Advanced CUBIC Protocols for Whole-Brain and Whole-Body

Home , 3DISCO, CUBIC, Nature Protocols

© 2015 Nature America, Inc. All rights reserved. whole organ or body. whole-organ scale. using multiple samples simultaneously. We previously used analysis can take <1 d, but it is dependent on the number of samples in the data set. on the sample type and the experimental purposes. analysis of multiple samples. T Here we describe a protocol for advanced Published online 8 October 2015; Majors, University Tsukuba,of Ibaraki, Japan. 5 Japan. Tokyo, AMED, 4 (CREST), Technology and Science Evolutionary by electrophoresis by lipids the of most removing and polymer hydrogel into tissue a both fluorescence retention and by high transparency embedding enabled CLARITY of introduction The capability. clearing low a have relatively however, they retained; to be signals fluorescent FRUIT and SeeDB including reagents, hydrophilic other use methods tissue-clearing oped Sca stabilizingfluorescent proteins to crucial are clearing during temperature and control pH that method development. A recent this addressed issue, publication further reporting to led issues safety and proteins fluorescent of analysis immunohis tochemical whole-mount with compatible be to shown were they and tissue, the of (RIs) indices refractive homogenizing and lipids a days few by removing within transparency high achieved solvent-cleared of benzoate purpose this imaging for (3DISCO)) organs 3D in alcohol–benzyl used solvents and benzyl (BABB), salicylate, alcohol–methyl Early tissue-clearing methods used organic chemicals (e.g., benzyl Development methods tissue-clearing of trend recent a become has technologies related and this multiple cells simultaneously in organs. Thus, the development of of the analysis enables that is lowed by one approach 3D imaging fol clearing Tissue networks. cellular and cells of activity and number position, type, about information to provide is expected organisms whole and organs in cells of analysis Comprehensive people have been seeking a way to observe all cells inside the body. Since the discovery of the cell as the basic unit of organisms, living I 1 Ueda R Hiroki Susaki A Etsuo whole-body clearing and imaging Advanced CUBIC protocols for whole-brain and its technical difficulty and limited scalability because of the need need the of because scalability limited and difficulty technical its Department of of AnatomyDepartment and Embryology, Medicine,Faculty of University Tsukuba,of Ibaraki, Japan. and Computer Engineering Science,Electrical School of Science Faculty, and Engineering Queensland University Technologyof (QUT), Brisbane, Queensland, Australia. Department of Systemsof Department Pharmacology, The University Tokyo,of Tokyo, Japan. NTRO he l e CU 9 , use a hydrophilic chemical urea, and more recently devel urea, more recently and , chemical a use hydrophilic A D BI rc-dVenus transgenic ( transgenic rc-dVenus UCT C protocol enables simple and efficient organ clearing, rapid imaging by light-sheet microscopy and quantitative imaging 1 I 4 . These methods are easy and safe, and they allow allow they and safe, and easy are methods These . ON 1 1 1 – – 0 1 3 3 T , 5 7 , hese hese protocols provide a platform for organism-level systems biology by comprehensively detecting cells in a . Possible drawbacks of CLARITY included included CLARITY of drawbacks Possible . 7 Clear . However, concerns about the quenching quenching the about concerns However, . , Kazuki Tainaka Kazuki , T doi:10.10 (ref. T T he he organ or body is cleared by immersion for 1–14 d, with the exact time required dependent g) mice. 8 . Alternative techniques, such as as such techniques, . Alternative 1 38/nprot.2015.08 1 , r 2,2 or ), 7 3– These These authors contributed equally to this work. Correspondence should be addressed to H.R.U. ( 6 . Some of these methods methods these of Some . CU 1 CU – BI 3 , BI 7 ` C -thiodiethanol , Dimitri Perrin Dimitri , informatics calculated the Venus signal subtraction, comparing different brains at a C ( 5 C lear, A single imaging set can be completed in 30–60 min. Image processing and CU 3 Laboratory forLaboratory Synthetic Biology, RIKEN Quantitative Biology Center (QBiC), Osaka, Japan. U 1, BI 2 nobstructed Brain/Body Imaging . C 12,1 to image whole-brain neural activities at single-cell resolution 2 Japan Agency for Medical Research and Development (AMED) – Core Research for - - - 3 4

, 7 , Hiroko Yukinaga Hiroko , and whole-body clearing by CUBIC. CUBIC also provides provides biological also extracting for images CUBIC 3D of CUBIC. analysis and by processing clearing whole-body and high-throughput images. 3D multiple a of collection facilitates method tissue-clearing efficient CLARITY-processedof brains imaging whole-brain-scale for used been has microscopy) sheet microscopy) (ultra unit light-sheet macrozoom-compatible a using brain a of BABB-cleared imaging whole-brain was rapid imaging brain calcium dynamics whole-brain or embryos developing such of imaging imaging, 4D time-lapse a and as 3D to applied been has it and manner, collect can imaging microscopy of for type This used been also has (LSFM) microscopy cence fluores light-sheet transparent, highly tissues render clearing methods above the of some Because microscopies. 3D optical in with imaged be can methods above the using cleared Tissues tissues cleared imaging for Methods information biological extracting for mouse analyses whole image for used and were images heart) These body. and lung (e.g., organs mouse whole hemisphere, marmoset whole a brains, mouse whole of preserves imaging 3D rapid and clearing which for CUBIC used Wehave clearing. reagents, body clearing hydrophilic whole- and tissue whole-organ reproducible enables It on fluorescence. device-free based and method high-performance a offers scalability the increased that been protocols clearing have passive of development the by difficulties addressed these However, device. specific a use to Rapid 3D imaging with LSFM can be used after whole-organ whole-organ after used be can LSFM with imaging 3D Rapid CUBIC CUBIC. perform to how describe we protocol, this In 6 4 PhD PhD Program in Human Biology, Integrative School of and Global . More recently, COLM (CLARITY-optimized light- (CLARITY-optimized COLM recently, . More T 20–2 3 he , Akihiro Kuno , Akihiro NATUREPROTOCOLS 3 CU C . One of the earliest cases of whole mouse mouse whole of cases earliest the .of One 18,1 ocktails and 16,1 BI 9 . C 7 . clearing protocol can process 1 6 . Thus, the use of LSFM after an an after LSFM of use the .Thus, C 1 omputational omputational analysis).

z , | 2 -stack images in a rapid rapid a in images -stack VOL.10 NO.11VOL.10 , 5 , 6 & [email protected]

PROTOCOL | 2015

1709 1, ). 2

- - .

