Resource

Whole-Body Imaging with Single-Cell
Resolution by Tissue Decolorization
Kazuki Tainaka,1,2,3,4 Shimpei I. Kubota,1,4 Takeru Q. Suyama,1 Etsuo A. Susaki,1,2,3 Dimitri Perrin,2Maki Ukai-Tadenuma,2 Hideki Ukai,2 and Hiroki R. Ueda1,2,3,*
1Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan
2Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047,
Japan
3CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
4Co-first author*Correspondence: uedah-tky@umin.ac.jp
http://dx.doi.org/10.1016/j.cell.2014.10.034

SUMMARY

The development of whole-body imaging at singlecell resolution enables system-level approaches
to studying cellular circuits in organisms. Previous
clearing methods focused on homogenizing mismatched refractive indices of individual tissues,enabling reductions in opacity but falling shortof achieving transparency. Here, we show that an
aminoalcohol decolorizes blood by efficiently eluting
the heme chromophore from hemoglobin. Directtranscardial perfusion of an aminoalcohol-containing
cocktail that we previously termed CUBIC coupledwith a 10 day to 2 week clearing protocol decolorized
and rendered nearly transparent almost all organs ofadult mice as well as the entire body of infant andadult mice. This CUBIC-perfusion protocol enables
rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent
microscopy. The CUBIC protocol is also applicable
to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest thatwhole-body imaging of colorless tissues at highresolution will contribute to organism-level systems
biology.
INTRODUCTION
Since the discovery of cells in the organism about 350 years ago,whole-body imaging of single cells in opaque organisms likemammals has been a fundamental challenge in biology andmedicine. Imaging intact structures at single-cell resolution will
enable a systems-level elucidation of cellular connectivity anddynamics. This will improve our understanding of the generationand progression of diseases with stochastic and proliferative
processes such as autoimmune and malignant neoplastic diseases, because single-cell events in these diseases eventuallyaffect the health status of the entire organism. However, eventhe 30 g body of an adult mouse comprises more than 30 billion
cells, which makes it difficult to comprehensively identify cellular
circuits and quantitatively analyze dynamics. Conventional histology techniques are laborious and require tissue sectioning,which are challenging barriers toward rapid three-dimensional(3D) visualization of organ structures.
By contrast, optical sectioning with light-sheet microscopy
in combination with recent advances in tissue-clearing techniques is a promising route toward visualizing single cells within
a whole-organ or whole-body context (Dodt et al., 2007; Ertürk
et al., 2012; Keller and Dodt, 2012; Susaki et al., 2014; Tomeret al., 2014). The pioneering work by Werner Spalteholz first
introduced the principle of transparent 3D specimens a century ago (Spalteholz, 1914). Up until now, a number of tissueclearing reagents (BABB, THF-DBE, Scale, SeeDB, ClearT)and protocols (3DISCO, CLARITY, PACT-PARS) have beendeveloped (Becker et al., 2012; Chung et al., 2013; Dodt
et al., 2007; Ertürk et al., 2012; Hama et al., 2011; Ke et al.,2013; Kuwajima et al., 2013; Tomer et al., 2011; Yang et al.,2014). However, they cleared tissues to different degrees oftransparency. In order to observe deep tissue structures, weaimed for a level of transparency of internal organs (such asheart, lung, kidney, liver, pancreas, spleen, stomach, and small
and large intestines) and soft tissues (such as muscles) to theextent that: (1) chest and abdominal backbones and the bonesof limbs through internal organs and soft tissues from both
ventral and dorsal sides of the body are visible, and (2) individual
cells within intact organs and tissues are resolved by wholebody or whole-organ imaging. We previously demonstrated
that aminoalcohol-based chemical cocktails, termed CUBIC(clear, unobstructed brain imaging cocktails and computational
analysis), efficiently cleared whole brains of adult mice byremoving lipids to homogenizing refractive indices (RIs) without
signal loss from fluorescence proteins (Susaki et al., 2014). All
previous tissue-clearing methods, including ours, have mainly
focused on homogenizing RIs, and some of them based on
the lipid removal (BABB, 3DISCO, CLARITY, CUBIC, PACTPARS) succeeded in the clearing and imaging of adult wholebrain (Becker et al., 2012; Chung et al., 2013; Dodt et al.,Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. 911

2007; Ertürk et al., 2012; Susaki et al., 2014; Yang et al., 2014),
which is a relatively lipid-rich tissue. However, when applied
to the whole body, thick tissues block photons in the visible
region (400–600 nm) not only because of light scattering byendogenous materials with mismatched RIs, but also becauseof the light absorbance by endogenous chromophores—mainly
hemoglobin and myoglobin (Faber et al., 2003; Weissleder,
2001). Thus, efficient decolorization of endogenous chromophores inside tissues (particularly heme, which is one of themost abundant endogenous chromophores) is needed to clear
whole-body tissues. Classical treatment by the peroxide
decolorizes tissues. However, this peroxide treatment causes
not only serious damage to outer tissues (Steinke and Wolff,
2001), but also a significant loss of GFP signal (Alnuami et al.,2008). Heme tightly binds to hemoglobin and can be released
only in highly acidic (%pH 2) or basic (RpH 11) conditions(Kristinsson and Hultin, 2004; Teale, 1959). This highly acidic
or basic treatment also results in significant loss of signals
from GFP-related fluorescence proteins (Haupts et al., 1998).
Thus, a clearing method is needed to efficiently elute heme
in moderately basic conditions to enable GFP-related fluorescence proteins to retain their fluorescence properties.
In this study, we demonstrate that aminoalcohols within
CUBIC cocktails decolorized the blood by efficiently eluting theheme chromophore from hemoglobin in moderately basic conditions. An accelerated clearing protocol with CUBIC perfusion
rendered nearly transparent almost all organs of adult mice, as
well as the entire bodies of infant and adult mice, and enabled
rapid whole-body and whole-organ imaging with single-cell resolution. We used CUBIC to diagnose the pathology of Langerhans islets in the diabetic pancreas, as well as to anatomicallyannotate various organ structures in 3D. We also applied CUBIC
to 3D immunohistochemistry of various organs. These results
suggest that whole-body imaging with single-cell resolution
achieved by tissue decolorization can contribute to the foundation of the organism-level systems biology.
RESULTS
Aminoalcohols in CUBIC Cocktails Decolorized the
Blood by Efficiently Eluting Heme Chromophore
To achieve whole-body imaging with single-cell resolution, it isimportant to decolorize endogenous chromophores such asheme. When we immersed various organs directly (i.e., without
perfusion) to paraformaldehyde (PFA) and subjected them to
ScaleCUBIC-1 (Susaki et al., 2014; in this study, we term it‘‘CUBIC-1’’ for simplicity), we found that the color of the solvent
immediately turned a dark green. This observation led us to hypothesize that CUBIC-1 could solubilize and elute endogenous
heme from blood-infused tissues. To test this hypothesis, wefirst investigated whether CUBIC reagents can directly decolorize blood. The PFA-fixed blood suspension was washed threetimes with CUBIC-1, resulting in transparent pellet and olivegreen supernatant (Figure 1A). On the other hand, the pellet
and the supernatant from PBS-treated blood suspension
remained dark red and colorless, respectively. Among CUBIC1 constituents, N,N,N’,N’-tetrakis(2-hydroxypropyl)ethylenediamine (termed here as ‘‘aminoalcohol #10’’) (Susaki et al.,912 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

2014) alone, but not other reagents, is sufficient to decolorize
the blood (Figures 1A and 1B). The visible spectra of chemicallytreated erythrocytes were recorded to investigate what kind ofolive-green molecule was eluted from the blood into the supernatant. The visible spectrum from the PBS-treated erythrocyte
suspension is quite different from that of erythrocyte suspension
treated by 0.1 M NaOH (initial pH = 13), aminoalcohol #10
and CUBIC-1 reagent (Figure 1C). We also recorded the visible
spectra from purified hemin (free heme chromophore released
from hemoglobin), purified biliverdin (which is a product ofheme catabolism), iron(II) chloride, erythrocytes, and supernatant of PFA-fixed blood treated by aminoalcohol #10 (Figure 1D)
or CUBIC-1 reagent (Figure S1A available online). The normalized spectra of the erythrocyte and supernatant overlappedalmost completely with hemin solutions in aminoalcohol #10
or CUBIC-1, which suggests that heme is released from hemoglobin in erythrocytes when treated by aminoalcohol #10 or the
CUBIC-1 reagent.
We noted that aminoalcohol #10 and CUBIC-1 reagent havebuffering capacity in pH 9–11 whereas other reagents did not(Figure S1B). If buffering capacity in basic pH is critical for thedecolorizing capability, we predicted that a basic solution containing 0.01 M NaOH, which usually elutes heme from hemoglobin, will lose its decolorizing capability at a higher concentrationof erythrocytes because the higher concentration of cells will
shift the pH from basic to neutral. To test this prediction, we prepared a mixture of erythrocytes and these chemicals at different
ratios (Figure 1E). Although urea and Triton X-100 in basic solution moderately promoted heme release at high chemical ratios,these were less effective at lower concentrations. On the other
hand, aminoalcohol #10 and CUBIC-1 reagent promoted heme
release even at high erythrocyte ratios. As shown in Figure 1C,
the absorbance ratio between 600 nm and 575 nm can be used
as a quantitative index for the efficiency of heme release.
OD600/OD575 values of samples from Figure 1E were therefore
plotted against erythrocyte/chemical ratio (Figure S1C). These
results quantitatively confirmed that aminoalcohol #10 in the
CUBIC-1 reagent can efficiently promote heme release regardless of the erythrocyte/chemical ratio and can work as adecolorizing buffer at moderate basic pH. We evaluated the
pH dependence on the efficiency of heme release for aminoalcohol #10, CUBIC-1 reagent, and non-buffered basic solution,
respectively (Figure 1F). In non-buffered basic solution, heme
was efficiently released from hemoglobin, but only at pH >11,
as previously reported (Kristinsson and Hultin, 2004). On theother hand, aminoalcohol #10 efficiently eluted heme even inmoderately basic conditions around pH 10, which is within
the optimal pH range for fluorescence signal intensity of GFPrelated fluorescence proteins (Haupts et al., 1998). CUBIC-1 reagent further promoted heme release even around pH 9, whichmight be attributed to a combinatorial effect with urea and/or
Triton X-100. These results suggested that aminoalcohol #10
in CUBIC-1 reagent expanded the pH window for efficientheme release. To clarify the chemical properties of aminoalcohol #10 associated with its decolorizing capability, we investigated erythrocyte with a series of aminoalcohol derivatives(Figures S1D and S1E). These results revealed the chemical
properties (i.e., higher amines without carboxylic groups)

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Figure 1. Aminoalcohols in CUBIC Cocktails Decolorized the Blood by Efficiently Eluting Heme Chromophore(A) Decolorizing of the PFA-fixed blood by the previously published CUBIC-1 cocktail (‘‘CUBIC-1’’) and its constituents. Numbering of aminoalcohol #10 was
derived from the previous chemical screening. Bright-field images of pellet and supernatant from fixed blood samples washed with PBS, CUBIC-1 reagent, 25 wt% aminoalcohol #10, 25 wt% urea, 15 wt% Triton X-100, or 0.01 M NaOH, respectively. pH of the original liquid and supernatant is shown at the bottom.
(B) Decolorizing PFA-fixed blood by CUBIC-related chemicals. Bright-field images of pellet and supernatant from PFA-fixed blood samples washed with PBS,
CUBIC-1 reagent (‘‘CUBIC-1’’), a mixture of 25 wt% urea and 15 wt% Triton X-100 (‘‘Urea + Triton X’’), 25 wt% glycerol (‘‘Glycerol’’), or a mixture of 25 wt% urea,
25 wt% glycerol and 15 wt% Triton X-100 (‘‘Modified ScaleA2’’), respectively. pH of the original liquid and supernatant is shown at the bottom.
(C) Visible spectra of a chemically treated 0.1% mouse erythrocyte suspension. Erythrocytes were mixed with PBS (gray), 0.1 M NaOH (black), aminoalcohol #10(magenta), or CUBIC-1 reagent (red), and were incubated at 37 C overnight. Inset: magnification of the Q-band region.
(D) Normalized visible spectra of 0.1% mouse erythrocyte (black), 1/10 diluted supernatant from (A) (red), 10 mM hemin (blue), 10 mM biliverdin (orange), and100 mM iron(II) chloride (green), in 25 wt% aminoalcohol #10.
(E) Aminoalcohol #10 in CUBIC reagents facilitates the release of heme independent of the mixing ratio with erythrocytes. Bright-field images of the 1% to 20%
erythrocyte suspension mixed with CUBIC-1 reagent, 25 wt% aminoalcohol #10, 25 wt% basic glycerol, 25 wt% basic urea, 15 wt% basic Triton X-100, and0.01 M NaOH. Indicated value in each well corresponds to pH.
(F) Aminoalcohol #10 in CUBIC reagents significantly expands the pH range of heme release. 1% erythrocyte suspension including 1-100 mM NaOH (black
diamond, n = 3), aminoalcohol #10 (pH = 9.4-11.0, magenta square, n = 3), CUBIC-1 (pH = 9.2-11.1, red circle, n = 3), and 25 wt% tris(hydroxymethyl)aminomethane (pH = 10.6-11.8, green triangle, n = 3) incubated at 37 C overnight. Visible spectra of these samples were recorded. Because OD600/575 correlates withthe efficiency of heme release, OD600/575 of these samples is plotted against pH.
Data represent the average ± SD. See also Figure S1.

associated with efficient decolorizing and might reflect the affinity of these chemicals to the heme chromophore. Overall, aminoalcohol in CUBIC reagents critically contributed to blood

decolorization, which is assisted by its chemical capacities tobuffer pH in moderately basic conditions and to expand thepH window for efficient heme release.
Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. 913

Whole-organ clearing protocol

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Figure 2. CUBIC Provides a Simple and Efficient Whole-Organ and Whole-Body Clearing Protocol(A) (Top) Whole-organ clearing protocol in 10 days. (Middle) Transmission images of whole organs (brain, heart, lung, kidney, liver, pancreas, spleen, and muscle)from adult C57BL/6N. In the CB-Perfusion protocol, deeply anesthetized mice were sacrificed and transcardially perfused with PBS, 4% PFA/PBS (w/v), and 1/2
diluted CUBIC-1 reagent. The excised organs were further cleared by CUBIC-1 reagent without postfixation in 4% PFA/PBS. (Lower-left) Bright-field images ofwhole stomach and intestine from C57BL/6N treated with the CUBIC, CB-Perfusion protocol, SeeDB, or PBS using 10-day clearing protocol (Scale-treated(legend continued on next page)

