Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain

Journal name:
Nature Neuroscience
Volume:
14,
Pages:
1481–1488
Year published:
DOI:
doi:10.1038/nn.2928
Received
Accepted
Published online
Corrected online

Abstract

Optical methods for viewing neuronal populations and projections in the intact mammalian brain are needed, but light scattering prevents imaging deep into brain structures. We imaged fixed brain tissue using Scale, an aqueous reagent that renders biological samples optically transparent but completely preserves fluorescent signals in the clarified structures. In Scale-treated mouse brain, neurons labeled with genetically encoded fluorescent proteins were visualized at an unprecedented depth in millimeter-scale networks and at subcellular resolution. The improved depth and scale of imaging permitted comprehensive three-dimensional reconstructions of cortical, callosal and hippocampal projections whose extent was limited only by the working distance of the objective lenses. In the intact neurogenic niche of the dentate gyrus, Scale allowed the quantitation of distances of neural stem cells to blood vessels. Our findings suggest that the Scale method will be useful for light microscopy–based connectomics of cellular networks in brain and other tissues.

At a glance

Figures

  1. Tissue clearing performance of ScaleA2.
    Figure 1: Tissue clearing performance of ScaleA2.

    (a) Transmission curves of ScaleA2 (blue), 60% sucrose/PBS (green), FocusClear (yellow) and MountClear (magenta). (b) Transmission curves of fixed brain slices (1.5 mm thick) in ScaleA2 (blue), 60% sucrose/PBS (green), Focus/MountClear (magenta, a slice treated with FocusClear was placed in MountClear) and PBS (violet) after treatment with the respective solutions. (c,d) A whole fixed and cleared brain of a mouse (P15) after treatment with ScaleA2 for 2 weeks. (c) A photo was taken with a black and white pattern as background. (d) The green light from a 1-mW, 532-nm laser beam pointer traversed the cleared brain. (e) A photo of two embryos (E13.5) taken with a black and white pattern as background. Left, embryo placed in PBS after fixation with 4% PFA. Right, embryo incubated in ScaleA2 solution for 2 weeks after fixation with 4% PFA. (fy) Characterization of the expansion of macroscopic structures in fixed brain slices of a YFP-H mouse during ScaleA2 treatment. A coronal slice (1 mm thick) containing the hippocampus was prepared from a 9-week-old mouse. The slice was split into two halves and the right half was incubated in ScaleA2 solution for 5 d while the left half was incubated in PBS. Before (0 d, fi) and 1 d (jm), 2 d (nq) or 5 d (ru) after these incubations, the pair of slices on a coverslip with a patterned background were imaged using a fluorescence stereomicroscope for transmission (f, i, j, m, n, q, r and u) and YFP fluorescence (g, h, k, l, o, p, s and t). The slice became transparent and expanded after a 1–2-d incubation in ScaleA2 solution (l, m, p, q, t and u). The extent of the linear expansion was calculated as 1.28. Ag, amygdala; Cp, cerebral peduncle (basal part); Cx, cortex; Dmn, dorsomedial nucleus; Hf, hippocampal formation; Pmc, posteromedial cortical amygdala nucleus. The outlines of the slices and their internal structures at 0 d and 5 d were drawn with blue and orange, respectively. The outlines of the PBS-treated slice at 0 d and 5 d overlapped substantially (v). Reduced drawings of the outlines of the ScaleA2-treated slice at 5 d also overlapped with the outlines at 0 d extensively (w). In addition, the outlines of the ScaleA2-treated half (green) at 0 d were inverted and overlaid to the outlines of the PBS-treated half (magenta) at 0 d. As the brain slice had been split slightly asymmetrically, the edges of each half were not precisely even, but proper alignment was achieved (x). A similar overlay was done between the size-normalized outlines at 5 d (y). In x and y, the difference between green and red traces indicates the inherent baseline left/right asymmetry of the slice. Notably, the degree and distribution of the asymmetry are almost identical between x and y. All scale bars represent 5 mm.

