technical reports

Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain
Hiroshi Hama1, Hiroshi Kurokawa1,2, Hiroyuki Kawano1,3, Ryoko Ando1, Tomomi Shimogori1, Hisayori Noda1,4, Kiyoko Fukami2, Asako Sakaue-Sawano1,3 & Atsushi Miyawaki1,3
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 microscopybased connectomics of cellular networks in brain and other tissues. An important challenge in biological research is the development of highresolution optical methods to label and image cell populations in three dimensions deep in intact tissue. For example, the ability to image and reconstruct intact neuronal networks would be valuable for understanding structure-function relationships in the brain. Methods for labeling specific cell types regardless of tissue depth and geometry have rapidly progressed with the advent of genetically encoded fluorescent proteins and transgenic marking methods such as Brainbow1. However, complementary optical techniques to image and reconstruct labeled threedimensional cell populations deep in intact tissue are also needed. Three-dimensional imaging of biological tissue typically involves mechanical sectioning to improve axial resolution and access to deeper structures. However, although this approach can have a high degree of optical resolution, promising current methods, such as array tomography and serial block-face scanning electron microscopy, are costly and laborious, require sophisticated data reconstruction procedures and are currently limited to smaller tissue volumes24. In contrast, optical sectioning provides a potentially fast, simple and inexpensive alternative for three-dimensional reconstruction of fluorescently labeled structures at subcellular resolution. However, the utility of optical sectioning for deep imaging is prevented by tissue opacity and light scattering. At present, standard laser-scanning confocal microscopy (LSCM) penetrates only to a depth of ~150 mm below the brain surface. Two-photon excitation fluorescence microscopy (TPEFM) offers improved depth, but it cannot, even under ideal conditions, penetrate more than 500800 mm from the brain surface59. Thus, optical sectioning of intact tissue is believed to be insufficient to image and reconstruct large brain projections and cell populations that are often several millimeters in scale and deep below the surface. Light scattering can be reduced by optical clearing, which aims to increase tissue transparency to achieve refractive uniformity throughout the specimen and allow greater depth of imaging. Although this approach is only applied to fixed specimens, it can in principle facilitate optical sectioning and enable three-dimensional imaging and reconstruction. Several clearing solutions have been described. The watersoluble reagent FocusClear has been used to treat insect brains1012. Unfortunately, this commercial reagent is prohibitively expensive for larger samples. Furthermore, because its contents are proprietary, FocusClear cannot be optimized for different biological samples. BABB (a mixture of benzyl-alcohol and benzyl-benzoate) is another clearing solution that has been used with ultramicroscopy13,14 and TPEFM15 to perform three-dimensional reconstructions in whole organs of mice and fruit flies. In this method, a fixed sample is incubated in BABB after dehydration with ethanol and hexane. However, the extent to which this organic chemical quenches fluorescent proteins inside specimens remains unknown. In addition, a technique was reported for clearing thick slabs of mouse cortex for TPEFM with an index-matched solution of 60% sucrose16. However, this sucrose-based method confers only modest transparency on tissue samples. We developed a clearing reagent called Scale that alleviates the major limitations of previously reported solutions. We found that Scale renders mouse brain and embryos transparent while completely preserving fluorescent signals from labeled cells. This combination allows the imaging of intact brain at a depth of several millimeters and large-scale reconstructions of neuronal populations and projections at subcellular resolution. We demonstrated proof of principle by reconstructing networks involving cortical, callosal, hippocampal and neurogenic populations. We also developed additional Scale reagents and protocol variants for specific experimental applications, and discuss potential future applications for quantitative three-dimensional brain reconstructions.

2011 Nature America, Inc. All rights reserved.

1Brain Science Institute, RIKEN, Wako-city, Saitama, Japan. 2School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, Japan. 3Life Function and Dynamics, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, Wako-city, Saitama, Japan. 4Tokyo Institute of Technology, Meguro-ku, Tokyo, Japan. Correspondence should be addressed to A.M. (matsushi@brain.riken.jp).

Received 21 March; accepted 12 August; published online 30 August 2011; doi:10.1038/nn.2928

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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 12-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.

RESULTS Development and properties of the Scale reagent The discovery of the Scale reagent was based on a serendipitous observation. We found that polyvinylidene fluoride membranes became transparent when soaked in 4 M urea, which promotes the hydration of biological samples (Supplementary Fig. 1). This result inspired us to search for an optimal reagent to clear fixed biological samples for light microscopy. We first treated mouse brain sections (60 mm thick) fixed with 4% paraformaldehyde (PFA) with solutions containing 18 M urea. After 48 h, sections treated with 48 M urea became transparent along with some expansion. To further optimize tissue clearance, we next combined ureacontaining solutions with other ingredients. The most effective solution, which we named ScaleA2, was composed of 4 M urea, 10% (wt/vol) glycerol and 0.1% (wt/vol) Triton X-100. Glycerol was predicted to prevent excess hydration and minimize tissue expansion. ScaleA2 has a pH of 7.7 and refractive indices of 1.382, 1.387 and 1.380 at 589, 486 and 656 nm, respectively. ScaleA2 is colorless; the solution absorbs light at 276 nm but is permissive to light greater than 300 nm (Fig. 1a). To examine tissue transparency quantitatively, we measured transmission in brain slices. We prepared 1.5-mm-thick slices from mouse brain samples that had been fixed and incubated in water-soluble reagents:
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phosphate-buffered saline (PBS), 60% sucrose/PBS, FocusClear, MountClear and ScaleA2. Transmission was measured in a spectrophotometer with water as a reference sample. The ScaleA2-treated slice was substantially more permissive to visible and infrared light (350920 nm) than slices incubated in other reagents (Fig. 1b). We next incubated intact fixed mouse brain in a ScaleA2 solution. Incubation for >2 weeks substantially cleared brain tissue; transparency was evident against a patterned background (Fig. 1c) or by penetration with a 532-nm laser light (Fig. 1d). The tissue-clearing effect of ScaleA2 was also prominent on whole mouse embryos (Fig. 1e). To estimate the extent of brain expansion, we calculated brain volume by liquid displacement before and after ScaleA2 treatment by slowly lowering samples into a graduated cylinder containing water. The mean sample volume was doubled (197 13%, n = 5) by ScaleA2 treatment. Taking the cube root of 1.97, we assume that ScaleA2 causes a 1.25-fold linear expansion. To characterize the expansion of macroscopic structures in two dimensions, we time-lapse imaged fixed brain slices (1 mm thick) of a transgenic mouse line, thy1-YFP line H (YFP-H)17,18 during a 5-d incubation in ScaleA2 solution (Fig. 1fy and Supplementary Fig. 2). We traced the outlines of the slices and several internal structures in transmitted-light bright-field and fluorescence images. Overlay
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50% ethanol Figure 2 Comparison of ScaleA2 with BABB. 80% ethanol (a,b) Sensitivity of EGFP fluorescence to ScaleA2 thy1-YFP line H ScaleA2 100% ethanol solution and a conventional chemical clearing Hexane 1.0 1.0 reagent (BABB). Cultured HeLa cells expressing BABB EGFP were fixed with 4% PFA and were timelapse imaged while being exposed to ScaleA2 0.5 0.5 solution (a) or BABB following dehydration with ethanol and hexane (b). Replacement of Hanks Balanced Salt Solution with ScaleA2 resulted 0.0 0.0 ScaleA2 BABB in a change in focus and a slight decrease in 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Time (min) Time (min) 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.