914 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

CUBIC Is a Simple and Efficient Whole-Organ andWhole-Body Clearing Protocol
In this study, we focused on the development of an immersiononly tissue-clearing protocol based on hydrophilic clearing reagents (Extended Experimental Procedures). We evaluated theclearing performance of Scale, SeeDB, and CUBIC protocols invarious organs (Hama et al., 2011; Ke et al., 2013; Susaki et al.,2014). Each protocol was based on the experimental conditionsof the original papers, as shown in Figure 2A, except for a daily
exchange of the clearing medium. We measured the temporaldevelopment of average transmittance of different organs (brain,heart, lung, kidney, and liver) with each protocol (Figure S2A) andobtained transmittance curves of these organs around the visible
region after 10 days (Figure S2B). The CUBIC protocol was superior to other methods in both clearing kinetics and plateau performance in transmittance for all organs studied. We confirmed that
the CUBIC protocol enhanced the transmittance of organs in the
480–680 nm range, as expected from the decolorizing capabilityof CUBIC reagents. Bright-field images of different organs (brain,heart, lung, kidney, liver, pancreas, spleen, muscle, stomach andintestine, and skin) after 10 days also demonstrated that all organs treated with CUBIC reagents resulted in the most transparent and decolorized images (Figure 2A).
Based on the blood decolorizing capability of the CUBIC-1
reagent, transcardial perfusion of this reagent (and hence
the blood decolorizaion through blood vessels) will enhancewhole-organ and whole-body clearing. We thus developed a
CUBIC perfusion (CB-Perfusion) protocol to achieve whole-organ and whole-body clearing. In the original CUBIC protocol, tissues were conventionally fixed by transcardial perfusion of 4%
PFA (25–50 ml) and were immersed in 4% PFA at 4 C overnight
as a postfixation. In the advanced CB-Perfusion protocol, tissues in the entire body were fixed by prolonged transcardial
perfusion of 4% PFA (150 ml) and were then cleared by subsequent transcardial perfusion of 1/2 diluted CUBIC-1 reagents(20 ml). Each organ was then extracted and further cleared by
CUBIC reagents. The CB-Perfusion protocol accelerated theclearing kinetics and further enhanced the plateau performancein transmittance when compared with the original CUBIC protocol (Figures S2A and S2B). In a bright-field imaging comparisonof all organs, the CB-perfused organs were the clearest (Figure 2A). These results indicate successful development of thewhole-organ clearing protocol, which finishes within 10 days.
We next attempted whole-body clearing of infant (postnatal
day 1 [P1] and P6) and adult mice (19-week-old) by using thisCB-Perfusion protocol. After transcardial perfusion of 4% PFA
and 1/2 diluted CUBIC-1 reagent and subsequent detachmentof skin, the entire body was cleared by the CUBIC-1 reagent.
We noticed that the clearing medium (CUBIC-1 reagent) immediately turned a dark green color probably due to decolorizationof endogenous chromophores (mainly heme). To facilitate thewhole-body clearing, clearing medium were refreshed daily for

2 weeks. The clearing medium gradually became less colored,
and after 2 weeks, the apparent transparency of the wholebody became saturated. Whole-body samples can be thenstored in CUBIC-1 reagent and even shaken at 37 C overmonths because the high pH of CUBIC-1 reagents prevents proliferation of microorganisms. Bright-field images of infant mice(P1 and P6) demonstrated that CB-Perfusion protocol transparentized and decolorized P1 and P6 infant mice (Figure 2B). We
confirmed the visualization of chest and abdominal backbones
as well as bones of limbs through internal organs and soft tissues
from both ventral and dorsal sides of the body. Bright-field images of adult mice (19-week-old) demonstrated that CB-Perfusion protocol also markedly transparentized and decolorized
adult mice (Figures 2B and S2C). The bones of forelimbs(including scapula, humerus, radius, and ulna) and hindlimbs(including femur, tibia, and fibula) and caudal vertebrae wereclearly visualized in either V-D or the dorsal-to-ventral (D-V) images of adult mice.
CUBIC Is Applicable to Whole-Body Imaging of Infantand Adult Mice with Single-Cell Resolution
The high transparency of CB-perfused samples prompted us
to perform light-sheet fluorescence microscopy (LSFM). As
previously described, the CUBIC protocol is compatible withwhole-brain nuclear counterstaining (Susaki et al., 2014). We
thus applied nuclear counterstaining by propidium iodide (PI)to whole-body samples and imaged PI-stained CAG-EGFP
transgenic (Tg) (Okabe et al., 1997) P1 mouse transparentizedand decolorized by the CB-Perfusion protocol. Whole-body images were obtained from two opposite directions (D-V and V-D)
(Figure 3A). Both images were sufficiently clear even at 4–5 mm
depth. Nuclear staining with PI tends to highlight non-muscularorgans and solid tissues, whereas CAG-EGFP signals weremore intense in the muscular organs such as heart and subcutaneous tissue. Magnified images from the same sample werealso obtained to visualize internal structures of head, chest,
abdominal, and pelvic organs as well as limbs (Figures 3B,
S3A, and S4). These images were then deconvolved by AutoQuant X3 software (we denoted the deconvolved images as prefix ‘‘DCV-’’ in the figures). For example, the granule duct in thesubmaxillary gland, coronary vessels in the heart, bronchiole
in the lung, gastric walls in the stomach, vascular structures in
the liver, renal cortex and medulla in the kidney, villus in the intestine, and penis and testis in the scrotum were clearly identified ineach z stack image (Figure S4). Surprisingly, detailed structuresin hippocampus and cerebellum were visible through the skull,central canal inside the spinal cords were seen through vertebrae, and internal structures of tibia in the leg were also visualized (Figures 3B and S4). We note that the magnified imagesof these organs resolved to single-cell levels (Figure 3C, top).
We also performed whole-body imaging of PI-stained CAGEGFP Tg P6 mouse transparentized and decolorized by thestomach and intestine were usually fragile and torn during the procedure due to proliferation of microorganisms; thus it was impossible to take comparison data).
(Lower-right) Bright-field images of skin from BALB/c-nu/nu treated with CUBIC reagents and PBS using the 10-day clearing protocol.
(B) Whole-body clearing protocol in 2 weeks. Bright-field images (ventral and dorsal view) of whole body (C57BL/6N P1, P6, and adult mice) stocked in PBS after
fixation or subjected to the CB-Perfusion protocol. 8-week-old mouse for the PBS sample and 19-week-old mouse for the cleared sample are shown.
See also Figure S2.

Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. 915

A CAG-EGFP Tg P1 whole body images with PI staining
V-D imageZ = 4.0 mm

C Magnified view

D-V imageZ = 3.5 mm

P1 Chest (V-D)
PI, Z = 2.5 mm

100 μmAdult Chest (V-D)
PI, Z = 3.0 mm

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B

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Figure 3. CUBIC Is Applicable to WholeBody Imaging of Infant and Adult Mice with
Single-Cell Resolution(A) 3D-reconstituted and X-Y plane (indicated withZ position), lower-resolution whole-body images of
PI-stained CAG-EGFP Tg P1 mouse. Images wereacquired with light-sheet fluorescence microscopy(LSFM) from the ventral-to-dorsal (V-D) and dorsalto-ventral (D-V) directions, respectively.
(B) 3D-reconstituted and X-Y plane body images of
PI-stained CAG-EGFP Tg P1 mouse. Head (D-V),
chest organs (V-D), abdominal, and pelvic organs(V-D and D-V) are shown. All images were deconvolved with AutoQuant X3 software. Prefix ‘‘DCV-’’
indicates the deconvolved image.
(C) Magnified images in (B) (Chest, Z = 2500) and(D) (Chest, Z = 3000) indicating these images takenwith single-cell resolution. Each dot is PI signals of
single cell nucleus.
(D) 3D-reconstituted and X-Y plane body imagesof the PI-stained CAG-EGFP Tg adult mouse(8-week-old). Chest organs (V-D), upper abdominalorgans (V-D), lower abdominal organs (D-V), andhindlimb are shown. Enlarged parts in (C) are indicated as a white box in (B) and (D). Prefix ‘‘DCV-’’
indicates the deconvolved image.
See also Figures S3 and S4.

2 mm

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D CAG-EGFP Tg 8w body images with PI staining
DCV-Chest (V-D)

Z = 3.0 mm

Abdominal (Upper, V-D)
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Z = 2.0 mm
4 mm

abdominal organs, vascular structure in
the liver, cortex, and medulla in the kidney, muscularis externa in the stomach,
2 mm
2 mm
and villus in the intestine were also visualized (Figure 3D). Tibia and fibula as well
Abdominal (Lower, D-V)
Z = 3.0 mm
Hindlimb
Z = 4.0 mm
as fiber orientation of gastrocnemius andmuscles in the hindlimb were also clearlyvisualized (Figure 3D). We note thatthe images of these adult organs also
4 mm
4 mm
4 mm
4 mm
achieved single-cell resolution (Figure 3C,
bottom). These results demonstrated that
CUBIC is applicable to the imaging of the
CB-Perfusion protocol. Because the P6 mouse was too large to internal structures of entire body regions (chest and abdominal
be captured in a single view, we obtained whole-body images organs, forelimb, and hindlimb) with single-cell resolution.
from three views (head, chest, and abdomen, Figure S3C). The
quality of whole-body images of P6 mice was almost compara- CUBIC Is Applicable to Whole-Organ Imaging with
ble to that of P1 mice and visualized the internal structures of Single-Cell Resolution
head, chest, abdominal, and pelvic organs as well as limbs.
For rapid whole-organ imaging, we used a knockin (KI) mouse
We next performed whole-body imaging of PI-stained CAG- strain expressing three tandem repeats of mKate2 with a nuclear
EGFP Tg adult mice (8-week-old) transparentized and decolor- localization signal, under control of the CAG promoter (Niwa
ized by the CB-Perfusion protocol. Adult mice bodies could et al., 1991) from the 30 -UTR of b-actin gene locus (Tanaka et al.,not fit into existing LSFMs, so we dissected the adult body into 2012). We used mKate2 because of its signal intensity, photofour parts (chest organs, abdominal organs, forelimbs, and hin- stability, and rapid protein maturation (Chudakov et al., 2010;
dlimbs) and then obtained their images (Figures 3D and S3B). Shcherbo et al., 2009), as well as its resistance to fluorescence
Adult mouse tissues exhibited much higher contrast between quenching by CUBIC reagents, as previously described (Susaki
PI and EGFP signals than those of infant mice. For example, et al., 2014). We then performed whole-organ imaging of various
EGFP signals were much higher in heart, pancreas, stomach, CB-perfused organs, including heart, lung, kidney, liver, pancreas,
and muscles probably because EGFP expression from CAG pro- spleen, muscle, stomach, and intestine from this b-actin-nuc-3 3
moter in these organs was elevated. In z stack images of chest mKate2 KI mouse (8-week-old) and counterstained with SYTO 16,
organs, ventricle structures in the heart and bronchiole in the a cell-permeable green-fluorescent nucleic acid stain. The resultlung can be clearly visualized (Figure 3D). In z stack images of ing 3D-reconstituted image of each organ in this mKate2 KI
916 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

A β-Actin-nuc-3×mKate2 KI #1/SYTO 16
DCV-HeartZ = 2.0 mm

X = 6.0 mm

Z = 4.0 mm

Y = 2.0 mm

X
Y

2 mm

2 mm

100 μmB DCV-Lung
2 mm

2 mm

2 mm

Z = 1.8 mm

Z = 4.2 mm

2 mm

2 mm

E

X = 17.0 mm

2 mm

2 mm

X

CAG-EGFP Tg #1/PI
Skin

100 μm2 mm

C DCV-KidneyX

Z = 1.9 mm

Z = 2.5 mm

X = 5.5 mm

Magnified

Y = 5.3 mm

1 mm

2 mm

2 mm

2 mm

2 mm

2 mm

Y

Ear
2 mm

100 μmD DCV-LiverZ = 2.5 mm

Z = 3.7 mm

X = 15.0 mm Y = 10.0 mm

X
2 mm

2 mm

Y

Magnified
2 mm

100 μm

2 mm

2 mm

1 mm

Figure 4. CUBIC Is Applicable to Whole-Organ Imaging with Single-Cell Resolution
The reconstituted 3D and section images of a heart (A), lung (B), kidney (C), and liver (D) from nuclear-stained b-actin-nuc-3 3 mKate2 KI mouse (8-week-old) andskin and ear (E) from CAG-EGFP Tg mouse (10-week-old) were acquired with LSFM. Raw X-Y section images and reconstituted Y-Z and X-Z section images areat the indicated positions. Prefix ‘‘DCV-’’ indicates the deconvolved image. In (A–D), enlarged images indicated by a white box on z plane images are shown,indicating that these images are taken at single-cell resolution. Each dot is nuclear-localized mKate2 signals from single cell nuclei.
See also Figures S5 and S6.

mouse, as well as its horizontal, coronal, and sagittal sections,enabled the visualization of spatial gene expression patterns(from the CAG promoter at b-actin 30 -UTR) and examination ofdetailed internal structures (Figures 4A–4D and S5A–S5F). For
example, 3D reconstitution images of the heart successfully visualized not only lumens of atria and ventricle, but also internal
Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. 917

papillary muscle, coronary vascular structures, and even valve
structures (Figure 4A). In lungs, the networks of trachea, bronchi,
and bronchiole in both lobes were clearly identified (Figure 4B). In
the kidney, 3D reconstitution images easily distinguished between
renal cortex, medulla, and pelvis (Figure 4C). In the liver, two kindsof vascular structures were identified as thicker vessels, which
would indicate the hepatic artery, and thinner vessels, which
would indicate the portal vein (Figure 4D). Langerhans islets andpancreatic ducts were identified in the pancreas (Figure S5A).
Splenic white pulp and red pulp were identified in the spleen (Figure S5B). Orientations of muscle fibers and blood vessels wereobserved in the muscle (Figure S5C). Corpus glands were significantly highlighted, and pylorus and cardia were clearly observedin the stomach (Figure S5D). Mesentery and villi were seen in theintestine (Figure S5E). We also obtained 3D reconstitution imagesof brain as well as kidney and muscle, which are contralateral tothose in Figures 4C and S5C from the identical mouse body (Figure S5F). In addition to imaging internal organs and muscles, wecollected skin and ears from pelage-removed CAG-EGFP Tg
mouse and subjected them to CUBIC reagents with nuclear-counterstain PI. We then performed whole-organ imaging of thesetissues (Figure 4E). EGFP signals highlighted epidermis, muscle
layer under hypodermis, and residual pelage, whereas PI signal
intensity was relatively higher in hair matrix cells. Taken together,
these results demonstrate that CUBIC enables whole-organ imaging with single-cell resolution of almost all organs by using various
fluorescence proteins.
The successful whole-organ imaging using mKate2 led us totest other fluorescence proteins, including EGFP and YFP. We
performed similar whole-organ imaging of each organ from threestrains of CB-perfused adult mice: CAG-EGFP Tg (Figure S6A),
an EGFP-fused histone-2B-expressing strain (R26-H2B-EGFP[CDB0239K]; Figure S6B) (Abe et al., 2011), and a Thy1-YFP-H
Tg strain (Figure S6C) (Feng et al., 2000), respectively. PI wasused as nuclear staining for whole-organ imaging of thesestrains. When we captured the section images of each organfrom CAG-EGFP Tg (Figure S6A) and b-actin-nuc-3 3 mKate2
KI mice (Figures 4 and S5), the spatial distribution of fluorescence proteins (EGFP and nuc-3 3 mKate2) was markedly
different. This is because EGFP was localized in the nucleus
and cytosol, whereas mKate2 was localized only in the nucleus.
Similarly, we could obtain 3D reconstitution images of whole organs from R26-H2B-EGFP and the neuronal marker Thy1-YFP-H(Figures S6B and S6C). These results demonstrate that CUBIC
protocol enables whole-organ imaging with various fluorescence
proteins (mKate2, EGFP, and YFP) and that whole-organ imaging with single-cell resolution can extract 3D single-cell information of internal anatomical structures in different organs.
CUBIC Is Applicable to 3D Pathology of Langerhans
Islets in the Diabetic Pancreas
One attractive application of whole-body and whole-organ imaging with single-cell resolution is 3D pathology of organs. To test
this potential, we investigated the spatial distribution of Langerhans islets (LIs) in diabetic and normal pancreases by using the
CB-Perfusion protocol. As a model, we first focused on diabetic
pancreases because BABB-based analysis combined with
optical projection tomography (OPT) has already been applied
918 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

