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Supplementary Information

A Chemical Approach for Fluorescence Imaging and 3D

Reconstruction of Transparent Mouse Brain

Hiroshi Hama, Hiroshi Kurokawa, Hiroyuki Kawano, Ryoko Ando,

Tomomi Shimogori, Hisayori Noda, Kiyoko Fukami, Asako Sakaue-Sawano,

and Atsushi Miyawaki

Nature Neuroscience: doi:10.1038/nn.2928

PBS 4 M urea

Supplementary Figure 1


Before(0 d)

1 d

2 d

5 d

Transmission TransmissionYFP YFPPBS ScaleA2

Before (0 d)5 d

a b c d

m n o p

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a b c d Transmission GFP fluorescence Transmission



o p q r

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k l m nTransmission TransmissionFluoroMyelinTM Red

PBS ScaleA2




4 M urea8 M urea

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(10) (10) (10) (8) (20)

(6) (6) (6) (6) (6) (6) (6) (6) (6) (6)


thy1-GFP line M

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1P

514-nm Laser

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Galvano mirrors

34ch PMT array

Femtosecond

pulsed laser

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thy1-YFP line H (3 weeks old)

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Excised hippocampus of thy1-YFP line H (13 weeks old)

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thy1-GFP line M PSA-NCAM BLBP

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hilusMF

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thy1-GFP line M VGLUT1

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Supplementary Table1

transparency

preservation of fluorescence

volume change

sample property

speed

economy

reversibility (transferrability to IHC)

composition

long-term storage

++++

++++

expansion

soft, fragile

slow

almost free

reversible

disclosed

possible

++++

++++

slight expansion

soft

very slow

almost free

reversible

disclosed

possible

++++

+

shrinkage

hard

relatively slow

less expensive

irreversible

disclosed

?

++

++++

shrinkage

firm

fast

expensive

reversible

undisclosed

precipitates

ScaleA2 BABB FocusClearScaleU2


Legends of Supplementary Figures, Table, and Videos Supplementary Figure 1: Urea solution renders polyvinylidene fluoride (PVDF) membranes transparent. PVDF soaked in PBS (left) and 4 M urea (right) solutions for 3 minutes and photographed using a digital camera. Scale bar, 1 cm. Supplementary Figure 2: Characterization of the expansion of macroscopic structures in brain slices of a transgenic mouse line, thy1-YFP line H (YFP-H) during treatment with ScaleA2 solution. A coronal slice (1 mm thick) containing the striatum was prepared from a YFP-H mouse (9 weeks old). 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) (a–d) and 1 d (e–h), 2 d (i–l), or 5 d (m–p) after these incubations, the pair of slices on a coverslip with a patterned background was imaged using a fluorescence stereomicroscope for transmission (a, d, e, h, i, l, m, and p) and fluorescence from YFP (b, c, f, g, j, k, n, and o). The pattern can be seen in the image background. It is evident in these images that the ScaleA2-treated slice was transparent and expanded (g, h, k, l, o, and p). The extent of the linear expansion in this experiment was calculated 1.28. (b) Cx, cortex; CPu, caudate putamen (striatum); LV, lateral ventricle; LS, lateral septum nucleus; Tu, olfactory tubercle. The outlines of the slices as well as the white matter at 0 d and 5 d were drawn with blue and orange (q, r), respectively. The outlines of the PBS-treated slice at 0 d and 5 d overlapped substantially (q). Reduced drawings of the outlines of the ScaleA2-treated slice at 5 d also overlapped with the outlines at 0 d extensively (r). 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 (red) at 0 d (s). A similar overlay was done between the size-normalized outlines at 5 d (t). Scale bar: 5 mm. Supplementary Figure 3: Characterization of the expansion of macroscopic structures in thin brain slices of a transgenic mouse line, thy1-GFP line M (GFP-M) after treatment with ScaleA2 solution. A coronal section (50 µm thick) containing the hippocampus was prepared from a GFP-M mouse (30 weeks old). To highlight the