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comparisons between Scaled and control samples revealed that the slice outlines and relative positions of internal structures (hippocampus, amygdala and white matter) maintained their overall shape and proportions, indicating that tissue expansion was isotropic and homogenous. Similar experiments were performed using 50-mm-thick brain slices of another transgenic mouse line, thy1-GFP line M (GFP-M)17 (Supplementary Fig. 3). Although ScaleA2-treated samples were typically soft and fragile, macroscopic structures that maintained their proportions in slice samples lacking full connective tension should show even better preservation of topology in intact, unsectioned whole brains. Fluorescence imaging with the Scale reagent A critical question for brain clearing solutions is whether the capability for fluorescence imaging is retained. It was reported that wild-type Aequorea green fluorescent protein (GFP) is sensitive to 8 M urea at acidic pH but not a 920-nm laser at neutral or alkaline pH19. We verified that the fluorescence of enhanced GFP (EGFP)
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and several GFP-like proteins was resistant to 4 M urea at pH 7.7 (Supplementary Fig. 4a,b). To examine the in vivo stability of EGFP fluorescence in ScaleA2, we transfected the protein into cultured HeLa cells. After fixation, fluorescent cells were time-lapse imaged in a ScaleA2 solution and no substantial decrease in fluorescence intensity was observed (Fig. 2a). In contrast, when cells were incubated in BABB following dehydration with ethanol and hexane13,14, EGFP fluorescence diminished over time (Fig. 2b). Next, we applied ScaleA2 to fixed brains of the YFP-H line17,18, in which yellow fluorescent protein (YFP) is expressed in a subpopulation of neurons confined to pyramidal neurons in the hippocampus and neurons with somata in layers V and VI in the cerebral cortex. The whole fixed brain showed homogeneous fluorescence under blue light.

Figure 3 Three-dimensional reconstructions of 2.0 mm YFP-expressing neurons in ScaleA2-treated brain samples of YFP-H mice. The actual imaging depth is shown in parentheses. Unsectioned thy1-YFP line H brains (am) and an excised hippocampus (n,o) (3 weeks old) were imaged. (ac) TPEFM imaging using a 25 objective (XLPLN25XWMP, numerical 2P 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 onephoton (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 mm.

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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-mm steps) using a 1 objective lens at scanner zoom 3, and z stacked them. (b) We acquired 17 confocal images (43-mm steps) using a 2 objective lens at scanner zoom 2, and z stacked them. (c) We acquired 34 confocal images (10.8 mm steps) using a 2 objective lens at scanner zoom 4, and z stacked them. All scale bars represent 500 mm.

(Fig. 3df). The imaging depth was sufficient to reach the dorsal tip of the hippocampus (Fig. 3g). We then imaged the same region with a 514-nm excitation and an internal descanned path detector through a confocal pinhole (~2 Airy disks; Supplementary Fig. 6b). This onephoton excitation imaging setup produced sufficiently bright images at depths permitted by the objectives working distance (~2.0 mm; Fig. 3hk), suggesting that very little scattering occurred inside the specimen. In deep regions, however, TPEFM yielded a better signal-tonoise ratio than LSCM. A similar three-dimensional reconstruction was performed by TPEFM using an older YFP-H mouse (13 weeks old; Supplementary Video 1). Axons that traveled horizontally through the dendritic trees of the cortex were identified and individual axons tunneling inside white matter was also discernable.