(Alanentalo et al., 2007). Streptozotocin (STZ) is a well-known
cytotoxic agent for pancreatic b cells, which disrupts the abilityof b cells to secrete insulin and stabilize blood sugar levels(King, 2012). High-dose STZ injection destroys b cells, which
are major constituents of LIs and induce type I diabetes mellitus.
LIs contract because of b cell destruction after the onset of diabetes. We therefore performed comprehensive and statisticalanalysis of diabetic LIs in the whole pancreas of adult mice. We
dissected and transparentized pancreases from saline-treatedcontrol mice (n = 3) and acute type I diabetic mice (n = 3, whose
blood sugar levels were over 400 mg/dl as shown in Table S1) by
the CB-Perfusion protocol with PI staining. Acquired images
enabled us to morphologically distinguish the characteristic clusters (LIs) from the characteristic tracts (pancreatic ducts) (Figure 5A). We identified and 3D reconstituted all LIs according to
Figure 5A and Extended Experimental Procedures. The image
analysis also provided a spatial distribution of LIs in the pancreas.
For example, larger LIs tend to be localized along pancreatic
ducts. By using the extracted LI data, we can compare the total
number of LIs between diabetic and healthy mice. As expected,
the average number of LIs was significantly reduced by the onsetof diabetes (Figure 5B, left, p = 0.005). The differences of average
LI volume between diabetic and healthy mice were also marginally significant (Figure 5B, center, p = 0.09), which is consistent
with the observed reduction in the existence probability of larger
LIs in diabetic mice (>1.0 3 107 mm3, Figure 5B, right). These data
indicate that larger LIs are more susceptible to b cell impairment
induced by STZ administration. Overall, these results indicatethat CUBIC enables 3D pathology of a diabetic pancreas.
CUBIC Is Applicable to 3D Anatomy of Various Organs
The successful 3D pathology of diabetic pancreases prompted
us to generalize this to 3D anatomy of various organs, which
may provide a versatile platform for elucidation of cellular mechanisms underlying the physiology and pathology of observed
anatomical structures. As a test case, we analyzed whole-heart
images from mKate2 KI mouse. We noted that mKate2 signals
are more intense in the surface of the tracts, which can bemorphologically annotated as coronary vessels (Figure 6A). We
extracted, by using the surface analysis in the Imaris software,
high-intensity signals in the heart and excluded the surfaces ofsmaller volume size, resulting in 3D images of coronary vessels
covering the entire heart (Figure 6A and Movie S1). Internal
structures such as tendinous cords and valves could be alsovisualized (Figure S7A). We also analyzed whole-lung images
from the same mKate2 KI mouse. We noted that mKate2 signals
in respiratory tract fall to undetectable levels, and thus we canextract low-intensity signals in the lungs, resulting in a bronchial
tree covering the entire lung (Figure 6B and Movies S2 and S3).
Interestingly, the 3D-reconstituted image of the tree includes
main stem bronchus, bronchi, bronchioles, and even alveoli
structures. We next analyzed whole-kidney images from thesame mKate2 KI mouse. Renal cortex, medulla, and pelvis
were clearly distinguished and annotated with characteristic
signal patterns of SYTO16 and mKate2 (Figure 6C and Movie
S4). We further analyzed whole-liver images from the same
mKate2 KI mouse (Figure 6D and Movie S5). We extracted two
vessel structures inside of the image, which were characterizedA#1 (Position 3)

LI identification (Z = 1750 μm)

LI / Duct (Surface + section)

1 mm

1 mm

1 mm

LI 3D-mapping

Merge

1 mm

LI counting and volume analysis

1 mm

1 mm

B
200
150
100
50

60
50
40
30
20
10

Saline i.p.

200 mg/kg STZ i.p.

4

12
10

2

8
6

0

100-120120-140140-160160-180180-200 > 200

4
2
0

0

0

14

Saline i.p.

200 mg/kg STZ i.p.

05- 5
10 1 0
15 15
20 20
25 25
30 30
35 35
40 40
45 45
50 50
55 55
60 60
6 5 6570 70
75 7 5
80 80
85 85
90 90
95 -95-100

250Volume probability (%)

70

Average islet volume

Average islet number

300

1

Islet volume (×105 μm3)

C

Extracted islets

Original image

1 mm

1 mm

Semiautomatical analysis

1 mmby high and low SYTO 16 signals, and were thus identified as hepatic arteries and portal veins, respectively. Finally, we extended
similar anatomical analysis to whole-body images of PI-stained
CAG-EGFP Tg P1 mouse (Figure 3). Each organ inside of thechest and upper abdomen were clearly visualized (Figure S8A
and Movie S6) with their inside structures, such as a bronchial
tree in the lung (Figure S8B), vascular structures in the liver (Figure S8C), villi in the intestine (Figure S8D), and spatial relations ofesophagus and esophageal gland (Figure S8E). These results
suggest that CUBIC enables 3D anatomy of various organs
even in whole-body images, which will provide a versatile platform for systems-level elucidation of cellular circuits underlying
the physiology and pathology of various organs of interest.
CUBIC Is Applicable to 3D Immunohistochemistry of
Various Organs
Another challenge for establishing whole-organ imaging is thedevelopment of 3D immunohistochemistry (3D-IHC). In our previous work, we have demonstrated that CUBIC was applicableto the 3D-IHC using large blocks of brain tissue (Susaki et al.,2014). According to the protocol, we performed 3D-IHC of

LI counting and volume analysis

Figure 5. CUBIC Is Applicable to the 3D Pathology of Langerhans Islets in the Diabetic
Pancreas(A) The procedures for image analysis of LIs inpancreases. First, LIs in each X-Y plane image
were manually identified. Seven LIs (white circles)
were identified in the center-top panel. Then, theseLIs were visualized in a 3D image with surface
analysis on the Imaris software. In the upper-rightpanel, the seven LIs (yellow) are in the plane. Fourother LIs (blue) are not in the same plane. The
pancreatic duct (green) is also indicated. The
spatial distribution of LIs (blue) as well as apancreatic duct (green) is displayed in the lowerleft and lower-center panels. Finally, LI counting
and volume analysis were conducted on the Imaris
software, shown in the lower-right panel.
(B) Statistical analysis of LIs in the whole pancreas
from control mice (n = 3) and diabetes mice (n = 3).
Data represent the average ± SE. The average
number of LIs was significantly reduced by theonset of diabetes (left, p = 0.005). The differencesof average LI volume between diabetic and healthy
mice were also marginally significant (center,p = 0.09), which is consistent with the observed
reduction in the existence probability of larger LIs
in diabetic mice (right, > 1.0 3 107 mm3).
(C) Computational, semi-automated identificationof LIs. Position 1 image of #1 mouse was used.
(Left and center) Raw and processed image withextracted islets (green circles) at plane 80. (Right)
Reconstituted 3D image with all identified islets.
See also Table S1.

nuclear-stained whole-organ samples(Figure 7A). Organ samples with CBPerfusion were subjected to CUBIC-1
treatment, followed by 3D-IHC with antibodies to two proteins expressed in theorgans. We used a-smooth muscle actin (a-SMA) antibody to label the heart, stomach, and intestine and pan-cytokeratin antibody to the lung. After immunostaining, organ samples weretreated with CUBIC-2 for several hours to 1 day, and the resulting
samples were observed with LSFM. The immunostained signals
were detected over 1 mm depth and were visualized as 3D-reconstituted images (Figure 7). In the heart, the immunofluorescence signal for a-SMA antibody clearly highlighted vascular
structures (Figure 7B). Epithelial cells in the lung were visualized
by immunostaining with the antibody to pan-cytokeratin (Figure 7C). Previously reported staining pattern of pan-cytokeratin
was recapitulated by CUBIC-based 3D-IHC (Xiang et al.,2007). a-SMA antibody also visualized smooth muscular cells
in the stomach and intestine (Figures 7D and 7E). The CUBIC
protocol is thus applicable to the 3D-IHC of various organs.
1 mm

DISCUSSION
Aminoalcohols in CUBIC Enable Decolorization of Blood
Almost all animals are not transparent because their body iscomposed of RI-mismatched materials as well as light-absorbing
Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. 919

β-Actin-nuc-3×mKate2 KI #1/SYTO 16

A

mKate2(3D)Coronary vessel (Surface)

mKate2(section)
Heart ventricles (Surface)

Coronary vessel (Surface)

RAu

Aorta
PA

RAu

RAt
LA

PV

Heart

RV

LV

LV

2 mm

2 mm

Reverse image

Front image

Bronchial tree
(Surface)

mKate2(3D)
Bronchial (Surface)B
Bronchial tree
(Surface + section)

2 mmAlveoliLung

mKate2 (section)(X = 7300 μm)

2 mm

C

SYTO 16 (section)(Y = 6300 μm)

Bronchial tree

Internal structures of kidney(Surface + section)
SYTO 16 (3D)Internal structures of kidney (Surface)

Internal structures of kidney (Surface)

2 mm

KidneyMedullaCortexRenal pelvis

2 mm

D

SYTO 16 (section)(X = 13800 μm)

Vascular structures(Surface + section)

SYTO 16 (3D)
Vascular structures (Surface)

2 mm

Vascular structures (Surface)

Liver

Hepatic artery

2 mm

Portal vein

Figure 6. CUBIC Is Applicable to the 3D Anatomy of Various Organs
Extraction of internal structures was performed with surface analysis of Imaris software. The reconstituted 3D images of the heart (A), lung (B), kidney (C), and liver(D) from the SYTO 16-stained b-actin-nuc-3 3 mKate2 KI mouse (8-week-old) in Figure 4 were used. After surface extraction by the software, each structure wasmanually curated and extra surface signals were eliminated.
(A) Structural identification of coronary vessels and ventricles in the heart. Contiguous mKate2 signals with high or low intensity within the heart were extracted ascoronary vessels (left two panels) or ventricles (right two panels). RAu, right auricle; RAt, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; PA,
pulmonary artery; PV, left ventricle.

(legend continued on next page)920 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

pigments. Although a number of tissue-clearing methods (BABB,
THF-DBE, Scale, SeeDB, ClearT) and protocols (3DISCO,
CLARITY, PACT-PARS) have been developed, the importanceof tissue decolorization may have been overlooked because
these methods were mainly optimized for pigment-less brains
and hence focused on the adjustment of mismatched RIs (Becker
et al., 2012; Chung et al., 2013; Dodt et al., 2007; Ertürk et al.,2012; Hama et al., 2011; Ke et al., 2013; Kuwajima et al., 2013;
Yang et al., 2014). However, when applied to whole body, thicktissues may block photons in the visible region due to the absorbance by endogenous pigments. Animals genetically lacking
endogenous pigments have see-through bodies such as the
medaka, in which superficial organs can be visualized across
body surfaces (Ohshima et al., 2013; Wakamatsu et al., 2001).
Therefore, to visualize detailed structures in deeper organs bysingle-photon excitation microscopy, we must overcome thelight absorbance by heme, which is one of the most abundant
chromophores in the body (Faber et al., 2003; Weissleder,
2001), in addition to light scattering by mismatched RIs. In theprevious study, we demonstrated that aminoalcohols in CUBIC
cocktails significantly enhanced whole-brain clearing via homogenizing mismatched RIs (Susaki et al., 2014). In this study, we
discovered another unexpected chemical nature of aminoalcohols: tissue decolorization. Q bands (500–700 nm) of heme inerythrocytes treated with aminoalcohol #10 or CUBIC-1 reagent
were considerably changed from those of hemoglobin-boundform (Figure 1C). These results imply that the aminoalcohols
could be tightly bound to heme porphyrin instead of oxygenand histidine in hemoglobin, which may facilitate heme releaseand explain the unexpected expansion of the effective pH window for heme release. Additionally, the buffering capability ofaminoalcohol #10 in moderately basic conditions should also
contribute to the decolorizing performance (Figures 1E and 1F).
Importantly, the buffering capability of aminoalcohol #10 isexerted in moderately basic conditions, which is the optimal pH
range for the fluorescence signal from GFP-related fluorescence
proteins (Haupts et al., 1998). In fact, treatment of individual organs by CUBIC reagents led to much higher transmittance inthe visible region than that of organs treated by other reagents(Figure S2B), whereas CUBIC reagents could preserve the fluorescence signal from various kinds of GFP-related fluorescence
proteins (Figure S6).
CUBIC Enables Whole-Body Clearing and Whole-Body
Imaging with Single-Cell Resolution
In this study, we demonstrated that the CB-Perfusion protocol,
which enabled both tissue decolorization and adjustment of mismatched RIs, achieved the visualization of chest and abdominal
backbones through internal organs as well as bones of limbs

through soft tissues from both ventral and dorsal sides of theadult and infant mice (Figure 2B). We also demonstrated that
the CB-Perfusion protocol achieved whole-body (Figure 3) andwhole-organ (Figure 4) imaging with single-cell resolution. We
note that CUBIC is also compatible with other protocols. For
example, we previously demonstrated that CUBIC reagentsare applicable to clear acrylamide-gel-embedded samples prepared according to the CLARITY protocol (Susaki et al., 2014).
Therefore, we expect that another CLARITY-inspired protocol,
PARS, can be a useful fixation method for the endogenous molecules before CUBIC treatment. Because the PARS method isbased on the intracardiac circulation of detergents (Yang et al.,2014), similar to the CB-Perfusion protocol, additional circulationof decolorizing medium (CUBIC cocktails) might increase transparency in the PARS method.
There are still some fundamental technical challenges remaining in both whole-body clearing and whole-body imaging.
Although in this study CUBIC cocktails facilitated the transparency of internal organs and soft tissues for the infant and adultmice body by tissue decolorization, these clearing reagentsare not effective for bones. Conventionally, decalcification isrendered by the continuous immersion of acids (such as formic
acids) or chelating agents (such as EDTA) (Mack et al., 2014).
Therefore, the combination of effective chelating solution with
CUBIC cocktails might provide further decalcification properties
for the current chemical cocktail. Alternatively, larger chemical
screening for tissue-clearing reagents (Susaki et al., 2014) could
afford a series of alternative candidates for tissue- and/or boneclearing reagents that may provide more clearing potency withfewer drawbacks. In whole-body imaging, further optimizationof LSFM setup is also required to seamlessly capture wholebody images of adult mice. Because the movable range of thestage was limited in the currently existing LSFM setup, thelargest field of view corresponded to a P1 mouse. An entire adultmouse is much thicker, wider, and longer than the body of an
infant mouse. Thus, extension of focus depth in the illuminatedlight-sheet, expansion of the movable range of the stage, andseamless tiling of the reconstituted images should be improvedin future LSFM setups.
CUBIC Is Applicable to 3D Pathology, Anatomy, andImmunohistochemistry
Whole-body and whole-organ imaging with single-cell resolution
will play a fundamental role in achieving 3D studies of pathology
and anatomy, which will aid in systems-level elucidation ofcellular mechanisms underlying abnormal and normal bodystates. In this study, we applied CUBIC-based whole-bodyclearing and LSFM-based whole-organ/whole-body imaging toachieve 3D pathology, anatomy, and immunohistochemistry.

