9330 • The Journal of Neuroscience, October 31, 2018 • 38(44):9330 –9337

Mini-Symposium

Whole-Brain Analysis of Cells and Circuits by Tissue
Clearing and Light-Sheet Microscopy
Tomoyuki Mano,1,2* X Alexandre Albanese,4* X Hans-Ulrich Dodt,8,9 X Ali Erturk,10,11,12 X Viviana Gradinaru,13Jennifer B. Treweek,13,14 Atsushi Miyawaki,15,16 Kwanghun Chung,4,5,6,7,17 and XHiroki R. Ueda2,3,18
1

Department of Information Physics and Computing, Graduate School of Information Science and Technology, 2International Research Center for
Neurointelligence, UTIAS, 3Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 113-0033 Tokyo, Japan,
4Institute for Medical Engineering and Science, 5Picower Institute for Learning and Memory, 6Department of Chemical Engineering, 7Department of Brain
and Cognitive Sciences, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, 8Department of Bioelectronics, Vienna Universityof Technology, FKE, 1040 Vienna, Austria, 9Center for Brain Research, Medical University of Vienna, 1090 Vienna, Austria, 10Institute for Stroke and
Dementia Research, Klinikum der Universität München, Ludwig Maximilians University of Munich, 80539 Munich, Germany, 11Graduate School of
Systemic Neurosciences, 80539 Munich, Germany, 12Munich Cluster for Systems Neurology, 81377 Munich, Germany, 13Division of Biology and BiologicalEngineering, California Institute of Technology, Pasadena, California, 91125, 14Department of Biomedical Engineering, University of Southern California,
Los Angeles, California 90089, 15Laboratory for Cell Function and Dynamics, Center for Brain Science, 16Biotechnological Optics Research Team, Center for
Advanced Photonics, RIKEN, Wako-City, 351-0198 Saitama, Japan, 17Broad Institute of Harvard University and MIT, Cambridge, Massachusetts 02142,
and 18Laboratory for Synthetic Biology, RIKEN Center for Biosystems Dynamics Research, 565-0871 Suita, Japan

In this photo essay, we present a sampling of technologies from laboratories at the forefront of whole-brain clearing and imaging forhigh-resolution analysis of cell populations and neuronal circuits. The data presented here were provided for the eponymous MiniSymposium presented at the Society for Neuroscience’s 2018 annual meeting.

Introduction
Microscopic analysis of tissues reveals that intricate organizationof cells underlies biological function. Tissues are not translucent
against visible light, so it is impossible to image far beyond thesurface. Thus, conventional tissue imaging applies a microtome
to cut samples into thin sections before staining with dyes andantibodies to visualize cells. Inferring 3D structure from thinsections is often problematic, and requires minimal sample distortion and precision alignment of serial sections.

Received Aug. 31, 2018; revised Sept. 27, 2018; accepted Sept. 27, 2018.
The Dodt laboratory thanks Dr. Saiedeh Saghafi and Dr. Klaus Becker for aspheric optics-based LSFM work, and
the Austrian science funding agency FWF and the German Hertie foundation for support; the Ertürk laboratory
thanks Ruiyao Cai for uDISCO figure and Synergy Excellence Cluster Munich (SyNergy), Fritz Thyssen Stiftung, and
DFG for support; the Miyawaki laboratory thanks Dr. Hiroshi Hama for ScaleS figure and Grant-in-Aid for Scientific
Research on Priority Areas (JSPS KAKENHI), the Human Frontier Science Program, and the Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) for support; the Ueda laboratory thanks Tatsuya Murakami for offering picture materials for CUBIC figure, and Brain/MINDS (AMED/MEXT), the Basic Science andPlatform Technology Program for Innovative Biological Medicine (AMED/MEXT)and Grant-in-Aid for Scientific Research (S) (JSPS KAKENHI) for support; the Chung laboratory thanks The Packard Award, the McKnight Foundationand the NIH (1-DP2-ES027992) for support; the Gradinaru laboratory thanks Sripriya Ravindra Kumar, Gerard M.
Coughlin, Rosemary Challis, and Collin Challis for CREATE and VAST images, Min Jee Jang for HCR images, and
J. Ryan Cho for GCaMP imaging, and the NIH BRAIN Initiative and NIH Director’s Office and NSF Neuronex for
support.
The authors declare no competing financial interests.
*T.M. and A.A. contributed equally to this work.
Correspondence should be addressed to either of the following: Dr. Hiroki R. Ueda, Department of Systems
Pharmacology, Graduate School of Medicine, The University of Tokyo, 113-0033 Tokyo, Japan, E-mail: uedahtky@umin.ac.jp; or Kwanghun Chung, Massachusetts Institute of Technology, Cambridge, MA 02139, E-mail:khchung@mit.edu.
https://doi.org/10.1523/JNEUROSCI.1677-18.2018
Copyright © 2018 the authors 0270-6474/18/389330-08$15.00/0

The analysis of large 3D volumes is necessary for mapping theconnections of far-reaching neurons inside the brain and determining the nature of cellular interactions underlying proper
function and behavior. In recent years, several techniques haveemerged to achieve optical transparency and enable highresolution microscopy of thick tissue sections and whole organs.
These techniques use different strategies to reduce light scatteringin tissues and improve image sharpness.
Scattering occurs in tissues due to light’s heterogeneous interaction with different molecules, subcellular structures, membranes, and cell populations inside the tissue. For example, theinterface between a cell’s lipid membrane and the cytoplasm
causes a significant drop in refractive index (RI). Heterogeneity at
the scale of molecules, cells, and tissues contribute to light scattering and requires homogenization via tissue clearing to increase
overall light penetration (Tainaka et al., 2016; Treweek and Gradinaru, 2016). Combining tissue clearing with light-sheet fluorescence microscopy (LSFM) has paved the road for current
whole-brain imaging by eliminating out-of-focus excitation(hence reduced background level and greatly preventing photobleaching), and by accelerating image acquisition.
Tissue clearing originated in the early 20th century, when
Werner Spalteholz experimented with high RI organic solvents (Spalteholz, 1914). Organic solvent-based clearing homogenizes a tissue’s RI by the removal of highly scattering
lipids and the displacement of water by high RI solvents.
Building on this approach, Dodt’s group revitalized organic
solvent-based tissue clearing for the modern era of neuroscience by coupling it with a new optical imaging method that

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Figure 1. The optical resolution of light-sheet microscopy is determined by both detection objective NA and thickness of the illuminating laser sheet. In conventional light-sheet
microscopy, there is a fundamental tradeoff between light-sheet thickness and the field-of-view; when a thin laser sheet is illuminated (hence a higher axial resolution), the effective
imaging area (determined by Rayleigh length) must decrease rapidly. To overcome this tradeoff, one approach is to use a beam that is generated by a sequence of aspheric lenses (Saghafi
et al., 2014). A, The optical design of this light-sheet generator, along with the measurements of the beam profile along the x-y plane for confirmation of the extended Rayleigh range.
Each optical element in the design are as follows: (a) first aspheric condenser lens, (b) Powell lens, (c) second aspheric condenser lens, (d) first achromatic cylindrical lens, and (e) second
achromatic cylindrical lens. B, A Scheme of the ultramicroscopy setup: (1) 488 nm laser; (2) light-sheet generator optics, duplicate arms are placed on the left and right side of thespecimen container; (3) specimen container with quartz windows; (4) detection arm with an exchangeable objective; and (5) scientific grade CCD camera. The objective for imaging is
dipped into the clearing solution from above so that the objectives can be exchanged easily for change of magnification. C–E, Using a custom-made ultramicroscope equipped with thenovel light-sheet generator, a cleared fruit-fly brain was imaged and reconstructed in 3D. A virtual cross-section through the head is presented in D. om, Ommatidia; m, medulla; lo,lobula. A virtual cross-section through the thorax is presented in E. oe, Esophagus; pv, proventriculus (cardia). F, G, The hippocampus area of Thy1-EGFP-M mouse brain was dissected andcleared, and imaged with ultramicroscopy. Close-up with 10⫻ magnification (F ) and 20⫻ magnification (G) are shown. Scale bars: F, G, 100 ␮m .

improves the resolution and field-of-view using LSFM (Fig. 1;
Dodt et al., 2007; Ertürk et al., 2012; Saghafi et al., 2014). This
technique allows rapid imaging of larger volumes than withconventional point scanning microscopy. Erturk’s group developed uDISCO method (Fig. 2) to image whole-mousebodies (Pan et al., 2016) and recently vDISCO method, awhole-mouse immunolabeling method to amplify the signal
two orders of magnitude for detecting single cells in clearedmouse bodies through intact bones and muscles (Cai et al.,2018). vDISCO enabled construction of the first whole-mouseneuronal connectivity map, study brain trauma effects in peripheral nerves, and image meningeal vessels and their cellular
contest through intact skull.

Some techniques have replaced organic solvents with aqueous
solutions to improve preservation of endogenous reporter proteins and simplicity of experimental handling, since a urea-based
clearing reagent ScaleA2 was developed (Hama et al., 2011). To
date, various mixtures and protocols have emerged that use aqueous chemicals for whole-tissue clearing. Miyawaki’s group has
additionally developed a urea/sorbitol-based clearing method
called ScaleS (Hama et al., 2015) to achieve mild clarification
where synaptic ultrastructure, observed by electron microscopy,
is well preserved. ScaleS was capable of imaging A␤ plaques inrodent models of Alzheimer’s disease and human clinical samples(Fig. 3). Ueda’s group has developed a clearing method called
CUBIC (clear, unobstructed brain/body imaging cocktails and9332 • J. Neurosci., October 31, 2018 • 38(44):9330 –9337

Mano, Albanese et al. • Tissue Clearing and Light-Sheet Microscopy

Figure 2. uDISCO enables investigation of long-range neuronal connections spanning the entire mouse body and entire CNS in unprecedented detail (Pan et al., 2016). A–D, The entire CNS (wholebrain and spinal cord) of a Thy1-GFP-M mouse was cleared by uDISCO and imaged by LSFM. Because of the superior preservation of the fluorescent protein signals achieved by uDISCO, detailed
neuronal structures are clearly visible. Zoom-in view of the brain hemisphere and spinal cord within boxed region in A are shown in B and D, respectively. Zoom-in view of B is shown in C, whereindividual pyramidal cell bodies and dendrites are clearly resolved. Scale bars: A, 10 mm; B, 1 mm; C, 100 ␮m; D, 1 mm. E–J, AAV2-Syn-GFP was transduced in the right motor cortex of mice, andAAV2-Syn-RFP in the left motor cortex. The brain was then cleared by uDISCO and imaged with LSFM. Fluorescence expression of the virally delivered proteins was detectable in the brain andthroughout the intact spinal cord (F–J ). Details of neuronal extensions of boxed regions in F are shown in H (thalamus and midbrain), I (cervical), and J (thoracic spinal cord regions). Arrowheads
in G indicate decussation of the descending motor axons. Scale bars: F, 5 mm; G, 2 mm; H–J, 1 mm.

computational analysis; Susaki et al., 2014, 2015; Tainaka et al.,2014; Kubota et al., 2017) and has recently reported controlled
expansion of cleared mouse brains using CUBIC-X (Fig. 4; Murakami et al., 2018). Physical expansion of a sample (or swelling)enables imaging of subcellular structures across the entire mousebrain. Using LSFM, authors generated a point-based mouse brain
atlas (called CUBIC-Atlas) with single-cell annotation, andtracked postnatal subregion development in mice.

Another important innovation in tissue imaging and phenotyping is the development of technologies that engineer
tissue physicochemical properties, tissue-molecular interaction, and molecular transport within intact tissue. Chung’sgroup has developed a suite of technologies that engineer tissue properties to maximally preserve tissue information usingsynthetic polymer (e.g., CLARITY, MAP) or crosslinker-based
tissue reinforcement strategies (e.g., SWITCH, Fig. 5; Chung

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J. Neurosci., October 31, 2018 • 38(44):9330 –9337 • 9333

Figure 3. Three-dimensional immunohistochemistry enables analysis of clinical samples, where transgenic or viral labeling strategies cannot be used. ScaleS and AbScale protocols ensure
reliable, reproducible, and homogeneous penetration of antibodies while conserving tissue ultrastructure (Hama et al., 2015). A–C, A 2-mm-thick brain slice from an Alzheimer’s disease (AD) model
mouse was stained with AlexaFluor 488-6E10 (green) and AlexaFluor 546-Iba1 (red) using the AbScale protocol, to visualize the distribution of A␤ plaques and microglia. High-magnification images
allow quantification of the distance between A␤ plaques and microglia in resting or activated states (B). Two representative A␤ plaques and microglia are shown, along with the distribution ofactivated and resting microglia distances (C). Scale bars: A–C, 50 ␮m. D, A 3D block of cerebral cortex from AD model mouse was stained with AlexaFluor 488-6E10 (green) and Texas Red lectin (red),
to reveal the distribution of A␤ plaques and blood vessels. Scale bar, 50 ␮m. E–J, A single plaque was tracked at multiple scales by successive imaging with light (LM) and electron microscopy (EM).
A 2-mm-thick slice from AD model mouse brain was cleared and stained by AbScale (E, F ). After restoration and re-fixation, 50 ␮m sections were made, followed by DAB staining with secondary
antibody (H, I ). Finally, 70-nm-thick ultrathin sections were imaged with TEM. The ScaleS protocol performs excellently in preserving the ultrafine structures at nanometer scale. Scale bars: F, G, 1mm; H, I, 50 ␮m; J, 2 ␮m.

et al., 2013; Murray et al., 2015; Ku et al., 2016). They have also
developed MAP (magnified analysis of proteome) that
expands entire brains fourfold linearly while preserving 3D
proteome for super-resolution molecular imaging. When thetransport of reagents into a large volume is diffusion-limited,chemical reactions are slow and uneven. To achieve uniform
tissue preservation and staining, the Chung group has developed SWITCH to control a wide range of chemical reaction
kinetics. In addition, they have developed stochastic electrotransport (Kim et al., 2015) that accelerates lipid removal andtissue staining to substantially improve the throughput of intact tissue phenotyping approaches. Together, these technologies enable rapid, integrated, multiscale phenotyping of
neuronal circuits.
Gradinaru’s group has developed hydrogel-tissue chemistry(HTC)-based strategies to stabilize endogenous fluorescentproteins and tissue biomolecules during successive rounds ofclearing, RNA labeling via single-molecule fluorescence in situ
hybridization or hybridization chain reaction (HCR), immunohistochemistry, and imaging at depth (Fig. 6; Yang et al., 2014;
Treweek et al., 2015; Shah et al., 2016; Greenbaum et al., 2017a,b).
Recent work on viral vector engineering has led to the generation ofnovel AAV capsids with enhanced tropism for specific cell types, andwith the ability to cross the blood– brain barrier when delivered systemically (Deverman et al., 2016; Chan et al., 2017). These vectors
permit noninvasive gene transfer and tunable multicolor labeling of
discrete cell populations across the CNS and PNS.
This sampling of current work demonstrates how the breathtaking complexity of the human brain and brains of otherpreclinical models continues to inspire technological advancements in whole-tissue clearing, fluorescent labeling, microscope
design, and analysis of large information-rich datasets. These9334 • J. Neurosci., October 31, 2018 • 38(44):9330 –9337

Mano, Albanese et al. • Tissue Clearing and Light-Sheet Microscopy

Figure 4. To acquire a fundamental understanding of tissue clearing by hydrophilic reagents, Ueda’s group strategically screened ⬎1600 chemicals, and identified key chemical properties that
contribute to the clearing. These include salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, andprotonation of phosphate ion for decalcification (Tainaka et al., 2018). By combining these insights, it is now possible to rationally design a new family of CUBIC protocols that are optimized, forexample, for large primate and human tissues. Further, Ueda’s group developed the CUBIC-X protocol, a fluorescent-protein-compatible, whole-organ clearing and homogeneous expansion method(Murakami et al., 2018), which can be combined with high-resolution LSFM to enable subcellular resolution imaging and digital identification of all cell nuclei in the adult mouse brain. A,
CUBIC-X-treated whole mouse brain (2.2-fold linear expansion) was stained with propidium iodide, and was imaged with high-resolution LSFM (NA ⫽ 0.6). Whole-brain 3D rendering (center) andzoom-in images of representative brain regions (side panels with two-step magnification) are shown. Each individual cell nuclei (and even its condensation pattern in some cells) were clearly
resolved across all brain regions, which were automatically detected by GPU-based high-speed algorithm. This allowed construction of the CUBIC-Atlas, a mouse brain atlas comprising an ensembleof ⬃72 million cells. Scale bars: 50 ␮m (after normalizing the sample expansion). B, Using a new CUBIC protocol optimized for preserving fluorescent protein (FP) signals (Tainaka et al., 2018), a
virtual-multiplex imaging of whole mouse brain was demonstrated. Seven individual brains with genetically encoded FPs were registered onto the reference brain, which revealed the brain-wide
distribution of unique cell types; pyramidal neurons (Thy1-YFP), astrocytes (Mlc1-YFP), oligodendrocytes (Plp-YFP), dopaminergic neurons (Th-EGFP), serotonin receptor 5B (Htr5b-YFP), anddopamine receptor D1 (Drd1-mVenus). Scale bars: macro view, 1500 ␮m; zoom-in view, 500 ␮m.

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Figure 5. A, CLARITY-processed mouse brains were stained with SYTO 16 (green), tomato lectin (red), and anti-histone H3 antibodies (white). Compared with standard diffusion (top row), Stochastic
electro-transport (SE) (bottom row; Kim et al., 2015) accelerates tissue labeling with fluorescent markers by applying a rotational electric field. SE achieves complete and uniform staining of an adult mousebrain in a single day. Scale bar, 1 mm. B, SWITCH (Murray et al., 2015) is a method that enables uniform tissue processing for large-scale tissues. Molecule transport is decoupled from its interaction with thesample. For example, DiD lipid dye under standard diffusion produces incomplete staining mostly saturated at the tissue surface (left). In SWITCH, the reactivity of fixatives, dyes, and antibodies is suppressed in
SWITCH-OFF buffer to allow the probes to readily diffuse deep into the tissue. For DiD, OFF buffer contains a low concentration of SDS detergent to block lipid binding until the tissue is saturated. Then, transfer
to “ON buffer” DiD dye binds to lipids (right). SWICTH technique produces rapid and uniform tissue staining compared with standard diffusion. Scale bar, 200 ␮m. C, Tissue processing in the Chung laboratory
enable multiple rounds of antibody staining. Here, we show a fully coregistered image showing various cell types in human visual cortex.D, MAP (Ku et al., 2016) physically expands tissues to achieve a fourfold
increase in image resolution. MAP is applicable to intact organs, such as mouse brain. Scale bar, 1 cm. E, Left, A 3D render of a cortical sample stained with neurofilament medium unit (NF-M; blue) and GABAB
receptor subunit-1 (GABABR1; red) antibodies; inset, synaptic neurofilaments colocalized with GABAergenic postsynaptic proteins. Middle, a maximum intensity projection image of calbindin (CB; magenta)
showing dendritic spines. Right, synaptic structures in MAP samples resolved with a presynaptic (bassoon) and postsynaptic marker (homer1) in the cortex. Yellow box shows a GFP-positive neuron; white box
highlightstheellipticalstructuresofpresynapticandpostsynapticproteinsdistributedatasynapticjunction.Theintensityofmarkersalongthesynapseaxisconfirmsseparationofpresynapticandpostsynaptic
markers. Scale bars: white, 50 ␮m; gray, 10 ␮m; yellow, 1 ␮m. F, SWITCH glutaraldehyde-reinforced human brain tissue shows a coregistered overlay of 9 of 22 different rounds of immunostaining.
Scale bar, 300 ␮m. G, Information-rich datasets obtained from multi-round staining can be used for quantitative analysis of 3D volumes. H, As a proof-of-concept soma diameter and distance tonearest blood vessel are compared for six different cell types (*p ⬍ 0.05, ***p ⬍ 0.001; ANOVA).

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Mano, Albanese et al. • Tissue Clearing and Light-Sheet Microscopy

Figure 6. Advancing modern neuroscience endeavors is the development of techniques for modifying chemically-distinct and/or genetically-specified cells and circuits for high-resolution
imaging at depth. Among these techniques are as follows: (A) HTC-based biomolecule stabilization and tissue-clearing (images adapted from Yang et al., 2014; Treweek et al., 2015; Treweek andGradinaru, 2016; Greenbaum et al., 2017a; Gradinaru et al., 2018), (B) viral vector engineering [left, Cre recombinase-based AAV targeted evolution (CREATE); right, vector-assisted spectral tracing(VAST); Deverman et al., 2016; Chan et al., 2017; Bedbrook et al., 2018; Challis et al., 2018], and (C, E) methodologies for cell-profiling in vivo and in situ. Recent advances in modern microscopy [e.g.,
LSFM and ultramicroscopy (Dodt et al., 2007); CLARITY-optimized light-sheet microscopy (Tomer et al., 2014); two-photon microscopy and microendoscopy (Jung et al., 2004; Barretto et al., 2011;
Marshall et al., 2016)], allow researchers to visualize deep tissue structures in real-time (C), and to image large tissue volumes rapidly and with subcellular resolution [D, E; HCR (Choi et al., 2014; Shahet al., 2016; Greenbaum et al., 2017b)]. Complementing these technologies are improved strategies for targeting individual cell populations and neuronal circuits, such as through the use ofgenetically engineered animal models (e.g., rodent fluorescent reporter and Cre driver lines; D, E) or through the systemic delivery (e.g., via retro-orbital (RO) injection) of viral vectors with uniquecell tropism (Deverman et al., 2016; Chan et al., 2017); B, Top left, For CNS targeting, AAV-PHP.eB was used to package single-stranded (ss) rAAV genomes expressing nuclear localized (NLS)
fluorescent reporters (XFP) from cell type-specific promoters for neurons (hSyn1, green), oligodendrocytes (MBP, red), or astrocytes (GFAP, blue), resulting in brain-wide gene expression upon RO
delivery; bottom left, for PNS targeting, AAV-PHP.S was used to package ss-rAAV genomes expressing XFPs from either neuron-specific (hSyn1) or tyrosine hydroxylase (rTH)-specific promoters, with
RO injection of ssAAVPHP.S:rTH-GFP and ssAAV-PHP.S:hSyn1-tdTomato-f (farnesylated) resulting in gene expression throughout TH⫹-containing cell bodies (green) and nerve bundles (red) of the
myenteric and submucosal plexus of the duodenum; images adapted from Challis et al., 2018) and/or multicolor labeling capabilities (Chan et al., 2017; e.g., VAST; B, right; E, left), the latter of which
promotes cell-sorting and long-range projection mapping endeavors (Bedbrook et al., 2018). D, E, Likewise, methodologies for labeling RNA transcripts and protein epitopes have been adapted foruse alongside tissue-clearing protocols (E; Shah et al., 2016; Greenbaum et al., 2017b). Here, HCR is uniquely suited to imaging RNA point-labels at depth with up to single-molecule precision (D),and to resisting label loss during immunohistochemistry (E) through its use of fluorescence signal amplification and direct in situ hybridization-based readout (Choi et al., 2014).

Mano, Albanese et al. • Tissue Clearing and Light-Sheet Microscopy

technological breakthroughs are paving the road to whole-brain
single-cell atlases and connectomes (maps of connected neurons)
for future neuroscientists.

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