white matter, we treated the section with the myelin-selective lipophilic dye, FluoroMyelinTM Red (Molecular Probes) for 20 min. Then the section was split into two halves, and the right half was incubated in ScaleA2 solution for 16 hours while the left half was incubated in PBS. Before and after these incubations, each section on a nylon mesh (Cell Stainer, 70 µm, BD Biosciences) was imaged using a Leica fluorescence stereomicroscope (MZ16F) equipped with a 1× objective lens (PLANAPO, NA 0.141) and a cooled CCD camera (DP30, Olympus) for fluorescence from GFP and FluoroMyelinTM Red as well as transmission. Since it is possible to introduce distortion due to handling in the cleared thin section, the nylon mesh was used to pick up every brain section floating in solution when it needed to be transferred for image acquisition. Fluorescence images acquired before and after a 16-h treatment with ScaleA2 solution were colored red and green, respectively. The extent of the linear expansion (E) was calculated by comparing the distances across highlighted structures such as the hippocampus between the red and green images. Then the green image was reduced in size by 1 – 1/E and superimposed on the red image to create a merged image. Scale bars: 10 mm. (a-j) Fluorescently-labeled macroscopic structures. Before (a–d) and after (e–h) these incubations, the pair of sections on a nylon mesh was imaged using a fluorescence stereomicroscope for transmission (a, d, e, and h) and fluorescence from GFP (b, c, f, and g). The fluorescence images before and after the incubations are colored red (b and c) and green (f and g), respectively. The mesh pattern can be seen in the background of the transmission images. It is evident in these images that the ScaleA2-treated section was transparent and slightly expanded (g and h). A reduced copy of the green image (g) was superimposed on the red image (c) to create a merged image (j). Likewise, a merge image (i) was created from the red and green images of PBS-treated section (b and f, respectively). In both merged images (i and j), green and red signals coexist almost everywhere. (k–t) FluoroMyelinTM Red-labeled macroscopic structures. The brain sections shown in (a–j) were stained with the myelin-selective lipophilic dye, FluoroMyelinTM Red (Molecular Probes) for 20 min. As was observed for GFP fluorescence, fluorescence images for FluoroMyelinTM Red (l, m, p, and q) were acquired. The same transmission images (k, n, o, and r) as shown in (a, d, e, and h) are presented. A reduced copy of the green image (q) was superimposed on the red image (m) to create a merged image (t). Likewise, a merged image (s) was created from red and green images of PBS-treated sections (l and p, respectively). In both merged images (s and t), green and red signals coexist essentially everywhere.


Supplementary Figure 4: Resistance of fluorescent proteins to high concentrations of urea. Bar graphs summarize the quantitative analysis of fluorescence from fluorescent proteins. Data represent means ± S.D. with the number of samples or cells assayed in parentheses. (a) Stability of purified fluorescent proteins (EGFP, mAG1, YFP, DsRed, or mCherry) in high concentrations of urea. Protein (5 µg/ml) was incubated in 4 M urea (pH 7.8) or 8 M (pH 8.7) at room temperature for 5 min, and its fluorescence intensity was measured using a fluorometer (Synergy H4, Biotek). The intensity was normalized to the control value obtained in urea-free solution at pH 7.8 (HEPES/NaOH) or at pH 8.7 (Tricine/NaOH), respectively. (b) Resistance of fluorescent proteins (EGFP, mAG1, YFP, DsRed, or mCherry) inside fixed HeLa cells to high concentrations of urea. Experiments were performed as described in Methods. The intensity was normalized to the initial value. Supplementary Figure 5: Application of ScaleA2 and BABB to the whole fixed hemispheres of a transgenic mouse line, thy1-GFP line M (GFP-M). Fluorescence images that compare the capability to preserve GFP signals between an aqueous (left, ScaleA2) and an organic (right, BABB) clearing agents. A brain of the GFP-M mouse was split into left and right halves. The left half was treated with ScaleA2. The right half was treated with BABB after dehydration. Brain slices (1 mm thick) were prepared and imaged for their fluorescence. The original shape of the fixed brain is drawn with broken lines. Scale bar, 5 mm. Supplementary Figure 6: Schematic diagrams of the optical configurations for image acquisition. (a) TPEFM (two-photon excitation fluorescence microscopy) using a non-descanned detector. (b) LCSM (laser scanning confocal microscopy) using a descanned detector. PD, photodiode; P, pinhole; DC, dichroic mirror; G, grating; PMT, photomultiplier tube.