(B) Structural identification of the bronchial tree (light blue) and peripheral alveoli (green) in the lung. Contiguous mKate2 signals with moderate intensity within oron the edge of the lung were extracted as the bronchial tree or alveoli, distinguished by their values of surface area.
(C) Structural identification of renal cortex, medulla, and pelvis in the kidney. High-intensity signals in SYTO 16 channel and high- or low-intensity signals ofmKate2 channel were extracted as the cortex, medulla, or pelvis, respectively.
(D) Structural identification of vessels in the liver. Two types of vascular structures (with or without high-concentrated nuclei signals around the vessel) weredistinguished. Contiguous SYTO 16 signals with low or moderate intensity were extracted as the portal vein (blue) or wall of the hepatic artery (red), respectively. In
mKate2 channel, similar contiguous signals with low intensity were also extracted and merged with the hepatic artery structure (red).
See also Figures S7 and S8.

Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. 921

Whole-organ immunostaining protocol with CB-Perfusion

A

Day 0

1

CB-Perfusion

2

3

4

5

6

7

8

9

FreezeThaw

CUBIC-1 refresh/day

10

11

12

13

Antibody
wash CUBIC-2, 4h to 1 day

Heart SYTO 16/anti-α-SMA Cy3

B

SYTO 16/α-SMA (3D)α-SMA (3D)

Magnified image (3D)Z = 700 μm (X-Y image)

X

1 mm

1 mm

500 μm500 μm

100 μmLung PI/anti-Pan-cytokeratin FITCC

PI/Pan-cytokeratin (3D)

2 mm

Pan-cytokeratin (3D)

Magnified image (3D)

2 mm

500 μm500 μmZ = 1000 μm (X-Y image)

100 μm

Stomach SYTO 16/anti-α-SMA Cy3DSYTO 16/α-SMA (3D)
2 mm

α-SMA (3D)

Magnified image (3D)

2 mm

500 μm

Z = 500 μm (X-Y image)500 μm200 μmE

Intestine SYTO 16/anti-α-SMA Cy3SYTO 16/α-SMA (3D) α-SMA (3D)
2 mm

Magnified image (3D)Z = 1000 μm (X-Y image)

2 mm

500 μm500 μm200 μmFigure 7. CUBIC Is Applicable to the 3D Immunohistochemistry of Various Organs(A) CUBIC protocol for 3D immunohistochemistry (3D-IHC) of CB-perfused various organs. The reconstituted 3D and section images of a SYTO 16- or PI-stained
heart (B), lung (C), stomach (D), and intestine (E) from C57BL/6N mouse (11-week-old) immunostained with Cy3-conjugated anti-a-SMA for heart, stomach, andintestine or FITC-conjugated anti-pan cytokeratin for lung were acquired with LSFM. Magnified 3D images and raw X-Y section images are at the indicatedpositions.

As a model case study, we analyzed entire pancreases in diabetic and normal mice to reveal the volume distribution of LIs between diabetic and healthy mice. As expected, the total number
922 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

and volume distribution of LIs were significantly different between normal and diabetic mice (Figure 5). We also note thatlarger LIs tend to be localized along pancreatic ducts andare more susceptible to b cell impairment induced by STZ
administration.
The application of whole-body and whole-organ imaging techniques is not limited to the pancreas but can be extended toother organs. In this study, we demonstrated that 3D anatomy
is feasible for other medically important structures from wholeorgan (Figure 6) or whole-body images (Figure S8) such as
coronary vessels in the heart; the bronchial tree in the lungs;
glomeruli distributed through the cortex and the medullary raysin the kidney, artery, and vein networks of the liver; and villi inthe intestine. Although such medically important structures areannotated manually in this study, automated or semi-automatedextraction of medically important anatomical structures will
facilitate 3D pathology and anatomy in future. As a pilot study,
we attempted and succeeded in semi-automated extraction of
medically important anatomical structures in abdominal andchest organs such as LIs in the pancreas (Figure 5C) and the coronary vessels in the heart (Figure S7B). We also note that CUBIC
application is not limited to transgenic mice with fluorescencereporter (Figure 7). Because 3D-IHC is compatible with CUBIC,
this whole-organ and whole-body clearing technique can be
widely applied to various mice without fluorescence reporter.
When combined with informatics as well as immunohistochemistry, CUBIC will thus contribute to the systems-level elucidationof cellular mechanisms underlying the generation and progression of diseases especially with stochastic and proliferative processes such as autoimmune diseases. New research fields suchas organism-level systems biology based on whole-body imaging with single-cell resolution lie ahead.
EXPERIMENTAL PROCEDURES
Additional details are available in the Extended Experimental Procedures.
Decolorization of the Blood by Aminoalcohols in CUBIC Cocktails
PFA-fixed blood was thoroughly mixed with each chemical, and resultant pellet and supernatant were imaged by bright-field microscopy. For taking visible
spectra data, erythrocytes were mixed with each chemical and incubatedat 37 C overnight. The mixtures and supernatant were diluted 10-fold witheach chemical. The visible spectra of samples was recorded by UV/Vis spectrometer (JASCO, V-550, Japan). In the decoloring capacity test, the mixturesof erythrocytes and each chemical with different mixing ratios (erythrocyte/
chemical = 0.01 to 0.20) were incubated at 37 C overnight. Bright-field imagesof these samples were captured. In the pH dependency of heme release,erythrocytes were mixed with each chemical and incubated at 37 C for 1 hr.
The OD575 and OD600 values of the mixtures were measured with thePowerWave XS and the attached operation software (Bio-Tek).
Mice
The b-actin-mKate2 knockin mouse (b-actin-CAG-nuc-3 3 mKate2) strain
was established in our laboratory. We also used the R26-H2B-EGFP KI(CDB0238K) (Abe et al., 2011), Thy1-YFP-H Tg (Feng et al., 2000), C57BL/
6-Tg (CAG-EGFP) (Okabe et al., 1997), BALB/cAJcl-nu/nu, and wild-type
C57BL/6N mouse strains. We fed DietGel Recovery (LSG Corporation,
72-06-5022, Japan) to mice for imaging the intestine. Diabetes was induced
in male C57BL/6N mice at 8 weeks by a single intraperitoneal administrationof saline or 10 mg/ml streptozotocin (total 200 mg/kg, Wako Pure Chemical
Industries, 197-15153) at day 0. Blood glucose values were measured by ablood glucose monitor (GLUCOCARD G Black; ARKRAY) after fasting for
6 hr at day 0 and 4. Mice with blood glucose levels over 300 mg/dl (n = 5) atday 4 and saline-administered mice (n = 4) were used for clearing with CUBIC
perfusion and PI staining. All experimental procedures and housing conditions

were approved by the Animal Care and Use Committee of Graduate School of
Medicine, the University of Tokyo or by the Animal Care and Use Committeeof the RIKEN Kobe Institute, and all of the animals were cared for and treatedhumanely in accordance with the Institutional Guidelines for Experiments
Using Animals.
The CUBIC Protocol and Other Clearing Methods
Two CUBIC reagents were prepared as previously reported (Susaki et al.,2014). For preparation of CUBIC-treated (non-perfused) samples, the fixedorgans were immersed in CUBIC-1 reagent for 5 days and were further
immersed in CUBIC-2 reagent. To make CUBIC-1 reagent penetrate
throughout the whole body, we also performed the CB-Perfusion protocol.
The anesthetized adult mouse was perfused with 10 ml of 10 U/ml of heparinin PBS, 150 ml of 4% (w/v) PFA in PBS, 20 ml of PBS (to wash out PFA), and
20 ml of 50% (v/v) CUBIC-1 reagent (1: 1 mixture of water: CUBIC-1) in thisorder via left ventricle of the heart. (The protocol was slightly modified for
infant mice.) The resulted whole animal or excised organs were continuously
immersed in CUBIC-1 reagent for 5 days to several weeks. Organs were
further immersed in CUBIC-2 reagent. These samples were stained with nucleic acid stains PI (Life Technologies, P21493) or SYTO 16 (Life Technologies,
S7578), when indicated. We also performed SeeDB standard protocol (Keet al., 2013) and ScaleA2 and ScaleB4 protocol (Hama et al., 2011). Light transmittance was measured with an integrating sphere (Spectral Haze Meter SH
7000, Nippon Denshoku Industries).
Microscopy and Image Analysis
Whole-body and organ fluorescence images were acquired with lightsheet fluorescence microscopy (LSFM) (Ultramicroscope, LaVision BioTec,
Germany) as reported previously (Dodt et al., 2007; Susaki et al., 2014). All
raw image data were collected in a lossless 16-bit TIFF format. 3D-rendered
images were visualized, captured, and analyzed with Imaris software (version
7.6.4 and 7.7.1, Bitplane). Blind 3D deconvolution for a set of our LSFM z stack
images was performed with software AutoQuant X3 (Media Cybernetics).
Anatomical structures and LI quantification were performed with surface
analysis of Imaris software. Automated detections of anatomical structure
were performed with Fiji (Schindelin et al., 2012) and using a filtering method
implemented in C++.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures, eight
figures, one table, and six movies and can be found with this article online at
http://dx.doi.org/10.1016/j.cell.2014.10.034.
AUTHOR CONTRIBUTIONS
H.R.U., K.T., and S.I.K. designed the study. K.T., S.I.K., and T.Q.S. performed
most of the experiments. E.A.S. developed CB-Perfusion protocol. D.P.
contributed to the image informatics. M.U.-T. and H.U. produced b-actinnuc-3 3 mKate2 KI mice. All authors discussed the results and commented
on the manuscript text.
ACKNOWLEDGMENTS
We thank the lab members at RIKEN CDB, QBiC, and The University of Tokyo,
in particular, H. Yukinaga, J. Hara, J. Yoshida, C. Imai, and Y. Niwa for theirkind help in preparing the materials; A. Millius for reviewing and editing themanuscript; K. Matsumoto for helpful suggestions in developing the CB-Perfusion protocol; Y. Abe, T. Sengoku, and S. Kondo for their kind help in developing the CB-Perfusion protocol and transmittance evaluation; and S. Shoi
for his kind help in statistical analysis. We also thank T. Abe and H. Kiyonari
for providing materials for targeting vectors and R26-H2B EGFP mice; LARGE,
RIKEN CDB for housing the mice; D. Asanuma and K. Hirose for their kind help
in measuring visible spectra; H. Sekiya, K. Kanemaru, D. Ino, J. Suzuki, K.
Miyazono, S. Ehata, K. Takahashi, H. Takayanagi, S. Sawa, G. Matteo, Y.
Mori, and Y. Yoshioka for their kind comments and suggestions; and Media

Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. 923

Cybernetics for supporting the usage of AutoQuant X3. This work was supported by the Program for Innovative Cell Biology by Innovative Technology
and the Brain Mapping by Integrated Neurotechnologies for Disease Studies(Brain/MINDS) from the Ministry of Education, Culture, Sports, Science andTechnology (MEXT) of Japan, a Grant-in-Aid for Scientific Research (S) (grantno. 25221004), a Grant-in-Aid for Scientific Research on Innovative Areas(grant no. 23115006), and a Grant-in-Aid JSPS Fellows (grant no. 26-1180)
from MEXT/Japan Society for the Promotion of Science (JSPS), the strategic
programs for R&D (President’s discretionary fund) of RIKEN, an intramural
Grant-in-Aid from the RIKEN Center for Developmental Biology and RIKEN
Quantitative Biology Center, a grant from Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency(JST), a Grant-in-Aid from Japan Foundation for Applied Enzymology, a
Grant-in-Aid from the Shimabara Science Promotion Foundation, and by the
RIKEN Foreign Postdoctoral Researcher Program.
Received: August 18, 2014
Revised: September 30, 2014
Accepted: October 11, 2014
Published: November 6, 2014
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Supplemental Information
EXTENDED EXPERIMENTAL PROCEDURES
Decolorization of the Blood by Aminoalcohols in CUBIC Cocktails
We hypothesized that CUBIC-1 reagent could solubilize and elute endogenous heme from blood-infused tissues. To test this hypothesis, we first investigated whether CUBIC reagents can directly decolorize blood. PFA-fixed blood was prepared by equivalent mixingof murine blood and 8% PFA-PBS, and incubated at 37 C overnight. Erythrocytes were purified by centrifugation of incoagulable
blood including 1 mg/ml EDTA at 3000 rpm for 30 min, supernatant wasting, and followed by three-times washing with 1 ml PBS
and centrifugation at 3000 rpm for 30 min. Both blood samples were stored at 4 C. In the decoloring experiments of PFA-fixed blood(Figures 1A and 1B), PFA-fixed blood (100 ml) was thoroughly mixed with each chemical [400 ml of PBS, CUBIC-1 reagent, 25 wt%
N,N,N’,N’-tetrakis(2-hydroxypropyl)ethylenediamine (aminoalcohol #10), 25 wt% urea, 15 wt% Triton X-100, 0.01 M NaOH, themixture of 25 wt% urea and 15 wt% Triton X-100, 25 wt% glycerol, or the mixture of 25 wt% urea, 25 wt% glycerol, and 15 wt%
Triton X-100 (modified ScaleA2)], and then immediately centrifuged at 15000 rpm for 5 min. Supernatant was collected and pellet
was washed with each chemical (500 ml) three times (we noted that since mixing efficiency is critical for the clearing performancein the washing step, the mixture should be not only mixed by a vortex mixer, but also vigorously stirred by the small spatula). The
pellet was suspended in each chemical (500 ml), and transferred in the dish. The collected supernatant (500 ml) was also transferredin the dish. Bright-field images of these samples were captured. As a result, aminoalcohol #10 alone, but not other reagents, is sufficient to decolorize the blood (Figure 1A). We also found that PFA-fixed blood could not be decolorized by glycerol, a mixture of urea
and Triton X-100, or modified ScaleA2 (the mixture of glycerol, urea, and Triton X-100) (Figure 1B), suggesting that aminoalcohol #10
in CUBIC-1 reagent is the active reagent in blood clearing. We also note that the decolorizing capability of aminoalcohol #10 is not
solely dependent on its high pH because PFA-fixed blood was not efficiently decolorized by highly basic solution (0.01 M NaOH, initial
pH = 12). Therefore, the blood decolorizing nature of aminoalcohol #10 in a moderately basic condition originates from its chemical
property.
We next investigated what kind of olive-green molecule was eluted from the blood into the supernatant. Thus, the visible spectra ofchemically-treated blood samples were recorded by UV/Vis spectrometer (JASCO, V-550) (Figures 1C, 1D, and S1A). Erythrocytes(5 ml) were mixed with each chemical (PBS, 0.1 M NaOH, 25 wt% #10, or CUBIC-1 reagent), and incubated at 37 C overnight. The
mixtures and supernatant from CUBIC-1- or #10-treated PFA-fixed blood were diluted 10-fold with each chemical. We also prepared
10 mM of hemin (Tokyo Chemical Industry), 10 mM of biliverdin (Sigma-Aldrich), and 100mM of iron(II) chloride (Nacalai Tesque), solubilized with CUBIC-1 reagent or #10. The normalized spectra of the erythrocyte and supernatant overlapped almost completely withhemin solutions in aminoalcohol #10 or CUBIC-1, which suggests that heme is released from hemoglobin in erythrocytes whentreated by aminoalcohol #10 or the CUBIC-1 reagent (Figures 1D and S1A).
Aminoalcohol #10 in the CUBIC-1 reagent can solubilize and elute the heme in the blood more efficiently than highly basic solution(0.01 M NaOH, initial pH = 12) (Figure 1A). What kind of chemical property in aminoalcohol #10 enables heme elution at moderate pH?
As shown in Figure 1A, we noticed that the pH variations were relatively small between the original liquid and the subsequent supernatant for samples treated by aminoalcohol #10 and CUBIC-1 reagent when compared with highly basic solution (0.01 M NaOH,
initial pH = 12). These results indicate that aminoalcohol #10 has buffering capacity at moderately basic conditions. To directly
test the buffering capacity of these reagents, 50 g of CUBIC-1 reagent, 25 wt% aminoalcohol #10, 25 wt% glycerol containing
0.01 M NaOH, 25 wt% urea containing 0.01 M NaOH, 15 wt% Triton X-100 containing 0.01 M NaOH, or 0.01 M NaOH were titrated
with 1 M HCl (Figure S1B). pH was measured by pH meter (HORIBA, LAQUAtwin). As a result, aminoalcohol #10 and CUBIC-1 reagent have buffering capacity in pH 9-11 whereas other reagents did not.
If buffering capacity in basic pH is critical for the decolorizing capability, we predicted that a basic solution containing 0.01 M
NaOH, which usually elutes heme from hemoglobin, will lose its decolorizing capability at a higher concentration of erythrocytes
because the higher concentration of cells will shift the pH from basic to neutral. To test this prediction, we prepared a mixture of erythrocytes and these chemicals at different ratios (Figure 1E). In the experiment, the mixtures (200 ml) of erythrocytes and each chemical(CUBIC-1 reagent, 25 wt% #10, 25 wt% glycerol containing 0.01 M NaOH, 25 wt% urea containing 0.01 M NaOH, 15 wt% Triton X100 containing 0.01 M NaOH, or 0.01 M NaOH) with different mixing ratio (eryhtocyte/chemical = 0.01 to 0.20) were incubated at 37 C
overnight. Bright-field images of these samples were captured. The OD575 and OD600 values of the mixtures were measured with
the PowerWave XS and the attached operation software (Bio-Tek). Since the absorbance was saturated at erythrocyte/chemical ratio = 0.20 in Figure 1E, the OD600/OD575 values at this ratio were not collected in Figure S1C. These results quantitatively confirmedthat aminoalcohol #10 in the CUBIC-1 reagent can efficiently promote heme release regardless of the erythrocyte/chemical ratio, andwork as a decolorizing buffer at moderate basic pH.
Since previous studies reported that heme can be released only in highly basic (RpH 11) or acidic (%pH 2) conditions (Kristinssonand Hultin, 2004; Teale, 1959), the remaining question is whether the decolorizing capability of aminoalcohol #10 in the CUBIC-1 reagent occurs only in highly basic conditions (pH > 11), or can occur in less basic conditions (pH < 11), in which GFP-related fluorescence proteins emit stronger fluorescence signal (Haupts et al., 1998). Thus, we evaluated the pH dependence on the efficiency ofheme release for 25 wt% aminoalcohol #10, CUBIC-1 reagent, 25 wt% tris(hydroxymethyl)aminomethane (Tris), and NaOH aqueous
solution, respectively (Figure 1F). pHs of CUBIC-1 reagent, #10, Tris solutions were adjusted by acidification with HCl or basification
with NaOH. Erythrocytes (5 ml) were mixed with each chemical (CUBIC-1 reagent, 25 wt% #10, 25 wt% Tris, or NaOH solution), andCell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. S1

incubated at 37 C for 1 hr. The OD575 and OD600 values of the mixtures were measured with the PowerWave XS. Haupts U. et al.
revealed that GFP fluorescence signals were more intensified in the moderate basic condition (pH 8-11) than in the neutral condition(pH 7.0) (Haupts et al., 1998). In our previous manuscript, we also demonstrated that GFP signals in some of 10 wt% basic aminoalcohols were higher than those in PBS (pH 7.4) (Susaki et al., 2014). Therefore, moderate basic condition between pH 8 to 11 was
optimal for fluorescence proteins. Additionally, aminoalcohol #10 has a strong buffering ability around pH 10 as shown in Figure S1B.
Therefore, pHs in the medium could be kept between 10 to 11 through the CUBIC clearing protocol due to the daily exchange of theclearing medium. Actually, we showed the final pH values of the mixtures of erythrocyte and #10/CUBIC-1 were maintained over 10
as shown in the revised Figure 1E. Thus, we focused on the pH range between 9 to 13 in the experiment. In non-buffered basic solution, heme was efficiently released from hemoglobin, but only at pH > 11. On the other hand, aminoalcohol #10 efficiently eluted
heme even in moderately basic conditions around pH 10. CUBIC-1 reagent further promoted heme release even around pH 9, whichmight be attributed to a combinatorial effect with urea and/or Triton X-100. These results suggested that aminoalcohol #10 in CUBIC1 reagent expanded the pH window for efficient heme release.
To clarify the chemical properties of aminoalcohol #10 associated with its decolorizing capability, we investigated erythrocyte witha series of aminoalcohol derivatives (Figure S1E), which include primary amine (#8, #15, and Tris), secondary amine (#4), tertiaryamine (#9, #10, #16, and #17) (Susaki et al., 2014) and amine bearing carboxylic acid groups (DHEG and EDTA) (Figure S1D). In
the blood-decoloring by a series of aminoalcohol-related chemicals shown in Figure S1D, the mixtures were prepared according
to the experiment of Figure 1E. Initial pH value in each chemical was pre-adjusted so that final pH ranges from 10.8 to 11.2. The decolorizing efficiency of primary amines is lower than those of secondary and tertiary amines (See also Figure 1F). Carboxylic groups also
suppressed the decolorizing capability. These results revealed the chemical properties (i.e., higher amines without carboxylic groups)
associated with efficient decolorizing and might reflect the affinity of these chemicals to the heme chromophore.
The reddish color of tissue originates mainly from the coordination of oxygen and histidine with heme in hemoglobin. Q bands(around 500-700 nm) of heme in erythrocytes treated with aminoalcohol #10 or CUBIC-1 reagent were considerably changed
from those of hemoglobin-bound form (Figure 1C). Those were also different from Q bands of alkaline-denatured free form ofheme. These results imply that the aminoalcohols could be tightly bound to heme porphyrin instead of oxygen and histidine in hemoglobin, which may facilitate heme release and explain the unexpected expansion of the effective pH window for heme release.
We also found that secondary and tertiary amines with higher coordination capability than primary amines significantly decolorized
the blood whereas related amines with a (charged) carboxyl group (DHEG and EDTA) exhibited less decolorizing capability (Figure S1D). We also note that the protonated (and hence charged) form of the aminoalcohol #10 (below its buffering pH 10) decreased
its decolorizing capability (Figure 1F). Since a heme molecule possesses an electron-deficient iron center and two negatively charged
carboxyl groups, we speculated that uncharged aminoalcohol #10, which possesses highly coordinating tertiary amino groups,
might have suitable chemical properties to efficiently coordinate to heme for blood decolorization.
In addition to this chemical property, the buffering capability of aminoalcohol #10 in moderately basic conditions should also
contribute to the decolorizing performance. For example, hemoglobin has been usually quantified in basic Triton X-100 (pH > 13)
because Triton X-100 could bind to heme in highly alkaline conditions (Wolf et al., 1984). However, the decolorization by basic
Triton X-100 without buffering capability was not effective due to its variable pH (Figure 1E). Therefore, the buffering capabilityof aminoalcohol #10 will assist the release of heme either by Triton X-100 or aminoalcohol #10 itself. If aminoalcohol #10 did
not have a buffering capability, the stable decolorizing capability may not be exerted (Figure 1F), as shown for Triton X-100 (Figure 1E). Importantly, the buffering capability of aminoalcohol #10 is exerted in moderately basic conditions, which is the optimal
pH range for the fluorescence signal from GFP-related fluorescence proteins (Haupts et al., 1998). In fact, treatment of individualorgans by CUBIC reagents led to much higher transmittance in the visible region than that of organs treated by other reagents(Figure S2B) while CUBIC reagents could preserve the fluorescence signal from various kinds of GFP-related fluorescence
proteins (Figure S6). Taken together, two chemical properties (a heme-coordinating capability and a buffering capability in moderately basic conditions) contribute to decolorize the tissue and simultaneously preserve fluorescence signal from GFP-related fluorescence proteins.
Mice
The b-actin-mKate2 knock-in mouse (b-actin-nuc-3 3 mKate2) strain was established in our laboratory. A back-bone vector fortargeting, pTVCI3, harboring all of the required elements, including the thymidine kinase (TK) promoter-driven cDNA encoding
Diphtheria Toxin A-fragment (DT-A) from ROSA26 targeting vector (Abe et al., 2011) and a synthetic sequence (50 -GCGGCCGCAGTTACGCTAGGGATAACAGGGTAATATAGGATCCCATTTCATTACCTCTTTCTCCGCACCCGACATAGATGATCCGTAACTATAACGGTCCTAAGGTAGCGAAGATCTGGCAAACAGCTATTATGGGTATTATGGGTGATCGAAAGAACCAGCTGGGGCTCGATCCCTCGAGATCGGATATC-30 ) as a multiple cloning region, was constructed by inserting these sequences at the EcoRV site of self-concatenatedpGEM-T-Easy (Promega) after removal of potential cutting sites for PI-PspI and PI-SceI from pGEM-T-Easy. The multiple cloning region of resulting pTVCI3 contains I-SceI, PI-SceI, I-CeuI, PI-PspI, XhoI and EcoRV site as single restriction site, respectively. The 30
homologous arm of b-actin was amplified from C57BL/6 mouse genomic DNA using PCR with the following primers: forward primer:(50 -CAGCTTGTGGCTCTGTGGCTTTGC-30 , Hokkaido System Science), reverse primer: (50 -ATGAAGAGATTGCTCAGTAGTTGAGAGAGTG-30 ). PCR product was treated with Mighty Cloning kit (TaKaRa) for 50 -end phosphorylation, and cloned into the EcoRV
site of pTVCI3, and the resulting vector was designated as pTVCI3-3arm. The 50 homologous arm of b-actin was ampliðed
S2 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