We then cut it at midplane and incubated the left half in ScaleA2 for 3 d while dehydrating the right half with ethanol and treating it with BABB13,14. Yellow fluorescence was preserved in the ScaleA2-treated half but not in the half exposed to ethanol and BABB (Fig. 2c). In addition, the ScaleA2-treated half expanded, whereas the BABB-treated half shrunk. A similar comparison with consistent results was made using a fixed brain sample from the GFP-M line17 (Supplementary Fig. 5). Three-dimensional reconstruction of neuronal structures We examined the three-dimensional architecture of neuronal networks comprised of fluorescent neurons from the fixed and cleared intact brain of a YFP-H mouse using TPEFM with 920-nm excitation (Fig. 3a). With a TPEFM system (Olympus FV1000MPE) employing a 25 objective (XLPLN25XWMP, numerical aperture (NA) = 1.05, working distance = 2.0 mm) and correction collar, an imaging depth of 2 mm was achieved (Fig. 3b). The three-dimensional reconstruction extended from the cerebral cortex to the dorsal tip of the CA1 region through the white matter (corpus callosum). Cortical layer V/VI pyramidal neurons and their dendritic networks were well resolved; individual dendritic spines were discernable in an expanded view at a depth of 0.9 mm (Fig. 3c). Given the estimated 1.25-fold expansion along one axis, the imaging depth in real tissue can be obtained by multiplying the measured depth value by 0.8. In these experiments, imaging at an xy position produced a data unit in the shape of a long quadratic prism and three-dimensional reconstructions were extended in the xy plane. To reconstruct cortical networks, multiple units of data were generated at neighboring xy positions and combined using the microscope systems tiling software (Fig. 3b). One- and two-photon microscopy of Scaled brain One-photon excitation fluorescence microscopy should benefit from tissue clearing to an even greater extent than TPEFM. To illustrate this, we employed LSCM to image a ScaleA2-treated brain sample. We examined the three-dimensional structures of a 3-week-old YFP-H mouse using a ZEISS LSM710-NLO system equipped with a 20 objective (W-PlanApochromat, NA = 1.0, working distance = 2.0 mm). With 920-nm excitation and an external non-descanned detector (Supplementary Fig. 6a), which is common to TPEFM20, three-dimensional reconstruction of the cortex was achieved to a depth of 2.0 mm. The high resolution of the reconstruction was demonstrated by xy images at various depths
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Beyond the current imaging depth limit With brains cleared by ScaleA2, the imaging depth limit was determined by the working distances of currently available objective lenses. Among commercially available lenses, for example, a 40 objective (LUMPLFLN40XW, NA = 0.80, working distance = 3.3 mm) has a longer working distance but a lower NA. Although the use of this lens permitted an imaging depth of 3 mm (Supplementary Fig. 7a,b), the resolution was lower. We therefore asked the manufacturer (Olympus) to develop a customized 25 objective lens, which has a longer working distance (4 mm) and a sufficiently high NA (1.0). Using this lens, we were able to generate very long quadratic prisms of the YFP-H line brain, with reconstructions that extended from the brain surface to the dentate gyrus (Fig. 3l,m and Supplementary Video 2). In addition to reaching new depth limits for brain reconstruction of fluorescent neurons, we were also able to optically reconstruct extensive neuronal networks. The hippocampal formation was excised from a fixed and cleared YFP-H brain to permit a comprehensive threedimensional reconstruction of the hippocampus containing the dentate gyrus and Ammons horn fields (Fig. 3n,o). Fine structures in the same excised hippocampal preparation were visualized by increasing the photomultiplier tube sensitivity (Supplementary Fig. 7ce). Surveying commissural axons in the intact mouse brain To determine whether the Scale system allows a comprehensive perspective of specific axon projections, we used a macro-confocal microscopy system (AZ-C1, Nikon) and imaged optically cleared brains in which specific neurons had been fluorescently labeled. We focused on axons in the corpus callosum, which connects the left and right cerebral hemispheres21,22. Tracing their axon bundles across the midline requires laborious procedures that produce a large number of sections. To visualize callosal axon projections of layer II/III pyramidal neurons, we electroporated plasmids encoding EYFP in utero into the dorsal ventricular zone of the mouse forebrain at embryonic day 15.5 (E15.5)22,23. The embryos were raised until postnatal day 10 (P10), when callosal connnections are almost fully established21,22, and their brains were removed and fixed. After incubation in ScaleA2 for 1 week, the entire EYFP signal in the brain sample was collected using a 1 objective (AZ-PlanApo, NA = 0.1, working distance = 35 mm). The z stack image (Fig. 4a) mapped the labeled pyramidal neurons in layers II and III in the ipsilateral (right) hemisphere and provided sweeping views of fluorescent
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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 Redlabeled blood vessels in the adult mouse hippocampus. 500 PNSC nuclei A schematic diagram showing the approach of TPEFM imaging (red arrow) Total nuclei 400 to a cleared excised hippocampus is presented in a. The imaged area is 300 shown by six quadratic prisms. A high-magnification volume rendering of 200 NSCs (green) and blood vessels (red) in the SGZ is shown in b. Volume 100 renderings generated from a large region in the hippocampus are shown 0 in c. Five perspective views were created from different angles (D, dorsal; 0 10 20 30 40 50 (8) (16) (24) (32) (40) V, ventral; C, caudal; R, rostral; F, front). GCL, granule cell layer; ML, Distance (m) 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 40 hippocampus for TPEFM imaging is shown in d. The quadratic prism that PNSC nuclei Total nuclei 30 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. 20 A series of perspective images of PNSC nuclei (green) and blood vessels 10 (red) when tunneling into the hippocampus are shown in e. Backward perspective images were created at different depths. After passing through 0 0 10 20 30 40 50 the SGZ, no PNSC nuclei were seen ahead. These images are animated (8) (16) (24) (32) (40) in Supplementary Video 4. RINZO automatically calculated the distance Distance (m) (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 mm (b,c) and 20 mm (e,f).

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callosal axons traveling into the contralateral (left) hemisphere. A series of confocal images from anterior to posterior (Supplementary Video 3) revealed that the commissural axons labeled at E15.5 appeared more dorsally in the posterior images. Using a 2 objective (AZ-PlanFluor, NA = 0.2, working distance = 45 mm), we then zoomed in on the projections of the labeled callosal axons in the primary somatosensory cortex (Fig. 4b,c). Neural stem cell association with blood vessels in the SGZ We explored the utility of Scale for imaging of discrete cell populations and quantitative measurement of their geometric properties. We chose to study the vascular niche for neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. Although BrdU labeling of mechanical z sections from the subventricular zone (SVZ)24,25 clearly shows that NSCs and blood vessels are proximal, the SGZ is less amenable to the type of within-z section measurements employed for the SVZ as a result of its more complex vascular geometry with many randomly oriented branches. Thus, we felt that the Scale method, by clearing the SGZ for optical rather than mechanical sectioning, could address this question, which has been difficult to study with current approaches. To first gain a comprehensive three-dimensional perspective of how NSCs interface with blood vessels in the SGZ, we imaged the Scale-treated
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brain of an adult transgenic mouse (7 weeks old) expressing GFP under the control of the Nestin gene regulatory region26. To fluorescently label blood vessels, we transcardially perfused the entire vasculature with Texas Redlabeled lectin. After subsequent fixation, the hippocampus was dissected out and treated with ScaleA2 for 2 d for TPEFM with 920-nm excitation (Fig. 5a). A relatively large number of NSCs were found exclusively in the SGZ and a majority were found to possess radial glia-like processes, whereas others had plump, short processes (Fig. 5b) that together probably represent type-1 and type-2a cells, respectively. Both cell types made considerable contact with blood vessels through their processes. We extended the three-dimensional reconstructions of GFP-filled NSCs and Texas Redlabeled blood vessels inside the hippocampus (Fig. 5c). A perspective image from the front of the hippocampus revealed that the density of NSCs varied substantially along the longitudinal axis of the hippocampus. This observation serves as a reminder that the number of NSCs in a single section does not necessarily reflect hippocampal neurogenic activity. To observe directly proliferating NSCs (PNSCs), we employed the Fucci (fluorescent ubiquitination-based cell cycle indicator) technique27. The Fucci probe consists of mKO2-hCdt1(30/120) and mAGhGem(1/110), which label nuclei of G1/G0 phase cells red and those in
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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 mm. 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 mAGhGem(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 mm (di).

S/G2/M phase green, respectively. A transgenic mouse line that almost ubiquitously expresses mAG-hGem(1/110), #504, was used previously to obtain in vivo information about proliferation patterns at various embryonic stages27. We labeled the vasculature of a #504 mouse with Texas Redlabeled lectin. After fixation, the hippocampus was dissected out and treated with ScaleA2 for 2 d. The cleared hippocampus was viewed with TPEFM at 920-nm excitation from its ventral surface (Fig. 5d) and a series of perspective images aimed toward the dorsal side were taken (Fig. 5e). Green nuclei of PNSCs were localized exclusively in the SGZ in apparent association with a network of blood vessels (Supplementary Video 4). We next examined the quantitative relationship between PNSCs of the SGZ lineage and the vasculature. Multiple hippocampi were fixed and cleared by ScaleA2 and treated with 4,6-diamidino-2-phenylindole (DAPI) to label nuclei. After performing TPEFM imaging on these preparations, we made three-dimensional reconstructions that localized PNSC nuclei and all nuclei in relation to blood vessels. The distances of the nuclei to the nearest vessels were measured in three-dimensional space using the RINZO algorithm (Fig. 5f), which we developed for this purpose. We analyzed 96 PNSC nuclei, out of 1,381 DAPI-positive nuclei in the SGZ (Fig. 5g,h). Of the PNSC nuclei, 37% (35 of 96) were situated within 10 mm of blood vessel surfaces. In contrast, only 22% of SGZ nuclei (298 of 1,381) were within 10 m of blood vessels; these nuclei mostly belonged to endothelial cells and pericytes, as well as PNSCs. The average distance of PNSC nuclei to blood vessels (16.38 12.10 mm) was significantly closer (P < 0.05) than the average for all SGZ cell nuclei (20.34 11.48 mm). Because these analyses involved automatic processing (except for manual tagging of PNSC nuclei) and were performed comprehensively in three-dimensional space, we conclude that PNSC nuclei are closely associated with blood vessels in the mouse SGZ.
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 mm.

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Scale reagent variants for targeted applications ScaleA2 is an optimal all-purpose solution for most brain clearing experiments. Nevertheless, we optimized other Scale variants that could provide better control of certain properties of cleared tissue for specific applications. An ideal clearing agent will preserve the volume of tissue for more accurate reconstruction. However, BABB and FocusClear make biological samples shrink, while ScaleA2 causes expansion. Thus, we developed a modified solution, ScaleU2, composed of 4 M urea, 30% glycerol and 0.1% Triton X-100. ScaleU2 requires longer incubation times to achieve clearing, but it reduces tissue expansion and the fragility of cleared brain samples and is therefore suitable for soft samples such as mouse embryos. To assess the utility of ScaleU2, we analyzed cellcycle profiles during mouse embryogenesis in a transgenic mouse line (#596/#504) that expresses the Fucci probe in which almost every cell nucleus exhibits either red (G1/G0) or green (S/G2/M) fluorescence27. We incubated fixed embryos (E11.5 and E13.5) in ScaleU2 for 6 months and performed three-dimensional imaging of the left half of each body using a Nikon AZ-C1 system (Fig. 6a,b). At both E11.5 and E13.5, the developing cerebral cortex contained more green nuclei than the
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technical reports
diencephalon, suggesting that cells in the diencephalon undergo cellcycle exit for differentiation before those in the cerebrum. Although neurogenesis in the diencephalon appeared to have reached completion by E13.5, there was a region rich in green nuclei at the posterior end of the diencephalon (inferior colliculus) (Fig. 6b). Local cell proliferation was also evident in a sectional image along the midline (Fig. 6c). The green nucleuscontaining cells were immunostained for Nestin (Fig. 6df), but not for PSA-NCAM (Fig. 6gi), which verified that the region was still mitotic. Thus, the ScaleU2 technique applied to Fucci transgenic mice provides a comprehensive perspective of proliferation versus differentiation in the developing brain. ScaleA2 takes days or weeks to clear large specimens; ScaleU2 takes weeks or months. However, we found that the clearing process could be accelerated by a transient tissue expansion induced by 8 M urea. We exploited this observation to devise a speedy protocol that incorporates a pulse of 8 M urea. In this protocol, fixed biological samples are incubated in ScaleA2 for 2 d, followed by ScaleB4 containing 8 M urea and 0.1% Triton X-100 for 2 d, and finally a ScaleA2 or ScaleU2 solution. As an added benefit, treating with ScaleB4 effectively depletes biological samples of background signals. The pH of a ScaleB4 solution is 8.7; the stability of fluorescent proteins in 8 M urea (pH 8.7) was verified (Supplementary Fig. 4a). It was not initially clear whether Scale treatment had an irreversible effect on tissue architecture. We investigated whether a ScaleA2-treated brain sample could be restored to its original state by simply washing with PBS. We split a fixed brain of the YFP-H mouse into two halves. The right half was kept in PBS while the left was cleared thoroughly with ScaleA2 for 3 weeks and then washed in 20 volumes of PBS. After two sequential washes for 15 min each, the sample shrank to the original size and became turbid. The two halves were embedded in optimal cutting temperature (OCT) compound, from which 30-mm-thick dentate gyrus sections were prepared for histology. The patterns of immunostaining for PSA-NCAM, as well as the cellular structures delineated with YFP fluorescence, were very similar between the two samples (Fig. 7af). Next, we examined subcellular structures in the CA3 region using a wildtype mouse brain. When immunostained for the pre- and postsynaptic markers Synaptophysin and GluR1, similar colocalization signals were observed in sections from the two halves (Fig. 7gl). Similar results were obtained with immunostaining for PSA-NCAM/BLBP (brain lipid binding protein) or VGLUT1 (vesicular glutamate transporter 1) using two neighboring sections from a GFP-M mouse brain (Supplementary Figs. 8 and 9). It is therefore easy to restore a cleared specimen to its original state in a manner that is compatible with immunohistochemistry and techniques such as array tomography2. DISCUSSION Comparison of Scale with other tissue-clearing techniques With the Scale system, we present a simple, but effective, technique for clearing mammalian brain tissue that has the potential to address questions in brain structure and function at unprecedented spatial detail and scale. Our results demonstrate the utility of Scale for reconstruction of neuronal populations and projections, in which labeled cells at subcellular detail can be viewed in three-dimensional networks at millimeter scales. As Scale is part of an emerging area of chemical technology for imaging, we evaluated the strength and limitations of Scale, compared its performance with that of other described clearing reagents and developed solutions to extend its range of utility. Here we discuss its potential roles among the emerging connectomics efforts to map the brain and its constituent structures. Given its simplicity and stability, we suggest that Scale will popularize high-resolution three-dimensional reconstructions in mammalian brain and other tissues, organs and animals.
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Our results indicate that ScaleA2 is superior to other described clearing agents in key properties necessary for performing detailed three-dimensional brain reconstructions at both fine scale and broad perspective (Supplementary Table 1). Unlike organic solventbased reagents, Scale allows signal preservation of fluorescent proteins. Scale is also superior to other aqueous reagents, including FocusClear, because it is inexpensive and its formula is simple and public, and to 60% sucrose, which allows only modest transparency. Thus, a large quantity of Scale can be used for clearing large tissue samples not only from rodent brains and embryos, but, we expect, from primate28 and human biopsy samples. Furthermore, researchers can modify the reagents composition according to the nature of the samples to be cleared. For example, the concentration of glycerol should be increased to preserve tissue volume and integrity, and the concentration of urea can be reduced for brain samples from fish and flies (data not shown). The drawbacks of ScaleA2 include the long time periods required for clearance and the fragility of cleared samples, but these problems may be largely solved by modifying reagent composition to ScaleB4 and ScaleU2, respectively. Scale applications for brain development and function Recent progress in gene targeting and genetic labeling methods has revealed the roles of specific genes in regulating axon guidance during the formation of topographic projections. The Scale method will facilitate the analysis of wild-type projection and screening of mutant mice to detect those with aberrant axon projections. Moreover, the high resolution of Scale may allow mechanistic insights into the nature of such defects. To follow up on our callosal findings, one could label axons with different colors at different embryonic days to survey the developmental structure of the corpus callosum29. The Scale technique can also be applied effectively to chemically stained samples, as we found with transcardial perfusion of Texas Redlectin to label blood vessels before fixation or in the future with in vivo labeling of astrocytes with Texas Redhydrazide, a paraformaldehyde-fixable analog of SR101 (ref. 30), and neuronal tracing with NeuroVue dyes in fixed brain tissue31. Further applications of the Scale system could involve studies of neuronal function. We labeled and reconstructed the proliferating NSC niche in the SGZ, which was made possible by Scale clearance of the hippocampus. It will also be useful to obtain projection images of Scaled brain samples using low-NA objective lenses to allow quantification of the spatial expression of neural activityregulated immediate early genes during behavior of transgenic mice in which the expression of GFP is controlled by the promoter of c-fos32 or Arc33. Scale will thus be valuable for the high-resolution immediate early gene mapping of behavior in intact large-scale brain networks. Scale applications for connectomics in intact brain Scale should be compatible with most light microscopy systems. In particular, we suggest the utility of Scale-treated samples for light sheet ultramicroscopy, a technique that can gather three-dimensional image data quickly13,14,34,35. In addition, in combination with Scale, it may be possible to observe larger animals via fluorescent protein tomography (FPT)36 using proteins that fluoresce at shorter wavelengths. Similarly, the Scale technique should be applicable to all organs. Very few techniques for three-dimensional reconstruction using light microscopy can penetrate tissue blocks thicker than 1 mm. In contrast, most tomographic techniques, including optical projection tomography37, FPT36, computer tomography and positron emission tomography, as well as magnetic resonance imaging, are capable of analyzing structural and quantitative features in a much larger mass of tissue, such as the whole body. Among the emerging approaches for brain connectomics that aim at detailed three-dimensional reconstructions of brain structures, the
7

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technical reports
Scale technique will likely provide a simple, inexpensive complement to array tomography and serial section electron microscopy efforts by enlarging the volume and depth of three-dimensional reconstructions that can be achieved using light microscopy data. In this way, the Scale system should help to bridge the imaging gap38 between the size of specimens that can be visualized with light microscopy versus other brain imaging techniques. The reversibility of Scale for retrospective immunohistochemistry will enable zooming out to observe neuronal circuits in a global three-dimensional reconstruction, followed by zooming in to image specific synaptic structures at both light and electron microscopy scales. Together with advances in microscopy and fluorescent proteins, Scale will contribute to the discovery of new biological principles in connectomics and circuit genetics3941. METHODS Methods and any associated references are available in the online version of the paper at http://www.nature.com/natureneuroscience/.
Note: Supplementary information is available on the Nature Neuroscience website.
7. Zipfel, W.R., Williams, R.M. & Webb, W.W. Nonlinear magic: multiphoton microscopy in the biosciences. Nat. Biotechnol. 21, 13691377 (2003). 8. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932 940 (2005). 9. Theer, P. & Denk, W. On the fundamental imaging-depth limit in two-photon microscopy. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 23, 31393149 (2006). 10. Chiang, A.-S. et al. Insect NMDA receptors mediate juvenile hormone biosynthesis. Proc. Natl. Acad. Sci. USA 99, 3742 (2002). 11. Liu, Y.-C. & Chiang, A.-S. High-resolution confocal imaging and three-dimensional rendering. Methods 30, 8693 (2003). 12. Lin, H.-H., Lai, J.S.-Y., Chin, A.-L., Chen, Y.-C. & Chiang, A.-S. A map of olfactory representation in the Drosophila mushroom body. Cell 128, 12051217 (2007). 13. Dodt, H.-U. et al. Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat. Methods 4, 331336 (2007). 14. Jhrling, N., Becker, K. & Dodt, H.-U. 3D-reconstruction of blood vessels by ultramicroscopy. Organogenesis 5, 145148 (2009). 15. Parra, S.G., Chia, T.H., Zinter, J.P. & Levene, M.J. Multiphoton microscopy of cleared mouse organs. J. Biomed. Opt. 15, 036017 (2010). 16. Tsai, P.S. et al. Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocaliztion of nuclei and vessels. J. Neurosci. 29, 1455314570 (2009). 17. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 4151 (2000). 18. Porrero, C., Rubio-Garrido, P., Avendao, C. & Clasc, F. Mapping of fluorescent proteinexpressing neurons and axon pathways in adult and developing Thy1-eYFP-H transgenic mice. Brain Res. 1345, 5972 (2010). 19. Alkaabi, K.M., Yafea, A. & Ashraf, S.S. Effect of pH on thermal- and chemical-induced denaturation of GFP. Appl. Biochem. Biotechnol. 126, 149156 (2005). 20. Denk, W., Piston, D.W. & Webb, W.W. Two-photon molecular excitation in laser scanning microscopy. in The Handbook of Confocal Microscopy (ed. J. Pawley) 445458 (Plenum Press, New York, 1995). 21. Lindwall, C., Fothergill, T. & Richards, L.J. Commissure formation in the mammalian forebrain. Curr. Opin. Neurobiol. 17, 314 (2007). 22. Wang, C.-L. et al. Activity-dependent development of callosal projections in the somatosensory cortex. J. Neurosci. 27, 1133411342 (2007). 23. Shimogori, T. & Ogawa, M. Gene application with in utero electroporation in mouse embryonic brain. Dev. Growth Differ. 50, 499506 (2008). 24. Tavazoie, M. et al. A specialized vascular niche for adult neural stem cells. Cell Stem Cell 3, 279288 (2008). 25. Shen, Q. et al. Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell interactions. Cell Stem Cell 3, 289300 (2008). 26. Yamaguchi, M., Saito, H., Suzuki, M. & Mori, K. Visualization of neurogenesis in the central nervous system using nestin promoterGFP transgenic mice. Neuroreport 11, 19911996 (2000). 27. Sakaue-Sawano, A. et al. Visualizing spatiotemporal dynamics of multicellular cellcycle progression. Cell 132, 487498 (2008). 28. Sasaki, E. et al. Generation of transgenic non-human primates with germline transmission. Nature 459, 523527 (2009). 29. Richards, L.J., Plachez, C. & Ren, T. Mechanisms regulating the development of the corpus callosam and its agenesis in mouse and human. Clin. Genet. 66, 276289 (2004). 30. Nimmerjahn, A., Kirchhoff, F., Kerr, J.N.D. & Helmchen, F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat. Methods 1, 3137 (2004). 31. Fritzsch, B., Muirhead, K.A., Feng, F., Gray, B.D. & Ohlsson-Wilhelm, B.M. Diffusion and imaging properties of three new lipophilic tracers, NeuroVue Maroon, NeuroVue Red and NeuroVue Green and their use for double and triple labeling of neuronal profile. Brain Res. Bull. 66, 249258 (2005). 32. Barth, A.L., Gerkin, R.C. & Dean, K.L. Alteration of neuronal firing properties after in vivo experience in a FosGFP transgenic mouse. J. Neurosci. 24, 64666475 (2004). 33. Wang, K.H. et al. In vivo two-photon imaging reveals a role of Arc in enhancing orientation specificity in visual cortex. Cell 126, 389402 (2006). 34. Voie, A.H., Burns, D.H. & Spelman, F.A. Orthogonal-plane fluorescence optical sectioning: three-dimensional imaging of macroscopic biological specimens. J. Microsc. 170, 229236 (1993). 35. Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J. & Stelzer, E.H. Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 10071009 (2004). 36. Zacharakis, G. et al. Volumetric tomography of fluorescent proteins through small animals in vivo. Proc. Natl. Acad. Sci. USA 102, 1825218257 (2005). 37. Sharpe, J. et al. Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 296, 541545 (2002). 38. Sharpe, J. Optical projection tomography as a new tool for studying embryo anatomy. J. Anat. 202, 175181 (2003). 39. Lichtman, J.W. & Sanes, J.R. Ome sweet ome: what can the genome tell us about the connectome? Curr. Opin. Neurobiol. 18, 346353 (2008). 40. Lichtman, J.W. & Smith, S.J. Seeing circuits assemble. Neuron 60, 441448 (2008). 41. Seung, H.S. Reading the book of memory: sparse sampling versus dense mapping of connectomes. Neuron 62, 1729 (2009).

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ACKNOWLEDGMENTS We thank H. Sakurai, H. Otsuka and M. Hirano for general assistance, F. Ishidate, B. Zimmermann, R. Wolleschensky, Y. Watanabe, E. Nakasho, H. Kimura, T. Tajima and S. Horie for help with acquiring and analyzing images, RIKEN BSI-Olympus Collaboration Center for technical support, Y. Yoshihara (RIKEN), M. Yamaguchi and K. Mori (The University of Tokyo) for the Nestin promoterGFP transgenic mice, J.R. Sanes (Harvard) for the YFP-H and GFP-M lines, E. Takahashi (RIKEN) for helpful advice on transgenic mice, S. J. Smith (Stanford) and J.W. Lichtman (Harvard) for helpful advice on tissue clearing, and D. Mou (Harvard), A. Govindarajan, K. Rockland and S. Tonegawa (Massachusetts Institute of Technology), A. Moore and C. Yokoyama (RIKEN) for critical comments. This work was partly supported by grants from Japan Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research on Priority Areas and the Human Frontier Science Program. AUTHOR 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 The authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/natureneuroscience/.
Published online at http://www.nature.com/natureneuroscience/. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html.

1. Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 5662 (2007). 2. 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). 3. Denk, W. & Horstmann, H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2, e329 (2004). 4. Helmstaedter, M., Briggman, K.L. & Denk, W. 3D structural imaging of the brain with photons and electrons. Curr. Opin. Neurobiol. 18, 633641 (2008). 5. Denk, W. et al. Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy. J. Neurosci. Methods 54, 151162 (1994). 6. Denk, W. & Svoboda, K. Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18, 351357 (1997).

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Scale solutions. Stock solutions containing high concentrations of urea (Wako Chemicals) or Triton X-100 (Wako Chemicals) were prepared. Scale solutions were made by mixing the stock solutions and glycerol (Sigma). The final concentrations of urea, glycerol and Triton X-100 were adjusted by diluting the mixed solutions with water. Sample preparation. Neonatal and adult mice (2-13 weeks old) were deeply anesthetized with pentobarbital (Nembutal) and killed by transcardial perfusion with 4% PFA/PBS (wt/vol). The whole brains were taken out and subjected to postfixation in 4% PFA/PBS at 4 C for 10 h and cryo-protection in 20% sucrose/PBS (wt/vol) at 4 C for 24 h. For observation of the entire hippocampal formation, the hippocampi were excised. Brains or hippocampal samples were embedded in OCT compound and frozen. They were thawed in PBS and fixed again with 4% PFA/PBS for 20 min at 25 C. Next, the samples were cleared in a ScaleA2 solution (20 ml per 0.5 g tissue) at 4 C for >2 d. Mouse embryos (E13.5) were transcardially perfused with 4% PFA/PBS, postfixed for 2 h and cleared with ScaleA2 for 2 weeks. Alternatively, mouse embryos (E11.5 and E13.5) were fixed with 4% PFA/PBS for 2 h and cleared with ScaleU2 for 6 months. For speedy processing, fixed samples were initially cleared with ScaleA2 or ScaleU2 for 2 d, with ScaleB4 for 2 d, and then with ScaleA2 or ScaleU2 again. The experimental procedures and housing conditions for animals were approved by the Institutes Animal Experiments Committee of RIKEN and all of the animals were cared for and treated humanely in accordance with the Institutional Guidelines for Experiments using animals. Imaging of fixed HeLa cells. HeLa cells grown on a 35-mm glass-bottom dish were transiently transfected with cDNA for EGFP using Lipofectin (Invitrogen). After fixation with 4% PFA for 10 min, cells were kept in Hanks Balanced Salt Solution containing 15 mM HEPES-NaOH (pH 7.4) and time-lapse imaged using an inverted microscope (IX81, Olympus) equipped with a standard 75-W xenon lamp, a 40 objective lens (UplanFlN Oil, NA = 1.30) and a cooled CCD camera (iXon EM+, Andor Technology). EGFP signals were obtained using an excitation filter (470 10 nm), an emission filter (517.5 22.5 nm) and a dichroic mirror DM485. The system was operated using MetaMorph 7.6 software (Molecular Devices). In utero electroporation. After anesthesia with sodium pentobarbital, pregnant mice at E15.5 were subjected to abdominal incision, and all the uterine horns were exposed onto PBS-moistened cotton gauze. Embryos were visualized through the uterine wall using a flexible fiber cable, and plasmid DNAs mixed with the non-toxic dye Fast Green were injected into the lateral ventricle through a pulled glass capillary. A pair of platinum electrodes was applied to the uterus, and a series of square-wave current pulses (38 V, 50 ms) was delivered five times at 1-s intervals using a pulse generator. Uterine horns were repositioned into the abdominal cavity, and the abdominal wall and skin were sutured. Image acquisition using TPEFM. The light source used for TPEFM was a femtosecond-pulsed Ti:sapphire laser. When YFP- or GFP-expressing neurons were imaged, an excitation wavelength of 920 nm was used. Emitted light was collected by an external non-descanned detector (Supplementary Fig. 6a). In the experiments shown in Figure 3ac,lo and Supplementary Figure 7ae, multiple neighboring regions in the brain were imaged using a TPEFM system (Olympus FV1000MPE + Coherent Chameleon Ultra-II + PreComp) equipped with a motorized xy stage module in addition to a motorized focus module (Z-drive). Adjacent regions overlapped by 10% to allow precise alignment. Lenses used included a 40 dipping objective (LUMPLFLN40XW, NA = 0.8, working distance = 3.3 mm), a 25 dipping objective (XLPLN25XWMP, NA = 1.05, working distance = 2.0 mm) or a custom-designed 25 objective lens (NA = 1.0, working distance = 4.0 mm). The brightness compensation function in the z direction was used to change the detector sensitivity and laser power. In the experiment represented in Figure 3dg, single regions were imaged using a ZEISS LSM710-NLO system (+ Spectral Physics MaiTai HP DeepSee) equipped with a 20 dipping objective (W-PlanApochromat, NA = 1.0, working distance = 2.0 mm) and z drive. The laser power was constant during image acquisition; the excitation intensity at the level of the specimen was 6 mW.

Image acquisition using LSCM. In the experiment appearing in Figure 3hk, brain samples of the YFP-H line were imaged using a ZEISS LSM710-NLO system equipped with a 20 dipping objective (W-PlanApochromat, NA = 1.0, working distance = 2.0 mm) and z drive. The z step size was 3.0 mm. YFP was excited with a 514.5-nm Argon laser. The laser power was constant during image acquisition; the excitation intensity at the level of the specimen was 2 mW. YFP fluorescence was descanned and collected confocally. The size of the confocal aperture was ~2 Airy disks (Supplementary Fig. 6b). Image acquisition using macro zoom LSCM. Brain samples containing EYFPpositive commissural connections were cleared with ScaleA2 and trimmed with a coronal cut at the midbrain and positioned on a cover glass with the anterior part uppermost. Transgenic mouse #596/#504 embryos (E11.5 and E13.5) were cleared with ScaleU2. These samples were imaged using a Nikon macro zoom confocal microscopy system (AZ-C1) equipped with a 1 objective lens (AZ-PlanApo, NA = 0.1, working distance = 35 mm) or a 2 objective lens (AZ-PlanFluor, NA = 0.2, working distance = 45 mm). EYFP and mAG were excited with a 488-nm laser diode. mKO2 was excited with a 561-nm laser diode. Pre-processing of images. Multiple xy images were tiled using the stitch function of a commercial program (Multi-Area Time-Lapse View of FV10-ASW, version 2.0c) at each z position. The correlation function was used to compute the xy positions in the overlapping regions. All tiled xy images were processed for shading correction by the custom-written filtering program JINARASHI. This program compensates signal intensity at the periphery of each image. We developed JINARASHI using the Open CV Library (http://opencvlibrary. sourceforge.net). JINARASHI is written in the C/C++ language and is available on our website (http://cfds.brain.riken.jp/). JINARASHI corrects signal gradients on the basis of the background intensity distribution. Shading-corrected images were stacked to achieve three-dimensional reconstruction using the volume-rendering function of the commercially available Volocity version 5.3 (Improvision/PerkinElmer). Volume-rendered images were displayed using fluorescence mode (three-dimensional opacity) and exported as AVI images. Measurement of distances. Nuclei emitting mAG-hGem(1/110) green fluorescence or those stained with DAPI in the SGZ regions were tagged manually. In contrast, blood vessels were automatically segmented using a custom-written program, Frame Level Threshold, which converts the signal into binary. This program is written in the C/C++ language and is available on our website (http:// cfds.brain.riken.jp/). The distance from each nucleus to the nearest blood vessel surface was measured using a custom-written program, RINZO. Different parameters were set depending on the voxel shape, that is, the ratio of the length of an x or y edge to that of a z edge. RINZO is written in Java and is available on our website (http://cfds.brain.riken.jp/). Data were pooled from six SGZ regions. Transmission measurement. Transmission curves were acquired using a U3310 spectrophotometer (Hitachi). Fixed brain slices (3 mm thick) were incubated in PBS, a 60% sucrose solution, FocusClear or a ScaleA2 solution. The slices equilibrated with sucrose or FocusClear shrank slightly, whereas the slices treated with ScaleA2 expanded slightly. After incubation, slices with a thickness of exactly 1.5 mm were generated from the equilibrated samples using a tissue slicer. These 1.5-mm-thick slices each were mounted in a cuvette for measurement of transmission. Immunohistochemistry. Adult mice (7-9 weeks old) were deeply anesthetized with pentobarbital (Nembutal) and transcardially perfused with 4% PFA/PBS. After postfixation in 4% PFA/PBS and cryo-protection in 20% sucrose/PBS, brains were embedded in OCT compound (Sakura). Restored brain samples from ScaleA2 treatment were also embedded in OCT compound. Sagittal sections (35 mm thick) were cut with a cryostat. Sections were permeabilized/ blocked for 30 min in 0.1% Triton X-100 (wt/vol)/10% horse serum (vol/vol)/ PBS and then processed by free-floating immunohistochemistry. For double staining, primary antibodies from different species were incubated simultaneously, followed by incubation with secondary antibodies. For primary antibodies, we used mouse monoclonal antibody to PSA-NCAM (Millipore), rabbit polyclonal antibody to GluR1 (Millipore) and mouse monoclonal antibody to synaptophysin (Sigma). For secondary antibodies, we used

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goat antibody to rabbit IgG conjugated to Alexa Fluor 546 (Molecular Probes), goat antibody to mouse IgG conjugated to Alexa Fluor 546 (Molecular Probes), goat antibody to rabbit IgG conjugated to Alexa Fluor 488 (Molecular Probes) and goat antibody to rat IgG conjugated to Alexa Fluor 633 (Molecular Probes). Observation of macroscopic structures in brain slices. Fixed brains were frozen in OCT compound, thawed in PBS and fixed again with 4% PFA/PBS for 20 min at 25 C. Then, coronal slices (1 mm thick) were cut with a razor blade from the brain sample placed in the Mouse Brain Slicer (MB-A1-C, Muromachi Kikai). Slices containing the hippocampus and the striatum were selected. These slices were cut near the midline and the right halves were incubated in ScaleA2 solution at 4 C for 5 d, while the left halves were kept in PBS. Intermittently, these slices were placed on a coverslip with a patterned background and imaged using a Leica fluorescence stereomicroscope (MZ16F) equipped with a 1 objective lens (PLANAPO, NA = 0.141) and a cooled CCD camera (DP30, Olympus).

Transmitted-light bright-field (transmission) images were acquired with oblique illumination and a reduced condenser aperture42, which can enhance structural differences in inherent absorption in the brain slices. Contrast was further enhanced in the final images by electronic image processing. Fluorescence (YFP) images were acquired using an excitation filter (480 20 nm) and an emission filter (510-nm long pass). The outlines of the slices and several internal structures in the images were traced. The extent of the linear expansion (E) was calculated by comparing the distances across highlighted structures between 0-d and 5-d images. At 5 d, the image was reduced in size by 1 1/E for superposition. Statistical analysis. The statistical analysis for Figure 5g,h was performed using the Mann-Whitney U test. Difference was considered to be significant when P < 0.05. The data described in the text represent means s.d.
42. Keller, H. E. Contrast enhancement in light microscopy. Curr. Protoc. Cytom. 2.1.1 2.1.11. (2001).

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