  2. Comparison of ScaleA2 with BABB.
    Figure 2: Comparison of ScaleA2 with BABB.

    (a,b) Sensitivity of EGFP fluorescence to ScaleA2 solution and a conventional chemical clearing reagent (BABB). Cultured HeLa cells expressing EGFP were fixed with 4% PFA and were time-lapse imaged while being exposed to ScaleA2 solution (a) or BABB following dehydration with ethanol and hexane (b). Replacement of Hanks' Balanced Salt Solution with ScaleA2 resulted in a change in focus and a slight decrease in fluorescence intensity. (c) Fluorescence images comparing the preservation of YFP signals between aqueous (left) and chemical (right) clearing agents. The brain of a YFP-H mouse (7 weeks old) was split into two halves. The left half was treated with ScaleA2 for 3 d. The right half was treated with BABB after dehydration. Then slices (1 mm thick) were prepared and imaged for fluorescence with a stereomicroscope. The original shape of the fixed brain is drawn with broken lines. Scale bar represents 5 mm.

  3. Three-dimensional reconstructions of YFP-expressing neurons in
                    ScaleA2-treated brain samples of YFP-H mice.
    Figure 3: Three-dimensional reconstructions of YFP-expressing neurons in ScaleA2-treated brain samples of YFP-H mice.

    The actual imaging depth is shown in parentheses. Unsectioned brains (am) and an excised hippocampus (n,o) were imaged. (ac) TPEFM imaging using a 25× objective (XLPLN25XWMP, numerical aperture (NA) = 1.05, working distance = 2.0 mm). The experimental setup for TPEFM imaging using the commercially available objective is shown in a. A three-dimensional reconstruction of YFP-expressing neurons in 16 (8 × 2) quadratic prisms located in the cerebral cortex and hippocampus is shown in b. A high-magnification xy image at a depth of 0.9 mm (a yellow box in b) is shown in c. (dk) Three-dimensional reconstruction of YFP-expressing neurons in a quadratic prism located in the cerebral cortex. The same brain region was imaged using a 20× objective (W-PlanApochromat, NA = 1.0, working distance = 2.0 mm) and taking both two-photon (920-nm excitation, dg) and one-photon (514-nm excitation, hk) approaches. For each volume rendering, three xy images at different z positions (df and ik) are presented. (lo) TPEFM imaging using a custom-designed objective with a working distance of 4.0 mm. The experimental setup for TPEFM imaging using the objective lens is shown in l and n. Three-dimensional reconstructions of YFP-expressing neurons in a quadratic prism located in the cerebral cortex and hippocampus (m) and in 24 (4 × 6) quadratic prisms located in the excised hippocampus (o) are shown. DG, dentate gyrus; GCL, granule cell layer; ML, molecular layer. All scale bars represent 50 μm.

  4. Visualization of labeled callosal connections in the intact mouse
                    brain.
    Figure 4: Visualization of labeled callosal connections in the intact mouse brain.

    A population of layer II/III pyramidal neurons was labeled by in utero electroporation of plasmids encoding EYFP into the dorsal ventricular zone on the right side (R) of the mouse forebrain at E15.5, and their axonal projections into the left side (L) were visualized at P10 using a macro-zoom confocal microscope after fixation and a 7-d treatment with ScaleA2. CC, corpus callosum; CPu, caudate putamen; Cx, cortex; HC, hippocampus; LV, lateral ventricle; ML, midline; TM, thalamus. (a) We acquired 18 confocal images (52-μm steps) using a 1× objective lens at scanner zoom 3×, and z stacked them. (b) We acquired 17 confocal images (43-μm steps) using a 2× objective lens at scanner zoom 2×, and z stacked them. (c) We acquired 34 confocal images (10.8 μm steps) using a 2× objective lens at scanner zoom 4×, and z stacked them. All scale bars represent 500 μm.

  5. Quantitation of the distances between proliferating neural stem cell (PNSC)
                    nuclei and blood vessels in the subgranular zone (SGZ) of adult mice.
    Figure 5: Quantitation of the distances between proliferating neural stem cell (PNSC) nuclei and blood vessels in the subgranular zone (SGZ) of adult mice.

    (ac) Visualization of GFP-labeled neural stem cells (NSCs) and Texas Red–labeled blood vessels in the adult mouse hippocampus. A schematic diagram showing the approach of TPEFM imaging (red arrow) to a cleared excised hippocampus is presented in a. The imaged area is shown by six quadratic prisms. A high-magnification volume rendering of NSCs (green) and blood vessels (red) in the SGZ is shown in b. Volume renderings generated from a large region in the hippocampus are shown in c. Five perspective views were created from different angles (D, dorsal; V, ventral; C, caudal; R, rostral; F, front). GCL, granule cell layer; ML, molecular layer. (dh) Hippocampi were excised from the fixed brains of #504 mice (7 weeks old) and cleared with ScaleA2 for 2 d. An excised hippocampus for TPEFM imaging is shown in d. The quadratic prism that was approached from the surface (red arrow) is shown. The objective was placed so that the z axis came into contact with the apex of the hilus. A series of perspective images of PNSC nuclei (green) and blood vessels (red) when tunneling into the hippocampus are shown in e. Backward perspective images were created at different depths. After passing through the SGZ, no PNSC nuclei were seen ahead. These images are animated in Supplementary Video 4. RINZO automatically calculated the distance (white lines) from each PNSC nucleus (green) to the nearest blood vessel (red) surface (f). Histograms show the distribution of distances to blood vessels for all SGZ cell nuclei (violet), and for PNSC nuclei (green). Cell numbers (g) or their frequencies (h) were plotted. The real distance is shown in parentheses. Scale bars represent 500 μm (b,c) and 20 μm (e,f).

  6. Three-dimensional reconstructions of Fucci transgenic mouse embryos treated
                    with ScaleU2.
    Figure 6: Three-dimensional reconstructions of Fucci transgenic mouse embryos treated with ScaleU2.

    (ac) Green and red signals are derived from the Fucci-S/G2/M marker and Fucci-G1/G0 marker, respectively. Transgenic mouse #596/#504 embryos (E11.5 and E13.5) were fixed with 4% PFA/PBS and then incubated in ScaleU2 for 6 months. The right halves of their bodies (heads) were imaged using macro-zoom LSCM (AZ-C1) equipped with a 2× objective lens (AZ-PlanFluor, NA = 0.2, working distance = 45 mm). The z step size was 5 μm. We used 488-nm and 561-nm laser diodes. Shown are maximum intensity projection (MIP) images at E11.5 (a) and E13.5 (b). A confocal image of the region indicated by a white box in the MIP image (b) is shown in c. (di) Immunohistochemical localization of Nestin (df) or PSA-NCAM (gi) on sections of the posterior end of the diencephalon of an E13.5 #504 transgenic embryo producing mAG-hGem(1/110). The immunostaining and mAG-hGem(1/110) signals are shown in white and green, respectively. High-magnification images of the regions indicated by yellow boxes in e and h are shown in f and i, respectively. IC, inferior colliculus; V, ventricle. Scale bars represent 1 mm (ac) and 100 μm (di).

  7. Immunohistochemistry on sections restored from ScaleA2.
    Figure 7: Immunohistochemistry on sections restored from ScaleA2.

    (af) A brain sample from the thy1-YFP mouse line H (7 weeks old) was used. Sections of the dentate gyrus were prepared from a fixed sample (ac) and a sample restored from ScaleA2 (df). Samples were stained with a mouse monoclonal antibody to PSA-NCAM. The YFP fluorescence and immunoreactivity for PSA-NCAM (with a secondary antibody conjugated to Alexa Fluor 546) were visualized. (gl) A brain sample from wild-type mouse (7 weeks old) was used. Sections of the CA3 region were prepared from a fixed sample (gi) and a sample restored from ScaleA2 (jl). Samples were stained with a rabbit polyclonal antibody to GluR1 and a mouse monoclonal antibody to synaptophysin. These primary antibodies were visualized with secondary antibodies conjugated to Alexa Fluor 488 and 546, respectively (Molecular Probes). Scale bars represent 20 μm.

Change history

Corrected online 13 October 2011
In the HTML version of this article initially published online, Greek μ characters were misformatted as the letter m and a prime sign was omitted. The errors have been corrected in the HTML version of this article.

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Author information

Affiliations

  1. Brain Science Institute, RIKEN, Wako-city, Saitama, Japan.

    • Hiroshi Hama,
    • Hiroshi Kurokawa,
    • Hiroyuki Kawano,
    • Ryoko Ando,
    • Tomomi Shimogori,
    • Hisayori Noda,
    • Asako Sakaue-Sawano &
    • Atsushi Miyawaki
  2. School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, Japan.

    • Hiroshi Kurokawa &
    • Kiyoko Fukami
  3. Life Function and Dynamics, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Wako-city, Saitama, Japan.

    • Hiroyuki Kawano,
    • Asako Sakaue-Sawano &
    • Atsushi Miyawaki
  4. Tokyo Institute of Technology, Meguro-ku, Tokyo, Japan.

    • Hisayori Noda

Contributions

H.H. and A.M. conceived and designed the study. H.H. performed all the experiments and analyzed the data. H. Kurokawa devised the algorithms and analyzed the data. H. Kawano constructed the TPEFM system. R.A. performed in vitro experiments using fluorescent proteins. T.S. designed and performed the experiments that imaged callosal connections. H.N. refined the algorithms. K.F. contributed to data analysis. A.S.-S. performed the experiments using Fucci transgenic mouse embryos. A.M. supervised the project and wrote the manuscript with the help of H.H.

Competing financial interests

H. Hama, H. Kurokawa, H. Kawano, R. Ando and A. Miyawaki hold the patent for the Scale technique.

Corresponding author

Correspondence to:

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Supplementary information

PDF files

  1. Supplementary Text and Figures (3M)

    Supplementary Figures 1–9 and Table 1

Movies

  1. Supplementary Video 1 (5.3M)

    Visualizing the 3D architecture of neuronal networks comprised of YFP-expressing neurons in a long quadratic prism (2 mm). A series of X − Y images through the 3D reconstruction data (500 × 500 × 2,000 μm volume) from the cerebral surface to the hippocampus of the YFP-H mouse (13 weeks old). TPEFM with a non-descanned detector and a 20× objective (NA 1.0, WD 2.0 mm) was used.

  2. Supplementary Video 2 (11.7M)

    Visualizing the 3D architecure of neuronal networks comprised of YFP-expressing neurons in a very long quadratic prism (4 mm). A series of X−Y images through the 3D reconstruction data (500 × 500 × 4,000 μm volume) from the cerebral surface to the dentate gyrus of the YFP-H mouse (13 weeks old). TPEFM with a non-descanned detector and a custom designed 25× objective lens (NA 1.0, WD 4.0 mm) was used.

  3. Supplementary Video 3 (2.3M)

    YFP-labeled pyramidal neurons in layers II and III in the right hemisphere and their callosal axons travelling into the left hemisphere. A series of X−Y images through the 3D reconstruction data (10 × 10 × 0.75 mm volume) from anterior to posterior of a brain (10 days old) containing the corpus callosum. A population of layer II/III pyramidal neurons on the right side is highlighted with EYFP fluorescence. A macro zoom confocal microscopy system was used.

  4. Supplementary Video 4 (2.9M)

    Nuclei of proliferating neural stem cells exclusively localized in the subgranular zone in association with a network of blood vessels. Animation (zooming in) of 3D image data (500 × 500 × 1,400 μm volume) in the hippocampal dentate gyrus of a #504 adult (7 weeks old) mouse extensively labeled with Texas Red-labeled lectin. Red, blood vessels; Green, nuclei of proliferating neural stem cells (PNSC) emitting mAG-hGem(1/110) fluorescence. TPEFM with two non-descanned detectors was used.

Additional data