from C57BL/6 mouse genomic DNA using PCR with the following primers: forward primer: (50 -AACACCTCCAGTTATTGGACC
ACTGG-30 ), reverse primer: (50 -GTGCACTTTTATTGGTCTCAAGTCAGTG-30 ). PCR product was treated with Mighty Cloning kit for
50 -end phosphorylation. pTVCI3-3arm was digested with I-SceI (New England BioLabs), and blunted with T4 DNA polymerase(TaKaRa). The 50 homologous arm fragment was ligated at the blunted I-SceI site of pTVCI3-3arm, and designated as pTVCI35arm-3arm. pTVCI3-5arm-3arm was digested with PI-SceI (New England BioLabs), blunted with T4 DNA polymerase. The Reading
Frame Cassette B fragment from the Gateway Vector Conversion System (Life Technologies) was cloned into the blunted PI-SceI siteof pTVCI3-5arm-3arm, and the resulting vector was designated as pTVCI3-b-actin-GW. pTVCI3-b-actin-GW was mixed with pENTR1A_CAG-nuc-3 3 mKate2-WPRE-PuroR (Susaki et al., 2014) to perform the LR recombination reaction using the Gateway LR Clonase II Enzyme mix (Life Technologies). In the resulting targeting vector, the insertion cassette was in the same orientation as theb-actin gene.
C-terminal-truncated (+63) TALENs (Miller et al., 2011) that bind target sequence (50 -GGCCTGTACACTGACTTGAGACCAATAAAAGTGCACACCTTACCTTACAC-30 , TALEN binding sequences are indicated with italics) containing a polyadenylation signal
sequence were designed using TALE-NT (Doyle et al., 2012). The targeting vector and TALEN-expression vector were purified
and introduced into HK3i C57BL/6 mouse embryonic stem (ES) cells (Kiyonari et al., 2010) using Xfect Transfection Reagent (Clontech Laboratories, Inc. A Takara Bio Company) according to the manufacturer’s instructions, and the homologous recombined, puroresistant ES cell clones were isolated for further culture and expansion. An aliquot of the cells was lysed and screened for successful
homologous recombination by PCR. The integrity of the targeted region was confirmed by PCR using primers annealing outside thehomologous recombination arms and within the inserted cassette. The primers used for the screening and the confirmation of
genome integrity were as follows: 1) forward primer annealing to the region upstream of the 50 homologous arm: 50 -CTGGCAGTCACCCAGAGACACTAGC-30 , 2) reverse primer annealing to the region downstream of the 50 homologous arm: 50 -GTTAGTGCA
GGCCAACTTGGCCTAGG-30 , 3) forward primer annealing to the region upstream of the 30 homologous arm: 50 -AGGATGG
TCGCGTCCATGCCCTGAG-30 , 4) reverse primer annealing to the region downstream of the 30 homologous arm: 50 -ACTTCCAGGAGCCCTATGTGGTAGC-30 , 5) reverse primer annealing to the region between 50 homologous arm and Gateway cassette of pTVCI3:
50 -GGTAATGAAATGGGATCCTATATTACCC-30 , and 6) forward primer annealing to the region between 30 homologous arm and
Gateway cassette of pTVCI3: 50 -GAAAGAACCAGCTGGGGCTCGATCC-30 . The copy number of the inserted cassette was confirmedwith a quantitative PCR assay using primers annealing to the coding sequences of the puro-resistance gene (forward primer:
50 -CTCGACATCGGCAAGGTGTG-30 , reverse primer: 50 -GGCCTTCCATCTGTTGCTGC-30 ) (Susaki et al., 2014), normalized to theamount of TATA-box binding protein gene amplification (forward primer: 50 -CCCCCTCTGCACTGAAATCA-30 , reverse primer:
50 -GTAGCAGCACAGAGCAAGCAA-30 ) (Tsujino et al., 2013) using the SYBR Premix Ex Taq GC (TaKaRa) and the ABI PRISM
7900 (Applied Biosystems). The selected ES cell clones were injected into 8-cell-stage ICR embryos to generate 100% ES cellderived chimeras (Kiyonari et al., 2010).
We also used the R26-H2B-EGFP (CDB0238K) mouse strain (Abe et al., 2011), the Thy1-YFP-H transgenic mouse strain (Fenget al., 2000), the R26-pCAG-nuc-3 3 mKate2 mouse strain (Susaki et al., 2014), and the C57BL/6-Tg (CAG-EGFP) mouse strain(Okabe et al., 1997) to observe static fluorescent gene-expression patterns in whole-organ imaging. Mouse strains were maintainedin the C57BL/6 background. We purchased the BALB/cAJcl-nu/nu mouse strain from CLEA Japan, Inc. and the C57BL/6-Tg (CAGEGFP) mouse strain from Japan SLC, Inc.
All experimental procedures and housing conditions were approved by the Animal Care and the Use Committee of Graduate
School of Medicine, the University of Tokyo or by the Animal Care and Use Committee of the RIKEN Kobe Institute and all of the animals were cared for and treated humanely in accordance with the Institutional Guidelines for Experiments using animals. We fed
DietGel Recovery (LSG Corporation, 72-06-5022, Japan) to mice for imaging the intestine.
Mouse Model of Streptozotocin (STZ)-Induced Diabetes
Diabetes was induced in male C57BL/6N mice at 8 weeks by a single intraperitoneal administration of saline (Otsuka PharmaceuticalCo., Ltd., 14987035081514, Japan) or 10 mg/ml streptozotocin (total 200 mg/kg, Wako Pure Chemical Industries, Ltd., 197-15153,
Japan) dissolved in saline at day 0. At day 0 and day 4, blood glucose values were measured by a blood glucose monitor(GLUCOCARD G Black; ARKRAY, Inc., Japan) after fasting for 6 hr. Body weight was also measured at the same days. After fourdays, blood sugar levels of 36% of the mice were elevated over 300 mg/dl, which is a commonly-used criterion for the diagnosis
of diabetes (Table S1). Mice with blood glucose levels over 300 mg/dl (n = 5) at day 4 and saline-administered mice (n = 4) wereused for clearing with CUBIC (CB)-perfusion and PI staining. Imaging was performed using mice with the highest blood glucose levelsof STZ group (n = 3) and corresponding number of control group (n = 3).
Comparison of Clearing Methods
A number of tissue-clearing methods (including BABB, THF-DBE, Scale, SeeDB, ClearT, 3DISCO, CLARITY, CUBIC, and PACTPARS) have been intensively developed. Because a simple and versatile protocol will facilitate a high-throughput approach to understanding cellular circuits in organisms, we mainly focused on the development of an immersion-only tissue-clearing protocol. Organic
solvent-based clearing media such as BABB, THF-DBE decrease signals from fluorescent proteins with short duration (Ertürk et al.,2012; Hama et al., 2011). In addition, benzylalcohol and THF inherently generates potentially explosive peroxide and this also
quenches the fluorescence signals of fluorescent proteins (Alnuami et al., 2008). Therefore, peroxides in benzylalcohol and THF
Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. S3

have to be removed by specific apparatus before use. Although organic solvent based clearing medium remains to be attractive in theclearing performance, these experimental drawbacks compelled us to focus on water-based clearing reagents. For Figure 2A, we
compared four chemical-based clearing methods. For collecting organ samples, adult mice (C57BL/6N and BALBc-nu/nu) weresacrificed by an overdose of pentobarbital (>100 mg/kg, Kyoritsu Seiyaku, Japan) and then perfused with 10 ml of 10 U/ml of heparin(Wako Pure Chemical Industries, 081-00136) in PBS (pH 7.4) and 20 ml of 4% (w/v) paraformaldehyde (PFA, Nacalai Tesque, 0289045) in PBS via left ventricle of the heart. The excised organs (brain, heart, lung, right lobe of the liver, both side of the kidneys,
pancreas, spleen, stomach, intestine, triceps surae muscle and skin) were post-fixed in 4% (w/v) PFA for 24 hr at 4 C. The specimens
were washed with PBS for 20 min x three times to remove PFA just before clearing. Uncleared organs (negative control) were stocked
in PBS with 0.01% Sodium Azide (Nacalai Tesque, 31208-82) at 4 C after fixation. In SeeDB standard protocol (Ke et al., 2013), thefixed organs were serially rotated (4 rpm) in 30 ml of 20%, 40%, 60% (w/v) fructose (Nacalai Tesque, 16315-55) for 8 hr each and in
30 ml of 80%, 100% (w/v) fructose for 12 hr each. From day 3, these organs were rotated in 30 ml of SeeDB (80.2% w/w fructose) at
25 C. SeeDB solution was replaced to the fresh one every day until observation. All fructose solutions contained 0.5% a-thioglycerol(Tokyo Chemical Industry, S0374). In ScaleA2 and ScaleB4 protocol (Hama et al., 2011), the fixed organs were immersed in 20% (w/v)
sucrose (Nacalai Tesque Inc., 30403-55, Japan) at 4 C for 24 hr. These organs were embedded in OCT compound (Sakura Finetek
Japan Co., Ltd., 4583) and frozen at 80 C. The next day, they were thawed in PBS and fixed again in 4% PFA. From day 3, theseorgans were rotated in 30 ml of ScaleA2 (4 M urea, Nacalai Tesque, 35904-45, 10% (w/v) glycerol, Nacalai Tesque, 17018-25, and
0.1% (v/v) Triton X-100, Nacalai Tesque, 25987-85) for 2 days, 30 ml of ScaleB4 (8 M urea, 0.1% (v/v) Triton X-100) for 2 days, and
30 ml of ScaleA2 for 3 days in this order. All of the Scale solutions were replaced to the fresh one every day until observation. Two
CUBIC reagents were prepared as previously reported (Susaki et al., 2014). Aminoalcohol #10 [N,N,N’,N’-tetrakis(2-hydroxypropyl)
ethylenediamine (Tokyo Chemical Industry, T0781)] is a constituent of previously published CUBIC-1 chemical cocktail. Aminoalcohol #16 [2,20 ,2’’-nitrilotriethanol (Wako Pure Chemical Industries, 145-05605)] is a constituent of previously published CUBIC-2 chemical cocktail. In brief, ScaleCUBIC-1 (CUBIC-1 reagent) was prepared as a mixture of 25 wt% urea, 25 wt% aminoalcohol #10, and15 wt% Triton X-100. ScaleCUBIC-2 (CUBIC-2 reagent) was prepared as a mixture of 50 wt% sucrose, 25 wt% urea, 10 wt%
aminoalcohol #16, and 0.1% (v/v) Triton X-100. We adopted two CUBIC protocols for this comparing experiment. For preparationof CUBIC-treated (non-perfused) samples, the fixed organs (from C57BL/6N) and skin (BALBc-nu/nu, to save plucking hairs before
clearing) were immersed in 30 ml of CUBIC-1 reagent at 37 C with gentle shaking for 5 days. These organs were then washed with
PBS and immersed in 50% glycerol at room temperature on day 6. They were immersed in 30 ml of CUBIC-2 reagent at room temperature on day 7 and at 37 C after day 8. In all four clearing methods, stomach and intestine were separately immersed from otherorgans to avoid contamination of sticky gastrointestinal contents.
To make CUBIC-1 reagent penetrate throughout the whole body, we also performed the CUBIC (CB)-Perfusion protocol as follows;
the anesthetized mouse was perfused with 10 ml of 10 U/ml of heparin in PBS, 150 ml of 4% (w/v) PFA in PBS, 20 ml of PBS (to wash
out PFA), and 20 ml of 50% (v/v) CUBIC-1 reagent (1: 1 mixture of water: CUBIC-1 reagent) in this order via left ventricle of the heart.
The resulted organs were excised and continuously immersed in 30 ml of CUBIC-1 reagent at 37 C with gentle shaking for 5 days.
These organs were washed with PBS, immersed in 50% glycerol in PBS at room temperature on day 5. Then, they were immersed in
30 ml of CUBIC-2 reagent at room temperature on day 6 and at 37 C after day 7. Bright-field images of adult mice (19-week-old)demonstrated that CB-Perfusion protocol markedly transparentized and decolorized adult mice (Figures 2B and S2C). Since theabdominal organs of adult mouse (including liver, pancreas, spleen, stomach, and small and large intestines) were sufficiently transparentized and decolorized, the backbones (the lumbar and sacrum vertebrae) and pelvis were easily visualized even in the ventralto-dorsal (V-D) image of adult mice (Figure 2B). Similarly, the chest organs of adult mouse (including lung, heart, aorta, pulmonary
artery and bronchi) were also highly transparentized and decolorized. Hence, the backbones (the cervical and thoracic vertebrae)and entire rib cages were easily visualized even in the ventral-to-dorsal (V-D) image of adult mice (Figure 2B). Furthermore, the muscles of limbs in adult mouse were markedly transparentized and decolorized. Therefore, the bones of forelimbs (including scapula,
humerus, radius and ulna) and hindlimbs (including femur, tibia and fibula), and caudal vertebrae were clearly visualized in either V-D
or the dorsal-to-ventral (D-V) images of adult mice (Figures 2B and S2C).
Measurement of Light Transmittance
For Figures S2A and S2B, we measured light transmittance of the brain, heart, lung, liver and kidney samples use in Figure 2A, from
380 to 780 nm at 5 nm intervals with an integrating sphere (Spectral Haze Meter SH 7000, Nippon Denshoku Industries). We put every
sample in the center of the optical cell (Nippon Denshoku Industries, 2277) without liquid to avoid sample floating. Finally, the heightof the sample and the aperture of integrating sphere were adjusted before measurement.
In the instrument, two values can be determined: (1) diffused (scattered) light transmittance (%) which is the value for the light transmitted through the tissue, (2) parallel (non-diffused) light transmittance (%) which is the value for the light detected without any
prevention. Diffused light transmittance was calculated as the value of (total light transmittance – parallel light transmittance) in
the integrating sphere. We also calculated (100 – parallel light transmittance) as a tissue size-related value, because the sample
makes a ‘shadow’ over the parallel light detector and the ‘shadow’ size is related to the sample size. Thus, the final transmittancewas calculated by the diffused light transmittance divided by the value of (100 – parallel light transmittance) to be compensatedwith the tissue size and compared directly even between the values from different tissues. Finally, the final transmittance fromfour samples was averaged.
S4 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

The CUBIC Protocol for Whole-Body and -Organ Imaging
For whole body and organ imaging, we slightly modified the above CUBIC protocol to improve the transparency and quality of imaging data (Figures 2B, 3, and 4).
For whole body imaging, adult (6 – 19 weeks old) mouse were subjected to the same CB-Perfusion protocol as above, and then the
CB-perfused mouse was immersed in 200 ml of CUBIC-1 reagent at 37 C with gentle shaking for at least two weeks. To facilitate thewhole-body clearing, the reagent was refreshed daily for two weeks. The clearing medium gradually became less colored, and after
two weeks the apparent transparency of the whole body became saturated. Whole-body samples can be then stored in CUBIC-1
reagent, and even shaken in 37 C over months until observation because the high pH of CUBIC-1 reagents prevents proliferationof microorganisms. The resulted sample was used for imaging and thus we did not perform further clearing with CUBIC-2 reagent.
For whole organ imaging, adult mice were subjected to the CB-Perfusion protocol as above. Organs were excised and continuously immersed in 30 ml (stomach, intestine) or 60 ml (brain, heart, lung, liver, kidneys, pancreas, spleen, muscle) of CUBIC-1 reagent
at 37 C with gentle shaking at least for 7 days. To facilitate the clearing, the reagent was refreshed daily for one week, and the transparency was saturated within 10 days as in Figure 2 and S2. The samples can be then stored in CUBIC-1 reagent, and even shaken in37 C over weeks to months to prevent proliferation of microorganisms as above. These organs were washed with PBS, immersed in
50% glycerol in PBS at room temperature, and then they were immersed in 30 ml of CUBIC-2 reagent for each group at room temperature on the first day and at 37 C after the second day. Images were acquired after 3 days incubation in CUBIC-2 reagent. For
skin imaging in Figure 4E, a CAG-EGFP Tg mouse (10-week-old) was also subjected to CB-Perfusion as above. The excised skinand ear were needed to remove subcutaneous tissues and hairs, and thus we washed the specimens with PBS several times andthen post-fixed and cleared according to non-perfusion protocol as above 10-day protocol. Images were acquired on day 12.
The 3D reconstitution image of heart from SYTO 16-stained nuc-3 3 mKate2 demonstrated that SYTO 16 signals highlighted
epicardium, papillary muscle, and right atrium whereas mKate2 signal was higher in coronary vessels (Figure 4A). The expression
level of mKate2 is dependent on the cell type in each tissue because mKate2 is expressed under the control of CAG promoter in
ROSA26 locus, and therefore distribution of the mKate2 fluorescent signals reflected the expression of the knocked-in expression
cassette. On the other hand, SYTO 16 indiscriminately stained all cell nuclei in the tissue. Thus, distribution of the SYTO 16 fluorescent signals was attributed to the cell density in each tissue. The spatial distribution of fluorescence proteins (EGFP in Figure S6A andnuc-3 3 mKate2 in Figures 4 and S5) was also markedly different. This is because EGFP was localized in the nucleus and cytosol
whereas mKate2 was localized only in the nucleus. Therefore, EGFP signals were useful for the visualization of the orientation of
muscular fibers in heart and muscle, whereas mKate2 signals could highlight the vascular and duct structures in heart, kidney,pancreas, and spleen. In addition, expression levels of these fluorescence proteins varied in each organ. For example, EGFP signals
were more intense in the heart and the pancreas whereas mKate2 signals were elevated in the brain, the kidney, and the pancreas.
For a neonatal (postnatal day 1 or day 6) mouse clearing, 3 ml of 10 U/ml of heparin in PBS, 10 ml of 4% (w/v) PFA in PBS, 3 ml of
PBS and 5 ml of 50% (v/v) CUBIC-1 reagent were perfused in this order via left ventricle of the heart after the anesthetization. Furthermore, 3 ml of CUBIC-1 reagent were additionally perfused for improving the transparency. Gastrointestinal tract was also flushedwith CUBIC-1 reagent from the mouth. The mouse was then immersed in 30 ml of CUBIC-1 reagent at 37 C with gentle shaking
for at least 2 weeks. To facilitate the clearing, the reagent was refreshed daily for two weeks. The samples can be then stored in
CUBIC-1 reagent, and even shaken in 37 C over weeks to months to prevent proliferation of microorganisms as above. This sample
was used for imaging and thus we did not perform further clearing with CUBIC-2 reagent. In the whole body imaging of P1 mouse, we
noted that PI fluorescence signal was enhanced where it binds not only with DNA in the nucleus of internal organs and soft tissues but
also in bone tissues (Figure 3A). Although light-sheet illumination was shaded to some extent by bone-rich structures such as paws,
fluorescence signal from shaded regions was still detectable. According to the whole-body images, organ transparency was notdiminished through the whole-body clearing procedure compared to clearing of individual organs separately.
Whole body and organs were stained with nucleic acid stain (propidium iodide (PI), life technologies, P21493 or SYTO 16, Life
Technologies, S7578), added in 50% (v/v) CUBIC-1 reagent (for CB-Perfusion), CUBIC-1 and -2 reagents (during clearing) at a concentration of 10 mg/ml PI or 0.5 mM SYTO16, when indicated. Note that 50% (w/v) Glycerol in PBS used before CUBIC-2 reagent canbe exchanged with 20% (w/v) sucrose in PBS in the original brain clearing protocol (Susaki et al., 2014).
Microscopy
Whole-body and organ fluorescence images were acquired with light-sheet fluorescence microscopy (LSFM) (Ultramicroscope,
LaVision BioTec) as reported previously (Dodt et al., 2007; Susaki et al., 2014). Samples were immersed in a 1:1 mixture of silicon
oil TSF4300 (Momentive Performance Materials, RI = 1.498) and mineral oil (Sigma-Aldrich, RI = 1.467), and were put on a glass plate
held with a customized sample holder. Images were captured at 0.63 3 to 5 3 zoom with the MVX-ZB10. Each plane was illuminatedfrom both the right and left sides, and a merged image was saved. The exposure times were adjusted according to the fluorescentsignal intensities of each sample. Detailed imaging conditions in each figure described as below; Figure 3 (A) Z-stack: 20-mm step,with 4.0 s 3 two illuminations for EGFP and with 0.1 s 3 two illuminations for PI. Zoom of the microscope: 0.63 3, (B) Z-stack: 20-mm
step, with 4.0 s 3 two illuminations for EGFP and with 0.1 to 0.15 s 3 two illuminations for PI. Zoom of the microscope: 2.0 3 except
2.5 3 for chest V-D images, (C) Z-stack: 20-mm step, with 0.4 to 2.0 s 3 two illuminations for EGFP and with 0.1 to 0.2 s 3 twoilluminations for PI. Zoom of the microscope: 0.8 3 except 1.25 3 for chest V-D images, Figure 4 (A) Heart images. Z-stack:
20-mm step 3 346 planes, with 0.05 s 3 two illuminations for SYTO 16 and with 0.5 s 3 two illuminations for mKate2. Zoom of
Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. S5

the microscope: 2.0 3 . (B) Lung images. Z-stack: 20-mm step 3 306 planes, with 0.05 s 3 two illuminations for SYTO 16 and with
1.0 s 3 two illuminations for mKate2. Zoom of the microscope: 1.0 3 . (C) Kidney images. Z-stack: 20-mm step 3 256 planes, with
0.05 s 3 two illuminations for SYTO 16 and with 0.5 s 3 two illuminations for mKate2. Zoom of the microscope: 1.6 3 . (D) Liver images. Z-stack: 20-mm step 3 266 planes, with 0.05 s 3 two illuminations for SYTO 16 and with 0.5 s 3 two illuminations for mKate2.
Zoom of the microscope: 1.0 3 . (E) Skin and ear images. Z-stack: 20-mm step, with 0.2 to 0.5 s 3 two illuminations for EGFP and with
0.1 s 3 two illuminations for PI. Zoom of the microscope: 1.6 3 for ear, 2.0 3 for skin, and 6.3 3 for both magnified images, Figure 7(B) Heart images. Z-stack: 10-mm step 3 501 planes (whole image) or 3 201 planes (magnified image), with 0.1 s 3 two illuminations
or 0.1 s 3 single illumination for SYTO 16 and with 2.0 s 3 two illuminations or 3.0 s 3 single illumination for Cy3, respectively. Zoomof the microscope: 2.5 3 or 6.3 3 . (C) Lung images. Z-stack: 10-mm step 3 511 planes (whole image) or 3 101 planes (magnified
image), with 0.1 s 3 two illuminations or 0.2 s 3 two illuminations for PI and with 2.0 s 3 two illuminations or 3.0 s 3 two illuminations
for FITC, respectively. Zoom of the microscope: 1.6 3 or 6.3 3 . (D) Stomach images. Z-stack: 10-mm step 3 381 planes (whole image) or 3 167 planes (magnified image), with 0.1 s 3 two illuminations or 0.1 s 3 single illumination for SYTO 16 and with 1.0 s 3 twoilluminations or 1.0 s 3 single illumination for Cy3, respectively. Zoom of the microscope: 2.0 3 or 6.3 3 . (E) Intestine images. Zstack: 10-mm step 3 371 planes (whole image) or 3 101 planes (magnified image), with 0.1 s 3 two illuminations or 0.1 s 3 single
illumination for SYTO-16 and with 2.0 s 3 two illuminations or 3.0 s 3 single illumination for Cy3, respectively. Zoom of the microscope: 1.25 3 or 6.3 3 .
Image Data Processing and Analysis
All raw image data were collected in a lossless 16-bit TIFF format. 3D-rendered images were visualized and captured with Imaris
software (version 7.6.4 and 7.7.1, Bitplane). Brightness, contrast, and gamma of the 3D-rendered images were manually adjustedwith the software at minimum when visualized.
We performed blind 3D deconvolution for a set of our LSFM Z-stack images with software AutoQuant X3 (Media Cybernetics).
Because the software does not have a preset for a macrozoom LSFM images, we searched parameters to significantly improve
the raw tiff images. Using the expert settings, we performed the 3D blind deconvolution with 50 iterations, noise manually set to0, and the original unfiltered image as initial guess. Montage was used both in the XY and Z directions, with 15-pixel sub-volume
overlap. RIs were to 1.00 and 1.49, with lens aperture at 0.15. Spacing, wavelength and magnitude settings are sample-dependent,
and all other parameters were kept to the default values for the ‘‘multi-photon fluorescence’’ modality. Results were saved as 16-bit
tiff images.
The reconstituted 3D images were then used for image analysis with Imaris software (Bitplane). In order to reliably identify all LIs in
the pancreas, we acquired 6-10 sets of the magnified z-stack images to cover the entire pancreas. LIs were identified as dense clusters of PI-stained cellular nuclei in the magnified images. These images also enabled us to morphologically distinguish the characteristic clusters (LIs) from the characteristic tracts (pancreatic ducts) (Figure 5A). We identified all LIs by visual annotation of all z-stackplanes in all sets of magnified images of a pancreas. The extracted LIs were then 3D-reconstituted by the surface analysis in the
Imaris software. For example, yellow (in the indicated z-stack plane) and blue objects (outside the indicated z-stack plane) indicate
LIs whereas green objects indicate pancreatic ducts (Figure 5A). We thus performed comprehensive and statistical analysis of diabetic LIs in the whole-pancreas of adult mice (Figure 5B). For the visualization of anatomical structures (Figures 6, S7, and S8), we
selected appropriate analytical segments in each organ and manually extracted signal intensities correlated with these structures on
the Surface analysis of the software. Then, each structure was manually curated and extra Surface signals were eliminated.
For extraction, counting, and volume calculation of pancreatic islets, pancreases derived from the STZ experiment and cleared by
CB-Perfusion with PI staining were used. Whole-tissue 3D images of each pancreas were acquired as 6 to 10-divided region images
with 4 3 zoom of MVX10. The merged areas were annotated manually. Each islet was morphologically detected in the entire X-Yplane images. Then, a 3D region including one to three islets was selected for Surface analysis of Imaris software. The size of automatically selected Surface was manually adjusted to the actual islet size by changing Threshold (Absolute intensity) parameter. To
remove Surfaces other than islet, Volume parameter was changed to 103 or 104 voxel number (depending on the islet size of theselected region), and manually removed the remaining Surfaces other than islet. Number and volume of islets were calculated by
Imaris. Further statistical analyses between saline- or STZ- administered groups were performed using R software (KolmogorovSmirnov test for difference in islet size distribution, and comparison of total islets, islets larger or smaller than 1.0 3 107 mm3 for t test).
As a pilot study, we considered the automatic extraction of relevant anatomical structures in the heart and pancreas. For heartsamples, analysis is performed in Fiji (Schindelin et al., 2012). We successively run the ‘‘Smooth’’ and ‘‘Find Edges’’ methods, followed by the ‘‘Tubeness’’ plugin (with sigma = 1). Finally, we apply a binary mask to keep only pixels with intensity higher than400. As shown in Figure S7B, the method successfully extracts coronary arteries and most of them were merged to the manual extracted Surface signal in Imaris. For the pancreas samples, the objective is to accelerate the identification of Langerhans islets. Our
method relies on two properties of these islets: (a) even though their intensity varies, the islets are generally brighter than their immediate surroundings; (b) islets have a roughly spherical shape. The first property is used in the pre-processing method, implemented in
C++. Using a sliding window, we measure the average intensity in each region of each Z-slice of the 3D image, and only keep pixels
with intensity at least 10% higher than this local average. All other pixels are set to 0. Each pre-processed 2D slice is then analyzed inFiji. We run the ‘‘Find Edges’’ method, followed by computing of the largest absolute eigenvalue of the Hessian matrix (using the corresponding FeatureJ plugin with smoothing = 1), to identify clusters that do not have the desired shape. At each pixel, the eigenvalue
S6 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

is then multiplied by seven and subtracted from the current intensity. We save the resulting file for each slice, and re-construct the 3D
image. While the method still needs to be refined, early results on the clearest slices are encouraging (Figure 5C). For darker slices,
curation of the result file removes inaccurate annotations. This process will be automatized in future versions.
3D Immunostaining of CUBIC Samples
3D IHC protocol for CUBIC samples was according to our previous paper (Susaki et al., 2014). Organ samples with CB-Perfusion
were treated with CUBIC-1 for 7 days, washed with PBS, immersed in 20% (w/v) sucrose in PBS, and frozen in O.C.T. compound
at –80 C overnight. The frozen samples were then thawed, washed with PBS, and subjected to immunostaining with the 1:100 diluted
fluorescent-labeled antibodies in 5 ml of 2% (v/v) Triton X-100 in PBS for 3 days at 4 C with rotation. The stained samples were thenwashed with 5 ml of PBS several times at 37 C with rotation. The stained samples were then immersed in CUBIC-2 for 4 to 15 hr. The
following antibodies were used for the staining: Cy3-conjugated anti-a-smooth muscle actin (a-SMA) antibody produced in mouse(Sigma, C6198) for heart, stomach, and intestine, and monoclonal FITC-conjugated anti-Pan cytokeratin antibody [C-11] (abcam,
ab78478) for lung. These antibodies were mixed with the samples after filtration with an Ultrafree-MC GV Centrifugal Filter (Millipore
UFC30GV00).
SUPPLEMENTAL REFERENCES
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tools for TAL effector design and target prediction. Nucleic Acids Res. 40 (Web Server issue), W117–W22.
Kiyonari, H., Kaneko, M., Abe, S., and Aizawa, S. (2010). Three inhibitors of FGF receptor, ERK, and GSK3 establishes germline-competent embryonic stem cellsof C57BL/6N mouse strain with high efficiency and stability. Genesis 48, 317–327.
Miller, J.C., Tan, S., Qiao, G., Barlow, K.A., Wang, J., Xia, D.F., Meng, X., Paschon, D.E., Leung, E., Hinkley, S.J., et al. (2011). A TALE nuclease architecture for
efficient genome editing. Nat. Biotechnol. 29, 143–148.
Tsujino, K., Narumi, R., Masumoto, K.H., Susaki, E.A., Shinohara, Y., Abe, T., Iigo, M., Wada, A., Nagano, M., Shigeyoshi, Y., and Ueda, H.R. (2013). Establishment of TSH b real-time monitoring system in mammalian photoperiodism. Genes Cells 18, 575–588.
Wolf, H.U., Lang, W., and Zander, R. (1984). Alkaline haematin D-575, a new tool for the determination of haemoglobin as an alternative to the cyanhaemiglobin
method. II. Standardisation of the method using pure chlorohaemin. Clin. Chim. Acta 136, 95–104.

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A

B
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CUBIC-1+ Erythrocyte+ PFA fixed blood(supernatant)+ Hemin+ Biliverdin+ Fe(II)

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Basic Glycerol

CUBIC-1

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1 M HCl (mL)

C

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1.0
NaOH
#10
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CUBIC-1

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N,N,N',N'-Tetrakis(2-hydroxypropyl)ethylenediamine (#10)

3-Amino-1,2-propanediol (#8)
Triethanolamine (#16)
2-Amino-1,3-propanediol (#15)

Tris(hydroxymethyl)aminomethane(Tris)

Triisopropanolamine (#17)

3-Methylamino-1,2-propanediol (#4)

Dihydroxyethyl glycine (DHEG)

N,N,N',N'-Tetrakis(2-hydroxyethyl)ethylenediamine (#9)

Ethylenediaminetetraacetic acid
(EDTA)

Figure S1. Aminoalcohols in CUBIC Cocktails Decolorized the Blood by Efficiently Eluting Heme Chromophore, Related to Figure 1(A) Normalized visible spectra of 0.1% mouse erythrocyte (black), 1/10 diluted supernatant from Figure 1A (red), 10 mM hemin (blue), 10 mM biliveridin (orange),and 100 mM iron(II) chloride (green), in CUBIC-1 reagent.
(B) 50 g of CUBIC-1 reagent (red circle, n = 3), 25 wt% aminoalcohol #10 (magenta square, n = 3), 25 wt% basic glycerol (blue triangle, n = 3), 25 wt% basic urea
(green cross, n = 3), 15 wt% basic Triton X-100 (orange bar, n = 3), or 0.01 M NaOH (black diamond, n = 3) were titrated with 1 M HCl. pHs are plotted against thetitrated volume of 1 M HCl. Data represent the average ± SD.
(C) OD600/575 values of 1% to 20% erythrocyte solution mixed with CUBIC-1 reagent (red circle, n = 3), 25 wt% aminoalcohol #10 (magenta square, n = 3), 25 wt% basic glycerol (blue triangle, n = 3), 25 wt% basic urea (green cross, n = 3), 15 wt% basic Triton X-100 (orange bar, n = 3), and 0.01 M NaOH (black diamond, n =3) in Figure 1E are plotted against erythrocyte/chemical mixture ratio. Data represent the average ± SD.
(D) OD600/575 values of 1% erythrocyte solution mixed with a series of aminoalcohols [primary amine (green, n = 3), secondary amine (orange, n = 3), tertiaryamine (magenta, n = 3), or amine bearing carboxylic acid (purple, n = 3)], NaOH (black, n = 3), basic glycerol (blue, n = 3) or CUBIC-1 reagent (red, n = 3) is shown.
Initial pH value in each chemical was pre-adjusted so that final pH ranges from 10.8 to 11.2. Data represent the average ± SD.
(E) Chemical structures of glycerol and a series of aminoalcohols examined in this study. Numbering of the chemicals was derived from the previous chemical
screening. Glycerol is a polyhydric alcohol without an amine group. 3-Amino-1,2-propanediol (#8), 2-amino-1,3-propanediol (#15), and tris(hydroxymethyl)aminomethane (Tris) are aminoalcohols with primary amine. 3-Methylamino-1,2-propanediol (#4) is a aminoalcohol with a secondary amine group. N,N,N’,N’Tetrakis(2-hydroxyethyl)ethylenediamine (#9), N,N,N’,N’-tetrakis(2-hydroxypropyl)ethylenediamine (#10), triethanolamine (#16), and triisopropanolamine (#17)
are aminoalcohols with a tertiary amine group. Dihydroxyethyl glycine (DHEG) and ethylenediaminetetraacetic acid (EDTA) are amines with carboxylic groups.

S8 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

A

B
Day 10CUBIC-1

CUBIC PFACUBIC-1

SeeDB PFA 20%-100%
Fructose

CUBIC-2PBS

PBS CUBIC-2
SeeDB

20% Freeze
Scale PFA Sucrose
Thaw ScaleA2 ScaleB4 ScaleA2

PBS

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Heart

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CB-Perfusion

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Brain

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Dorsal view

Brain

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Day 0

CB-Perfusion

C WT adult (C57BL/6N)

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Figure S2. CUBIC Is a Simple and Efficient Whole-Organ and Whole-Body Clearing Protocol, Related to Figure 2(A) Temporal development of transmittance of different organs (brain, heart, lung, kidney, and liver) with each protocol. Average light transmittance of fixed wholeorgans treated with PBS (black, n = 4), the Scale protocol (green, n = 4), the SeeDB protocol (blue, n = 4), the CUBIC protocol (pink, n = 4) or the CB-Perfusion
protocol (red, n = 4), were measured. Data represent the average ± SD.
(B) Transmission curves of different organs (brain, heart, lung, kidney, and liver) with each protocol after 10 days. Light transmittance around the visible region(380-780 nm) of fixed whole organs treated with PBS (black, n = 4), Scale protocol (green, n = 4), SeeDB protocol (blue, n = 4), CUBIC protocol (pink, n = 4) or the
CB-Perfusion protocol (red, n = 4) were measured. Data represent the average ± SD.
(C) Bright-field images (dorsal view) of fixed whole body (C57BL/6N adult mice) stocked in PBS or subjected to the CB-Perfusion protocol, also shown in Figure 2B. 8-week-old mouse for the PBS sample and 19-week-old mouse for the cleared sample are shown.

Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. S9

A CAG-EGFP Tg P1 body images

B CAG-EGFP Tg 8w body images

with PI staining

DCV-Chest (D-V)

2 mmwith PI staining

Z = 2.5 mm

Forelimb

2 mm

Z = 7.0 mm

4 mm

4 mm

C CAG-EGFP Tg P6 magnified body images with PI staining
Head (D-V)

Z = 2.0 mm

Chest (V-D)

Z = 3.0 mm

2 mm

2 mm

4 mm

Chest (D-V)

4 mm

Z = 2.6 mm

4 mm

Abdominal & Pelvic (D-V)

4 mm

4 mm

Abdominal & Pelvic (V-D)

4 mm

Z = 4.8 mm

4 mm

Z = 3.6 mm

4 mm

Figure S3. CUBIC Is Applicable to Whole-Body Imaging of Infant and Adult Mice with Single-Cell Resolution, Related to Figure 3(A) 3D-reconstituted and X-Y plane body images of PI-stained CAG-EGFP Tg P1 mouse. Chest organs (D-V) are shown. The image was deconvolved with
AutoQuant X3 software. Z-stack: 20-mm step, with 4.0 s 3 two illuminations for EGFP and with 0.1 to 0.15 s 3 two illuminations for PI. Zoom of the microscope:
2.0 3 . Prefix ‘‘DCV-’’ indicates the deconvolved image.
(B) 3D-reconstituted and X-Y plane body images of the PI-stained CAG-EGFP Tg adult mouse (8-week-old). Forelimb is shown. Z-stack: 20-mm step, with 0.4 to
2.0 s 3 two illuminations for EGFP and with 0.1 to 0.2 s 3 two illuminations for PI. Zoom of the microscope: 0.8 3 .
(C) 3D-reconstituted and X-Y plane (indicated with Z position) body images of the PI-stained CAG-EGFP Tg P6 mouse. Head (D-V), chest organs (V-D and D-V),
abdominal and pelvic organs (V-D and D-V) are shown. Z-stack: 20-mm step, with 4.0 s 3 two illuminations for EGFP and with 0.1 s 3 two illuminations for PI.
Zoom of the microscope: 1.25 3 except 2.0 3 for head D-V image.

S10 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

CAG-EGFP Tg P1 magnified organ images with PI staining
Z = 1.0 mm

DCV-Submaxillary Gland
1 mm

DCV-Kidney

Z = 1.7 mm

1 mm

1 mm

DCV-Heart

Z = 1.2 mm
1 mm

1 mm

DCV-IntestineZ = 1.0 mm

1 mm

1 mm

DCV-Lung

Z = 1.4 mm

1 mm

DCV-Spinal Cord

1 mm

DCV-Stomach

1 mm

1 mm

1 mm

Z = 0.6 mm

1 mm

DCV-Scrotum

1 mm

DCV-LiverZ = 1.8 mm

1 mm

Z = 1.5 mm
1 mm

Z = 1.0 mm

1 mm

DCV-Leg

Z = 0.9 mm

1 mm

1 mm

1 mm

Figure S4. Magnified Organ Images of CAG-EGFP Tg P1 Mouse, Related to Figure 3
3D-reconstituted magnified organ images (submaxillary gland, heart, lung, stomach, liver, kidney, intestine, spinal cord, scrotum, and leg) of the PI-stained CAGEGFP Tg P1 mouse were acquired with LSFM. All images were deconvolved with AutoQuant X3 software. Z-stack: 20-mm step, with 4.0 s 3 two illuminations for
EGFP and with 0.1 to 0.2 s 3 two illuminations for PI. Zoom of the microscope: 5.0 3 . Note that the structures inside the spiral canal or leg bones were detected(panels of spinal cord and leg). Prefix ‘‘DCV-’’ indicates the deconvolved image.

Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. S11

A

β-Actin-nuc-3×mKate2 KI #1/SYTO 16
DCV-Pancreas

Z = 3.6 mm

X = 13.0 mm

DCV-Spleen

Z = 3.5 mm

X

X

2 mm

C

B

2 mm

DCV-Muscle

2 mm

X = 8.0 mm

X = 6.5 mm

Z = 4.0 mm

2 mm

2 mm

2 mm

X

2 mm

F

Brain
2 mm

2 mm

2 mm

D

DCV-Stomach

Z = 5.0 mm

Y = 9.5 mm

2 mm

2 mm

2 mm

KidneyY

E

2 mm

DCV-IntestineZ = 6.0 mm

X = 16.5 mm

XMuscle

2 mm

2 mm

2 mm

2 mm

Figure S5. CUBIC Is Applicable to Whole-Organ Imaging with Single-Cell Resolution, Related to Figure 4(A–F) The reconstituted 3D and section images of the SYTO 16-stained mouse pancreas (A), spleen (B), soleus muscle (C), stomach (D), intestine (E), brain,
another kidney (contralateral to the kidney shown in Figure 4C), and another soleus muscle (contralateral to the soleus muscle shown in Figure S5C) (F), of b-actinnuc-3 3 mKate2 KI mouse (8-week-old) were acquired with LSFM. In (A)-E, images were deconvolved with AutoQuant X3 software. Raw X-Y section images, andreconstituted Y-Z and X-Z section images are at the indicated positions.
(A) Pancreas images. Z-stack: 20-mm step 3 321 planes, with 0.05 s 3 two illuminations for SYTO 16 and with 0.5 s 3 two illuminations for mKate2. Zoom of the
microscope: 1.25 3 .
(B) Spleen images. Z-stack: 20-mm step 3 231 planes, with 0.05 s 3 two illuminations for SYTO 16 and with 1.0 s 3 two illuminations for mKate2. Zoom of the
microscope: 1.6 3 .
(C) Soleus muscle images. Z-stack: 20-mm step 3 331 planes, with 0.10 s 3 two illuminations for SYTO 16 and with 2.0 s 3 two illuminations for mKate2. Zoom of
the microscope: 1.6 3 .
(D) Stomach images. Z-stack: 20-mm step 3 396 planes, with 0.05 s 3 two illuminations for SYTO 16 and with 1.0 s 3 two illuminations for mKate2. Zoom of the
microscope: 1.25 3 .
(E) Intestine images. Z-stack: 20-mm step 3 366 planes, with 0.05 s 3 two illuminations for SYTO 16 and with 2.0 s 3 two illuminations for mKate2. Zoom of the
microscope: 1.0 3 .
(F) The reconstituted 3D images of brain, contralateral kidney, and contralateral muscle. Zoom of the microscope: 1.25 3 for brain, 2.0 3 for kidney, and 1.6 3 formuscle. Prefix ‘‘DCV-’’ indicates the deconvolved image.

S12 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

B R26-H2B-EGFP/PIA CAG-EGFP Tg/PI
3D image

Magnified section image

Lung

Kidney

Heart

2 mm

1 mm

2 mm

2 mm

Liver

Pancreas

Lung

2 mm

1 mm

2 mm

2 mm

KidneyC Thy1-YFP-H/PI
SpleenMagnified image

2 mm
1 mm

Pancreas

2 mm

300 μm

1 mm

Intestine

Magnified image

Intestine

2 mm

2 mm

1 mm

2 mm

500 μmFigure S6. CUBIC Is Applicable to the Whole-Organ Imaging of Various Fluorescent Proteins, Related to Figure 4(A) The reconstituted 3D whole-organ and magnified section images of heart, lung, kidney, pancreas, and intestine from CAG-EGFP Tg mouse (8-week-old) wereacquired with LSFM. The magnified section images of EGFP/PI merged signals at the approximate 1 mm depth of each organ. Z-stack: 20-mm step, with 0.3 to
2.0 s 3 two illuminations for EGFP and with 0.1 to 0.4 s 3 two illuminations for PI. Zoom of the microscope: 0.8 3 for intestine, 1.0 3 for pancreas, 1.25 3 forlung, 1.6 3 for heart and kidney, and 5.0 3 for all magnified section images.
(B) The reconstituted 3D whole-organ images of lung, kidney, liver, and pancreas from R26-H2B-EGFP KI mouse (4-month old) were acquired with LSFM. Zstack: 20-mm step, with 2.0 s 3 two illuminations for EGFP and with 0.1 to 0.3 s 3 two illuminations for PI. Zoom of the microscope: 1.0 3 for pancreas, 1.25 3 forlung, liver, and kidney.
(C) The reconstituted 3D whole-organ images of spleen and intestine from Thy1-YFP-H Tg mouse (8-week-old) were acquired with LSFM. Z-stack: 20-mm step,with 0.7 to 2.0 s 3 two illuminations for YFP and with 0.1 to 0.3 s 3 two illuminations for PI. Zoom of the microscope: 0.8 3 for intestine and 1.25 3 for spleen.
Magnified 3D images are at the indicated positions. Arrow indicates neurons.

Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. S13

A

mKate2

mKate2/Tendinous cords

mKate2

mKate2/Aortic valve
Aorta

Aortic valve

LV
Tendinous cords

400 μmmKate2400 μmmKate2/Tricuspid valve

mKate2

mKate2/Pulmonary
arterial valve (PA valve)RA
PA valve

Tricuspidvalve
400 μmPA
400 μmRV

B β-Actin-nuc-3×mKate2 KI #1/SYTO 16
Heart

mKate2/Coronary vessels
automatic extraction

Merge

mKate2/Coronary vessels
manual extraction

2 mm

Figure S7. CUBIC Is Applicable to the 3D Anatomy of Organs, Related to Figure 6
Manual extraction of internal structures was performed with Surface analysis of Imaris software. The reconstituted 3D images of the heart from the SYTO 16stained b-actin-nuc-3 3 mKate2 KI mouse (8-week-old) in Figure 4 were used. After Surface extraction by the software, each structure was manually curated andextra Surface signals were eliminated.
(A) Detailed views of heart valves and surrounding structures. Cross sectional mKate2 images and surrounding structures (Surface volume, 3D) are shown.
(B) Automatic identification of coronary vessels. Left panel: automated extraction of coronary vessels with Fiji-based pipeline. Right panel: manual extraction ofcoronary vessels with Imaris in Figure 6A (view from different direction). Extracted vessels with these two methods are merged in the center panel.

S14 Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc.

A CAG-EGFP Tg P1 body
DCV-Chest (V-D)

PI (3D)

Merge

Surface
Heart

Lung

Liver
Stomach2 mm

BIntestine

SpleenPI (section)(Z = 4200 μm)

Bronchial tree
(Surface + section)

Bronchial tree
(Surface)PI (section)(Z = 1800 μm)

Vascular structure(Surface + section)

Vascular structure(Surface)PI (section)(Z = 2700 μm)

Intestinal villi(Surface + section)

Intestinal villi(Surface)PI (section)(Z = 3600 μm)

Esophagus and Stomach(Surface + section)

Esophagus and Stomach(Surface)

Lung

1 mm

C
Liver

1 mm

IntestineD

1 mm

Esophagus& StomachE

1 mm

Figure S8. CUBIC Is Applicable to the 3D Anatomy of Infant Body, Related to Figure 6(A–E) Extraction of internal structures was performed with surface analysis of Imaris software, similar in Figure 6. The reconstituted 3D images of the chest (A),lung (B), liver (C), intestine (D), and esophagus and stomach (E) from the PI-stained CAG-EGFP Tg P1 mouse in Figure 3B were used. After surface extraction by
the software, each structure was manually curated and extra surface signals were eliminated. (A) Overview of organ identification using the chest 3D image withsingle-cell resolution. Prefix ‘‘DCV-’’ indicates the deconvolved image. (B) – (E) Extraction and visualization of the bronchial tree (yellow) with organ surface (lightblue) of lung in (B), vascular structure (olive) with organ surface (green) of liver in (C), intestinal wall (purple) and villi (peachpuff) in (D) and stomach (brown),
esophagus (gray) and esophageal gland (light gray) in (E).

Cell 159, 911–924, November 6, 2014 ª2014 Elsevier Inc. S15