© 2015 Nature America, Inc. All rights reserved. In rapid whole-organ imaging, a single, cleared, whole mouse mouse whole cleared, single, an a imaging, use whole-organ We rapid In LSFM. in purpose. this used BioTec)for (LaVision Ultramicroscope be optimized can samples CUBIC-cleared informatics and image imaging or CUBIC for whole-body whole-organ signal intensities comparing for samples different of alignment and tures the in cells labeled whole-organstructure,specificanatomical strucextractionof genetically of positioning precise dye enables nucleic a with counterstaining whole-organ Furthermore, and it is scalable from subcellular structures (e.g., neuronal neuronal (e.g., structures subcellular axons or spines) from to marmoset brains to whole animal bodies scalable is it and experiment, single a in samples multiple to applied be can tocol tissues but also whole-body clearing of infant and adult mice dissected of clearing faster only not enabled CB- protocol The perfusion procedure. and decoloring body and clearing mouse the the accelerate to penetrate efficiently to reagent CUBIC (CB-perfusion) the of perfusion used We tissue. decolorize to tissues and blood in heme remove can lenge in tissue clearing. We accidentally found that aminoalcohols needed to elucidate tissue-clearing mechanisms. are studies further and thus the procedure, during roles different d ~14 within (ref. transparent became brain mouse whole a Thus, tissue. the within scattering light further-reduced which reagent, the and sample the between RIs the matched reagent second the reagent to the SeeDB of that similar is which ~1.49, to close RI an has which reagent, second the into the with immersed and then buffer for with ~1 week, reagent washed first treated was brain fixed A transparency. of degree the scattering material inside tissue, and its removal is correlated with remover.lipid potential a light- main the be to thought is Lipid as works reagent first The tissue. the inside scattering light mize Sca Sca and (reagent-1) reagents, two prepared we protocol, clearing clear tissue with minimal fluorescence quenching Sca in TritonX-100 and urea to addition in aminoalcohols, brain-clearing of chemicals. We discovery 40 screened Sca nonbiased enables screening This solution chemical candidate a with mixing after and before measured was suspension a fixed-brain of turbidity of reduction in which method screening a chemical new we pose, constructed Sca the criteria,by modifying westarted method with hydrophilic reagents had the potential to fulfill these a .clearing Because samples multiple of analysis for comparative LSFM, and, with two, imaging reproducibility whole-brain/body and transparency with the preservation of fluorescence for a efficiency rapid one, criteria: main two considered we imaging, body whole- and whole-brain for technique clearing a developing In whole-organ or whole-body clearing CUBIC Development for of efficient and reproducible samples. multiple with layers organ and cellular targeting which experiments perform to users informatics, of range wide image a enables and imaging whole-body or organ whole- of platform a presents CUBIC Therefore,information. 1710 PROTOCOL Because of its efficiencyitsreproducibility, and Becauseof proCUBICthe chal is another absorption light to scattering, In light addition

1 | VOL.10 NO.11VOL.10 8 ). However, some chemicals seem to have additional or or additional have to seem chemicals some However, ). 18,1 9 . | 2015 l CBC2 raet2, hc as mini also which (reagent-2), eCUBIC-2 |

NATUREPROTOCOLS l e-related e-related chemicals and found that 1 0 . from Moving to the buffer 1 l 9 e recipee ; thus, CUBIC is able able is CUBIC thus, ; 1 8 9 . In the CUBIC . For this pur this For . l eCUBIC-1 eCUBIC-1

18,1 1 1 l 9 e, 8 9 . - - - - - . .

with with open-source software such as Advanced Normalization Tools was performed analysis This samples. between subtraction signal These aligned images could be merged with each other to calculate the corresponding signal images (transgenes, etc.) for alignment. were to applied parameters Next,parameters. the transformation transformation calculate to brain reference a to registered were MRI analysis (fMRI) functional in used often informatics image implemented pancreases diabetic and normal in islets Langerhans of comparison for images 3D the in structures anatomical of extractions Weperformed extraction. spot and counting including surface ous functions image analysis depicting the reconstituted 3D image. Imaris implements numer directions. (V-D) side-up we took imaged in two imaging,orientations;whole-brain in the case of is sample the regions, deeper in detected are signals weaker that fluorescence in regions. Todeeper than red green fluorescence, particularly ensure with obtained are results imaging better thus and tissue, in deeper penetrate can wavelengths red general, in results; imaging of quality the affects wavelengthFluorescence brain can be imaged within 30–60 min per color and orientation. in our second CUBIC report CUBIC second our in ( protocol CB-perfusion The ( report CUBIC first our in protocol immersion The clearing. whole-body for tocol pro CB-perfusion the 2C, Step and faster organs; for whole of protocol clearing immersion and CB-perfusion the 2B, Step Step 2A, a simple immersion protocol for dissected whole organs; clearing. Tissue counterstaining. nuclear with together proteins fluorescent previously to weeks, as described applicable to staining with small chemicals or antibodies over days also see 7–15, (Steps samples brain different of comparison and preprocessing for informatics CUBIC (iii) and 3–6); (Steps a LSFM with imaging whole-organ and whole-brain (ii) 1–2); simple (Steps by CB-perfusion and protocols immersion clearing CUBIC advanced the (i) lowing: fol the describing body.on or focus Herewe organ whole a in cells of analysis a for comprehensive a platform provides CUBIC the CUBIC of pipeline Overview scale. organ and whole-body delineate structural abnormalities in disease samples at the whole- help will it and activities, neural timing-dependent or stimulus- ics fluorescent 3D images was first reported using whole-brain CUBIC informat using by analysis comparative direct Such stimuli. light the to responded neurons which in brain whole the in cells tion with or without light stimulation and calculate the signal subtrac in we analysis, scripts demonstrate comparative3D image analysis the of Arc-dVenus prepared of Tg example mouse with brains an As manuscript. pipeline this analysis easier an provide we convenience, user’s the For skills. science computer and ics (ANTS) Fig. Image visualization software such as Imaris can be used for for used be can Imaris as such software visualization Image 1 8 18,2 . CUBIC informatics enables quantitative identification of of identification quantitative enables informatics CUBIC . 2 ) in which the clearing speed and efficiency are increased. increased. are efficiency and speed clearing the which in ) 6 2 . The final subtraction data clearly depict regions and and regions depict clearly data subtraction final The . z 4 -stack images of the dorsal-side-up (D-V) and ventral- and ITK-SNAP 1 Here we provide three clearing procedures: 8 . First, structural images via counterstaining counterstaining via images structural First, . 1 8 has been improved to an advanced version version advanced an to improved been has 1 9 2 . For more complicated analyses, we we analyses, complicated more For . 5 , but it requires advanced informat advanced requires it but , Fig. 1 Fig. 9 18,1 , but more detail is given here. here. given is detail more but , 3 9 1 ) is almost identical to that that to identical almost is ) , we focus here on imaging of of imaging on here focus , we ). Although CUBIC is also also is CUBIC Although ). ------

© 2015 Nature America, Inc. All rights reserved. reagent-1. Samples are subsequently treated with reagent-1 and and reagent-1 with treated subsequently are Samples reagent-1. diluted with Alternatively, (wt/vol) perfused the fixed can body be further 1). 4% (Step postfixation for with dissected are organs perfused then and PFA, transcardially are animals Thus, needed. is fixation (PFA) paraformaldehyde a option, either In purposes. experimental their for transparency final desired the ( and it is on dependent the organ and to methods imaging be used because of a short fixation time. Incubation period can be varied, kidney or liver) muscle, (heart, organs heme-rich in particularly protocol, sion The clearing performance of CB-perfusion surpasses the immer in samples the to correspond samples actual of Images manuscript. imaging can be performed with an LSFM. The collected data are processed and analyzed, such as by signal comparison between samples, as describedhybrid in protocolthis (Step 2B), in which dissected organs after CB-perfusion can be continuously cleared according to the simple immersion protocol. experimentalRapid 3D purpose and organs); a CB-perfusion protocol for the whole adult mouse (Step 2C), which takes ~14 d; and the CB-perfusion we andprovide immersion three protocols: a simple immersion protocol (Step 2A), which takes ~11 d for a whole brain from an adult mouse (but Figure varies1 according to the reagent-2.Counterstaining isperformed during and after reagent-1 Committee of the RIKEN Kobe Institute and The University of Tokyo, and all animals were cared for in accordance with institutional guidelines. Figs. 1–

Clearing by Clearing by Image analysis CB-perfusion simple immersion

| 3 Overview of the CUBIC pipeline. CUBIC is composed of three major stages (clearing, imaging and analysis). For efficient and reproducible clearing, ). Users may select either of these options and determine Steps 1–2C(i) Steps 1–2B(iv) Fixation /CB-perfusion Step 1–2A(ii) Fixation Day Day (viewed inITK-SNAP) Convert toNIfTI-1/ Perfused withPBS Steps 7and Perfused withPBS,4%PFA downscaling and 4%PFA 0 0 NIfTI-1 data Preprocessing and 1/2-dilutedreagent-1 5–10 mi ~20 min 1 Raw TIFF 9 , but it tends to cause decreased signal intensity intensity signal , decreased to cause it but tends 30 min~ n 8 Acquision Acquision Postfixation Day 1 Steps 9and10 in 4%PFA 1 d Create D-V+V-D composite ~3.5 h V-D images D-V images Dissected organs Day 1 Whole body 1–7 side ventral Use side dorsal Use refresh/2–3 Step 2B(v–vii) Step 2A(iii–vi) Lipid removal Reagent-1 composite Create Figures d Lipid removal/decolorization Step 2C(ii,iii) Two-photon imaging,etc. refresh/1 Reagent-1 7 d d Signal subtraction 2 - Days 2–8

and and Steps 11and12 Structure Signal d fluorescence, and 50 mW of 588-nm laser-ET650/60 emission emission laser-ET650/60 588-nm of mW 50 and fluorescence, green for filter emission laser-ET525/50 488-nm of mW 100 use We installed. also is typically filters emission and laser excitation ( organs abdominal soft and samples A customized sample holder is also used for larger brain and body ductor (sCMOS) camera (Andor NEO 5.5, 2,560 × 2,160 pixels). Olympus) and a scientific complementary metal oxide semicon supplied with an optimized macrozoom microscope (MVX-ZB10, BioTec) LaVision (Ultramicroscope, instrument LSFM available imaging. whole-body or Whole-organ PAUSE as POINTS. indicated are which PROCEDURE, treatment 3 Wash . All animal experiments here were approved by the Animal Care and Use ~1 Day d Alignment 8 ~Day 9 ~3 h Step 2B(viii,ix) Step 2A(ii,iii) RI matching refresh/2–3 7 18,1 Reagent-1 refresh/1 d Reagent-2 Reference 2 d 9 . Samples can be stored at various points in the the in points various at stored be can Samples . d d NATUREPROTOCOLS Steps 13–15 Sample Normalization andsubtraction ~Day 11 Day 15~ between samples A Subtracted 0.5–1.5 Immersion Immersion ~10 mi h oil 1 oil Sample

Fig. d | VOL.10 NO.11VOL.10 n We use a commercially commercially a Weuse 4 B ). A suitable pair of of pair suitable A ). PROTOCOL 1–3 hpersample with LSFM Imaging Steps 3–6 | 2015 |

1711 -

© 2015 Nature America, Inc. All rights reserved. in 2 weeks of reagent-1 treatment. C57BL/6 male mice (8 weeks old) were used bones and gastrointestinal content become sufficiently transparent after tend to become transparent by CB-perfusion. Major abdominal organs except treatment. Organs such as pancreas, submaxillary gland and spleen quickly CB-perfused whole body just after CB-perfusion ( mm. 5 and bars, afterScale 2 reagent-2. in weeks than ofrather reagent-1 Some of the organs such as pancreas are more transparent in reagent-1 procedure are markedly transparent after 1 d of reagent-1 treatment. reagent-1 and at day 10 in reagent-2. CB-perfused organs from a successful 5 mm. ( is indicative of how well researchers succeed at CB-perfusion. Scale bars, scopically cleared and decolored at this point. The clearance of these organs CB-perfusion. Organs such as the pancreas, spleen or kidney are macro clearing through the vascular system. ( fixation, PBS for flushing PFA and 1/2-diluted reagent-1 for accelerative for flushing the blood, 4% (wt/vol) PFA on ice with peristaltic pump for CB-perfusion. The transcardiac perfusion line is connected to heparin-PBS Figure 3 and transgenes) (e.g., images signal both analysis, image further 10 set and LSFM used the of sheet thinnest the select Wetypically sheet. laser the of thickness the needed.resolutionis finer if were 1 Video they cerebral when the structures as sparsely subcellular labeled, such as discussed below from ( regions even in or cells cortex, single from signal ing 6.5 5.2 × ~5.2 is pixel one that such microscope, the of zoom the on dependent is pixel per filter for red to far-red fluorescence.guidelines. The size of the acquired image institutional with accordance in for cared were animals the of all and Tokyo, were approved by the Animal Care and Use Committee of the RIKEN Kobe Institute and The University of swollen, but the size is recovered after immersion in reagent-2. Scale bar, 5 mm. All animal experiments A brain from C57BL/6 male mouse (8 weeks old) was used. Reagent-1–treated sample is temporally shaker used for the PBS washing step at room temperature. ( clearing procedure. Inset: five brain samples treated with reagent-1 in a single tube (day 5). ( ( steps. degassing the for dissolved by heat and stirring with a microwave and a hot stirrer (bottom). ( contains a high concentration of urea (25 wt%) and sucrose (50 wt%; top). These can be completely Figure 2 1712 guidelines. institutional with accordance in for cared were animals the of Committee of the RIKEN Kobe Institute and The University of Tokyo, and all PROTOCOL e ab

b Complete Before – M d dissolution dissolution Fixed brain zoom. These pixel sizes are sufficient for detect zoom.forpixel sufficient1.6× are sizes These at m | . . All animal experiments were approved by the Animal Care and Use VOL.10 NO.11VOL.10 (day 1) c

) ) Clearing performance of CB-perfused dissected organs at day 1 in ). Users can also use higher magnified zoom values (~6.3×) | | Procedure of the simple immersion protocol. ( protocol. immersion simple the of Procedure Procedure of the CB-perfusion protocol. ( protocol. CB-perfusion the of Procedure 1/2 reagent-1 | (day 1,6h) 2015 c ) An incubator with a shaker (a hybridization incubator) that we use for the M | × ~6.5 is pixel one and zoom × 2 at m

NATUREPROTOCOLS Z -step size is selected according to to according selected is size -step b Reagent-1 c ) ) Dissected organs just after (day 7) Fig. 4b M d ) ) Clearing performance of a m as the as m – f a and and ) ) Surgical setup for (day 8and9) PBS washed a ) Preparation of reagent-2. This reagent Supplementary Supplementary z e -step size. For For size. -step ) Appearance of a brain sample in each step.

- 1/2 reagent-2 (day 9,6.5h) d

- b

) A vacuum desiccator L25×/1.00, WD = 6 mm, n n mm, 5.6 = WD 20×1.0 d c a Day 10inreagent-2 Day 1inreagent-1 After 2weeksofreagent-1treatment After CB-perfusion Heart 1/2 reagent-1 4% PFA Reagent-2 (day 11) Head d ) A table Lung

PBS Peristaltic pum /0.6, working working /0.6, × 10 Olympus XLPLN10XSVMP, (e.g., imaging tissue lenses deep objective for released have vendors purposes imaging for these developed methods clearing other the of clearingperformance surpasses that ofsome microscopy with two-photon imaging region deep to applicable one-step by for 1–3 d in are reagent-1 even immersion samples cleared used. Partially be also can microscopes two- or photon confocal used widely available, D-V images ( in are sharper slices horizontal dorsal and horizontal slices are sharper in V-D images ventral the because recommended is This of imaging) whole-brain are also collected. ferent directions (D-V and V-D, in the case fromsample dif same the 3D image,of data the throughout signals of sharpness To collected. ensure be should terstaining coun whole-organ via images structural n XLSLPLN25XGMP, and 25×/1.0, WD 1.33–1.52 = 8 mm, = range) index refractive n mm, 8 = (WD) distance Dorsum e = 1.41–1.52;= Zeiss LDPlan-Aphochromat If a proper LSFM instrument is not not is instrument LSFM proper a If PBS/heparin e Liver e = 1.457). = 14–.7 Lia C FLUOTAR HC Leica 1.43–1.47; = Butterfly needle p Kidney Fig. b Chest Kidney Spleen Pancreas 5 PFA perfusion ) 1 8 . Pancreas 1 8 1 , because the the because , 9 e . Microscope . Microscope (adjustable (adjustable CB-perfusion Pelvis Spleen - -

© 2015 Nature America, Inc. All rights reserved. the current software tools, files need to be downscaled to 25% by to 25% downscaled to be need tools, files software current the ware such as ITK-SNAP n ( (NIfTI) Initiative Technology Informatics Neuroimaging image in the NIfTI-1 data format (.nii extension) introduced by the TIFF stack ( raw data, 7–15). of each For(Steps preprocessing processing data CUBIC informatics. Imaris with and 690, GTX GeForce installed. software NVIDIA and RAM of GB 64 GHz, 3.50 at CPU i7-3970X Core(TM) Intel(R) with PC ( reconstitution 3D for used be should board graphics good a and size memory large a images with PC signal and structural acquired in two contains D-V directions, and V-D). Therefore, a high-spec (which set single a data for GB brain 25–30 thus and direction and color brain, per LSFM, for images (16-bit institutional guidelines. of the animals were cared for in accordance with Kobe Institute and The University of Tokyo, and all the Animal Care and Use Committee of the RIKEN All animal experiments here were approved by value of images in protocol. Brightness/contrast and minimal gamma- stained with PI (red) according to the CB-perfusion time = ~45 min). The samples were cleared and two illuminations from each side, total acquisition planes, zoom = 0.8×−1.6×, expose = 100 ms to (imaging2 condition:s × (GFP, green) stained with PI (male, 8 weeks old) 3D whole-organ images from a CAG-EGFP Tg ( ). × 5 = (zoom organ each of mKate2 and merged signals at the ~1 mm depth protocol. The magnified images of SYTO 16, stained with SYTO 16 according to the CB-perfusion time = ~45 min). The samples were cleared and two illuminations from each side, total acquisition planes, zoom = 0.8×−1.6×, expose = 100 old) (imagingms condition: to 2 s × B ( image of the right hippocampus, as indicated in ( viewed from the midline to lateral, as indicated in ( ( in data ( (sharpness, brightness and contrast) with ImageJ. the left. These images were minimally processed Right, a magnified image of the indicated area on to the immersion protocol in this manuscript. time = ~30 min). The sample was cleared according two illuminations from each side, total acquisition step × 749 planes, zoom = 1.6×, expose = 50 ms × 23 weeks old) (imaging conditions: a cleared Thy1-YFP-H Tg mouse brain ( sample holder (inset) used in this manuscript. ( Figure 4 discarding three of every four images of the TIFF g f e d c b a -actin-nuc-3×mKate2 KI mouse ifti.nimh.nih.gov/ni ) A magnified image of the reconstituted ) The reconstituted 3D image of the acquired ) The reconstituted 3D whole-organ images from ) A magnified image of the right hippocampus, ) The microscope setup and the customized ) A magnified image of the indicated area in ) Left, a raw TIFF image (2,560 × 2,160) from All raw image data collected in an uncompressed TIFF format format TIFF uncompressed an in collected data image raw All b

. A view from the dorsal side is shown. | Whole-organ imaging with LSFM. Fig. c – 5 z h = 20 20 = , ‘Collect raw images’) is first converted to a 3D 3D a to converted , first is images’) raw ‘Collect were adjusted with Imaris. Here we show the step-by-step procedures for z fti-1 = 20 20 = M m step × 350–500 h 2 ) The reconstituted 5 / . Because of the memory limitations of of limitations memory the of Because . M ). NIfTI-1 files are visualized using soft 1 m step × 350–500 9 Fig. 4 Fig. (male, 8 weeks z 3 = 10 10 = 4 (male, Fig. 4c Fig. b ) are typically ~7 GB in total total in GB ~7 typically are ) x 3 M - 5 z

c –

. c

. h

). We use a Windows Windows a use We ). a h g c 1 mm z 2 mm -stack series and

Liver Kidney Lung Heart Cover glass 2 mm 2 mm Sample Heart 3D 2 mm 2 mm 3D e http:// 2 mm 1 mm 1 mm Holder SYTO 16 nat Illumi Lung f i o - n

- d 1 mm 1 mm 200 ine operations only. Then, using the ‘WarpImageMultiTransform’ or it Normalization) can example, to be Symmetric restricted aff (for deformation allow can transformation The software. ANTS the of ‘ANTS’the function by calculated is This image. V-D the to image D-V the align to needed is which parameter, formation is processed. image is used The tostructural calculate the trans images, signal and structural images,the data NIfTI-1 of pair a directions opposite from (D-V and V-D), the acquired files need to be ( aligned brain same the of channel (which depends on the acquisition direction) is needed. raw and image data the parameters) and orientation downscaling of number pixel the (given spacing correct the of specification ( ITK-SNAP from tool Convert3D the using file NIfTI-1 a to converted is stack PNG each Next, stack. TIFF original each for files PNG 16-bit of stack temporary a create to procedure is done using ImageMagick ( tools. downscaling The informatics image of development future ( 540 × 640 to × 2,160 from 2,560 images these of resolution the by changing d µ In preparation for merging NIfTI-1 data in the same color color same the in data NIfTI-1 merging for preparation In 2 mm mKate2 m Kidney 3D Fig. Merged 5 Liver ). This limitation should be overcome with the the with overcome be should limitation This ). b Raw TIFFimage 500 NATUREPROTOCOLS 2 mm e µ

Intestine Stomach Spleen Pancreas m 2 mm 2 mm 2 mm Pancreas 3D 2 mm 3D 2 mm ( z 2 mm =3.5mm Spleen 1 mm 1 mm SYTO 16

| http: VOL.10 NO.11VOL.10 1 mm 1 mm ) 2 mm 500 f Stomach //imagemagick.org µ m 10 pixels Fig. Fig. mKate2 PROTOCOL 5 5 ). In this step,this In ). ). In this step, x - | z 2015 2 mm Intestine Merged |

1713 / ) - -

© 2015 Nature America, Inc. All rights reserved. NIfTI-1 data. 3D reconstitutions were performed with Imaris software as in values. The reconstituted 3D images and plane images in the ‘Align’ and ‘Combine’ panels were prepared by using exportedthreshold TIFF values imagesat the from the corresponding as an example again). To do so, the ‘edge content’ based on the Prewitt operator is calculated for both the D-V and V-D theimages. following This stepis (alignment).used to define We two then merge the aligned images in order to ensure sharpness throughout the resultingcounterstaining 3D imageare registered(YFP channel to is theshown corresponding V-D data. This step is to calculate transformation parameters, which is applied to to the25% signaland convertedD-V todata NIfTI-1 in files (shown as capture images of ITK-SNAP). Next, structural D-V data (shown as reconstituted 3D images)(here viaonly YFP nucleichannel shown) in the panel are as an example. The ‘Collect raw images’ part of the figure shows the scheme of brain 3D imaging of two different directions (D-V and V-D). Raw TIFF images composite NIfTI-1 data for both structural and signal images. signal and structural both for data NIfTI-1 composite functions of FSL vidual pixels in the images, indi of we values usethe access to theorder In image. ‘fsl2ascii’NIfTI-1 merged the and ‘fslascii2img’ create we and thresholds, the and pairs image the take Wethen images. two the of combination linear a are two the between in image,slicesabove thresholds, images (as a proxy for image sharpness). This is used to define two operator Prewitt the ness throughout the resulting 3D image ( image. V-D the to image D-V the of images signal and structural ref. to both align from parameters transformation permission the apply with we ANTS, in Adapted function guidelines. institutional with accordance in for cared were animals all and Tokyo, V-D images, respectively. All animal experiments here were approved by the Animal Care and Use Committee of the RIKEN Kobe Institute and The University of Figure 5 1714 Dorsal Dorsal Ventral Ventral PROTOCOL Next, we merge the aligned images in order to ensure sharp ensure to order in images aligned the merge we Next,

| VOL.10 NO.11VOL.10 Ventral Dorsal

| Preprocessing of acquired 3D image for comparison analysis. Here we use the data set of the Thy1-YPF-H Tg mouse brain acquired in ref. ref. in acquired brain mouse Tg Thy1-YPF-H the of set data the use we Here analysis. comparison for image 3D acquired of Preprocessing Collect rawimages V-D image D-V image n and D-V image First plane Last plane 2 m, 8 . The steps above produce . a D-V + above produce of The steps pair V-D | 2015 z m so that slices below -slice position 2 only come from the D-V image and slices slices and image D-V the from come only 7 to calculate the ‘edge content’ of the two two the of content’ ‘edge the calculate to Aligned NIfTI-1files(signal) | z z

=2.25mm =2.25mm z z NATUREPROTOCOLS =5.5mm =5.5mm n m -th plane -th plane n Combine D-VandV-D and and

z = ~700 planes 2,560 voxelresolution 25% reductionof TIFF m n and to create the merged composite NIfTI-1 image (shown as capture images of ITK-SNAP) according to these z only come from the V-D = 2.25 mm (sharp) and 5.5 mm (blurred) for top and bottom, respectively. The raw data sets are downscaled Fig. V-D image First plane Last plane PNG 2,160 Downscaling 5 ). To do so, we use 640 n m -th plane -th plane 540

z = ~175 planes

Figure Signal Structure Signal Structure - - Horizontal Horizontal Use weighed (First ~ ( m D-V +V-D provide the aligned 3D images to an atlas ( atlas an to images 3D aligned the provide brain atlas such as the Allen Brain Atlas to a is registered reference Next, the internal experiment). that of internal reference (i.e., one of the brain images among the samples an to experiment same the from sets data brain all We align first ‘ANTS’‘WarpImageMultiTransform’.on and relies process the and images), signal and (structural in pairs are processed images ( aligned are samples different from images in as preprocessed are the of control Venusunder protein fluorescence yellow of version destabilized Tg mouse brains with or without light stimuli signal images to be calculated. We show an example of Arc-dVenus of subtraction permits which files, NIfTI-1 merged these align Use V-D Use D-V 4 +1 ~last) ( Convert toNIfTI-1 , and they are shown as views from the dorsal and ventral side in D-V and n Next, to facilitate analysis across different brain data sets, we we sets, data brain different across analysis facilitate to Next, – m n V-D image D-V image ) –1) Sagittal Sagittal Coronal Coronal Arc gene promoter Horizontal Figure Figure V-D image D-V image structural Register image D-V +V-DNIfTI-1file(signal) 1 8 Structure Structure . Composite NIfTI-1file 5 . Then, the composite NIfTI-1 NIfTI-1 composite the Then, . 2 6 ( Align D-VandV-D Sagittal Fig.6 2 9 . thus These calculations transformation (alignment)

signal and Applyto structural

parameter Calculate .... n n images a 21 11 ). First, the raw data data raw the First, ). Fig. 6 Fig.

n Fig. 6 Fig. 12 1 ... 8 , which express a ,express which c b ). Finally, the the Finally, ). ). As before,As). Coronal Signal Signal 1 8

© 2015 Nature America, Inc. All rights reserved. tigated. So far, we have tested a hemisphere of infant marmoset, marmoset, infant of hemisphere a tested far,have we So tigated. sufficient is d 1–3 for reagent-1 in immersion microscopy, a one-step by two-photon to image plan users for if for example, the user’s dure can be purpose: modified and takes several days to 2 weeks to process. However, the proce ( chemicals five comprises reagent CUBIC optimal the application, LSFM for clearing efficient To achieve microscopy. are cence can be tissues preserved, signals by imaged fluorescence but also other organs inside brain ( the only not for applications of range wide a enables previously used acetone or peroxidase with methods decolorization harsh or flushing simple the to opposed as pro teins, for milder is method This ability. decoloring tissue active The is first methods. the clearing other to with compared CUBIC prehensive pipeline. Thus, there are advantages several important ing, and the informatics analysis was developed to enable a com developed CUBIC. and optimized for whole-organ and whole-body imag of version current the of Limitations Allen Brain Atlas data. in shown mice Arc-dVenus Tg ( github up-to-date versions also available on a GitHub repository ( as guide user brief a and steps all one image from the other ( subtracting by brains of pairs compare to used is function Next, the ‘fslmaths’ brains. all across same the is brain the inside intensity median the that so images aligned these normalize we signal channel images of different brains are compared. To do so, with institutional guidelines. Adapted with permission from ref. and The University of Tokyo, and all animals were cared for in accordance approved by the Animal Care and Use Committee of the RIKEN Kobe Institute Imaris software as in using exported TIFF images from the corresponding NIfTI-1 data and with in images 3D reconstituted The images. downscaled single cells in the sparsely labeled regions can be detected even in the are merged and indicated in blue. As seen in the magnified panel (top right), and light blue, respectively. Standardized PI signals of the Light (+) sample shown. Signals observed in Light (+) or (−) conditions are shown in magenta the dorsal side (left and top right) and the dorsolateral side (lower right) are ( corresponding NIfTI-1 data. Views from the dorsal side are shown. Venus channel (yellow) and aligned PI channel (blue) images from the in dorsal view. ( Light (+) or (−) samples, after alignment to the internal reference, are shown The 3D reconstituted images from NIfTI-1 data for structure and signal of Brain Atlas. All images are then aligned to the atlas and normalized. The internal reference is also registered to a brain atlas such as the Allen by registering the first structural data to the second (internal reference). as in ( PI. blue, Yellow, Venus.; respectively. are shown as views from dorsal and ventral sides in D-V and V-D images, for PI, respectively). The reconstituted 3D images from raw TIFF stacks expose = 3 s × two illuminations for Venus and 300 ms × two illuminations in ref. of the Arc-dVenus Tg mouse brains with or without light stimuli, acquired Figure 6 samples was efficient: for example, in the case of cleared infant infant cleared of case the in example, for efficient: was samples and infant and adult whole mice d http://cubic.riken.j ) Results of subtraction, shown as 3D reconstituted images. Views from The actual scalability of CUBIC has not yet been fully invesfully been yet not has CUBIC of scalability actual The fluores and tissue, to clear used are reagents aqueous Because Figure 1 8 .com/SystemsResearch

, as an example ( | Calculation of signal subtraction. ( subtraction. signal of Calculation 5 . Next, we align one data set, Light (+), to the other, Light (−), c ) Reconstituted 3D images of the aligned and normalized Figures 4 p ), we also share example raw TIFF data of of data TIFF raw example share also we), z = 10 10 = Fig. 6 Table Table and M m step × 625–675 planes, zoom = 2 ×, d

/CUBIC_npro 5 ). We provide the set of scripts for for scripts of set ). the We provide . All animal experiments here were 1 Figure Figure b 18,1 ). ) We first preprocess these data sets 1 8 9 . Supplementary Data Supplementary ( a Table Table ) Here we use the data set 6 and NIfTI-1-converted NIfTI-1-converted and b 1 t – ). On our website website our On ). ). Clearing of these these of ). Clearing d were prepared by CUBIC was was CUBIC 1 30,3 8

. Table Table https://

1 , with with , . This This .

2 ) ------

eventhrough theskull directly imaged be could brain the of structures internal mouse, yet been optimized to fully clear bone and melanin pigments pigments melanin and not ( bone has clear fully CUBIC to optimized addition, been In transient yet cause procedure. and the lipids during the swelling of proportion large a remove such distortion maystructural happen given that CUBIC reagents spine and axon the including structures subcellular detailed the in even tissue brain in changes obvious observe not did we Although con methods. other be the with to tissues clearing need when sidered may it and SeeDB, as such methods, clearing (e.g., may be a drawback to CUBIC,This clearing. in during which removed case be othermay clearingthey thus methods and structures, chemical their of PFAbecause by fixed readily be not may Dil as can be observed in the cleared tissue, whereas lipophilic dyes such ing. In this sense, fluorescent proteins fused to a membrane protein be by fixed PFA should clear or before Dyes proteins remove lipids. dyes), may exist given that the reagents CUBIC clearing massively dye(e.g., relatedand lipophilic Dil labeling methods, particularly test other variations of dyes, possible limitations on some labeling procedure clearing CUBIC the using cal and annotation, image analyses has registration been achieved anatomi for staining nuclei Whole-organ method. clearing the ticular dyes or stains is an important consideration when selecting alternative. an as considered be may it and scalability, similar a achieved apparently has CLARITY of release agent (PACT)/perfusion-assisted technique clarity passive The issue. this address may perfusion CB- and times incubation longer difficult, more be might brains primate adult larger of clearing Although studies. future in this to investigate we plan although marmoset), (e.g., brains primate c a Table Table Aligned andnormalized

Structural distortion has been carefully addressed in some some in addressed carefully been has distortion Structural par with combined be can method tissue-clearing a Whether Light (–) Light (+) Light (–) Light (+) 1 mm 1 mm D-V image D-V image Clear D-V 1 ). However, this issue remains unaddressed by other other by unaddressed remains issue this However, ). + V- T (ref. D V-D image V-D image 1 1 d ), SeeDB Subtraction, light (+/–) NATUREPROTOCOLS 1 mm 1 9 . However, we have not yet tested adult adult tested yet not have However,we . b Venus, highinLight(–) Venus, highinLight(+) Light (–) Light (+) 1 D-V D-V 0 , FRUIT , (PI) (PI) + + V-D V-D 1 18,1

4 | ) should be considered. VOL.10 NO.11VOL.10 to atla Register reference to internal and alig Register Align allimagestoatlas 9 in situ . Although we did not not did we Although . / / PI s n NIfTI-1 AB (PARS) protocol (PARS) protocol A PROTOCOL 0.5 mm 1 mm | Magnified 2015 Rotated D-V D-V (Venus) (Venus) + + |

V-D V-D 1715 1 8 - - - - ,

© 2015 Nature America, Inc. All rights reserved. voxel volume per cell calculated voxel from cell density,volume per cell calculated and average the (EV), volume exclusion considered: be to need eters pixel)used.is To cells, two distinguish neighboring two param ing to the Rayleigh criterion) when 2× zoom (5.2 accord lens of blurring + optical half-diameter (actual diameter sphere of a stained nucleus is detected as 2.5 2.5 + a 3.7 image, nuclei-stained typical a In respectively. The thickness of the light sheet is <10 <10 thinnest region, is which is smaller sheet than the typical step light size (10 the of thickness The respectively. 4.2 has manuscript this in use we that microscopy of resolution optical the specifications, vendor’s the to According resolution. optical In our opinion, this criterion is supported overall by the calculated (hippocampal cells of Thy1-YFP-H Tg; by in set data the in software evaluated Imaris of analysis spot roughly using is criterion This resolution’. ‘single-cell providing as this define we therefore manuscript this in and cell, single a from signals counted and We detected have considered. further needs investigation in future studies. issue this Thus, scenario. this in alternative able suit a suggest to unable are we also, thus and methods clearing c a T 1716 So far wetestedabrain sampleof postnatal day 3.Adult brain willbetestedinfuture studies. We performed imaging of postnatal day-1 samples day-1 postnatal of imaging performed We A Mouse stomach Mouse liver Mouse spleen Mouse lung Mouse heart Mouse hemisphere Marmoset brain Mouse body whole Mouse T (eye and hair) Tissues with melanin bone Mouse skin Mouse muscle Mouse node lymph Mouse pancreas Mouse kidney Mouse intestine Mouse PROTOCOL issue As discussed earlier, imaging resolution is also a point to be be to point a also is resolution imaging earlier, discussed As B LE

| VOL.10 NO.11VOL.10 1 M | m and 3.7 and m

Tissues to which CUBIC has been successfully applied. e, 1 1 1 18,1 1 9 9 1 9 9 9 1 1 1 1 9 9 9 9 1 9 1 1 9 9 9 a, d | 2015 1 9 M

c, m in m 1 8 |

NATUREPROTOCOLS x - y CB-perfusion CB-perfusion Simple immersion or CB-perfusion Simple immersion or CB-perfusion Simple immersion or CB-perfusion Simple immersion or CB-perfusion Simple immersion or CB-perfusion Simple immersion or CB-perfusion Simple immersion or CB-perfusion Simple immersion or CB-perfusion Simple immersion or CB-perfusion Simple immersion or CB-perfusion Simple immersion or CB-perfusion Simple immersion Simple immersion or CB-perfusion CB-perfusion images with 1.6× and 2× zoom, 1 9 . Imaging of cleared adult mouse wasnot testedbecauseof sizelimitation of current LSFMsetup. C learing protocol Supplementary 1 Video Supplementary M m in a half-diameter half-diameter a in m M m × 5.2 M m in the half- s M , the voxel m at the the at m Figure Figure M m per M d m). E.A.S., unpublished result. ). 4 - - -

R cell resolution, these issues will need to be further addressed addressed further be to studies. future in need will issues these resolution, cell downscaled. be must data Although CUBIC has the collected potential to detect all signals and with single- data, image large support not does manuscript this in used software analysis the addition, In field. imaged entire the cover not does is and sheet limited the of area layer.thinnest the that granule is consideration cerebellar further or A hippocampus denser the in as such resolution regions single-cell for sufficient not is size the voxel whereas density, cell similar with resolution regions and single-cell cortex the has in best its at image acquired the Thus, the the of in limit resolution of examples the manuscript. current (ref. cell 3,900 this is CA1 in hippocampal mouse used of EV study,whereas volume voxel the than larger sufficiently is which is neuron 11,000 cortex cerebral mouse of EV The half-diameter). its 4,867.2 By our calculations, nucleus is and if EV detectable separable is larger than sufficiently Each above). as blurring, optical the plus diameter nuclei or cell the considering (again camera the by acquired nuclei of volume eagent before imaging Reagent-2 or reagent-1 Reagent-2 Reagent-2 Reagent-2 Reagent-2 Reagent-2 Reagent-2 Reagent-2 Reagent-2 Reagent-2 Reagent-1 Reagent-1 Reagent-2 Reagent-2 Reagent-2 M M 3 m 3 m — e ) = (15.7 Needs hair removal. removal. hair Needs b 3 3 per cell (ref. , enough to include a sphere with 2.5 + 3.7 3.7 + 2.5 with sphere a include to enough , M s m) 20 20 × 15.6 × 15.6 (= voxels 2 × 3 × 3 = f 3 Bone clearing of infant mice wassufﬁcient for imaging = 3 × 3 × 2 to 4 × 4 × 2 voxels, which is at 3 b 2 The cleared body specimen can be stocked in the reagent. Good Good Good Good Good Good Good Good Good Good Good Not cleared with the current reagents current the with cleared Not investigation Partially cleared but needs further Good Good Good ) = (22.2 = ) C learing efