.
  13. Video 13: 3D visualization of 3rd round immunostaining on the same tissue shown in Videos 9-11. (5.72 MB, Download)
    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.
  14. Video 14: Hippocampus region showing networks of YFP-expressing neurons (green), distribution of PV-positive neurons (red) and astrocytes (blue). (8.84 MB, Download)
    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.
  15. Video 15: Animation illustrating single axonal tracing in intact postmortem human brain tissue shown in Fig. 5. (12.84 MB, Download)
    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.
  16. Video 16: 3D Visualization of PV-positive neurons in the neocortex of the autism case shown in Fig. 5. (8.68 MB, Download)
    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.
  17. Video 17: Animation illustrating tracing of the PV-positive neurons in layer 6 with abnormal dendritic bridging shown in Fig. 5. (11.88 MB, Download)
    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).

PDF files

  1. Supplementary Information (7.3 MB)

    This file contains Supplementary Figures 1-14 and Supplementary Tables 1-3.

Comments

  1. Report this comment #57396

    Ryuji Yamaguchi said:

    I am wondering if this technology can give us the precise locations of glucose uptake in the stimulated brain using NBDG and such. Currently we only have FDG-PET Scan which can provide us with live but fuzzy images.

Subscribe to comments

Additional data