Supplementary Figure 7: Visualization of YFP-expressing neurons in the intact brain of thy1-YFP mouse line H after fixation and a 2- to 3-week treatment with ScaleA2. (a) The experimental setup for TPEFM imaging of an intact mose brain using an objective lens with a working distance of 3.0 mm is shown in the schematic diagram. (b) 3D reconstruction of YFP-expressing neurons in 16 (8 × 2) quadratic prisms located in the cerebral cortex and hippocampus. (c)The arrangement for TPEFM imaging of an excised hippocampus using an objective lens with a working distance of 4.0 mm is shown in the schematic diagram. (d) 3D reconstruction of YFP-expressing neurons in 24 (4 × 6) quadratic prisms located in the excised hippocampus; a high magnification image in the CA1 (yellow box) is shown in (e). SC, subiculum; MF, mossy fiber. The PMT sensitivity was enhanced for visualizing fine structures. Scale bar: 20 µm. Supplementary Figure 8: Comparison of immunosignals for PSA-NCAM/BLBP in the ScaleA2/restored and reference sections prepared from thy1-GFP line M. Two neighboring sagittal sections (30 µm thick) were prepared from a fixed brain of a GFP-M line mouse (3 weeks old). One section was kept in control PBS (a and c) while the other section was incubated in ScaleA2 solution for 16 hours and then restored by incubation in PBS for 2 hours (b and d). After incubation in 0.05% Triton X-100/PBS for 30 min, these two sections were reacted with anti-PSA-NCAM mouse monoclonal antibody and anti-BLBP (brain lipid binding protein) rabbit polyclonal antibody and the immunosignals were visualized using secondary antibodies conjugated with Alexa Fluor 546 and Alexa Fluor 633, respectively. Using a confocal microscopy system (Olympus FV500), we obtained both low- and high-magnification images of the facing plane. No substantial alterations of architectural structures subcellular distribution, or the density of immunosignals were observed in the ScaleA2/restored section when compared with the reference section (PBS). DG, dentate gyrus; MF, mossy fiber. Scale bar, 20 µm. Supplementary Figure 9: Comparison of immunosignals for VGLUT1 in the ScaleA2/restored and reference sections prepared from thy1-GFP line M. Two neighboring sagittal sections (30 µm thick) were prepared from a fixed brain of a GFP-M line mouse (3 weeks old). One section was kept in control PBS (a and c) while the other section was incubated in ScaleA2 solution for 16 hours and then restored by incubation in PBS for 2 hours (b and d). After incubation in 0.05% Triton X-100/PBS for 30 min, these two sections were reacted with anti-VGLUT1 (vesicular glutamate


transporter 1) rabbit polyclonal antibody and a secondary antibody conjugated with Alexa Fluor 633. Using a confocal microscopy system (Olympus FV500), we obtained both low- and high-magnification images of the facing plane. The punctate signals (red) can be observed in the both sections, again with no apparent qualitative changes in fluorescence intensity or subcellular distribution. Scale bar, 20 µm. Supplementary Table 1: Comparison of properties of tissue clearing reagents. Supplementary Video 1: Visualizing the 3D architecure 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. Supplementary Video 2: 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. Supplementary Video 3: 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.


Supplementary Video 4: 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.


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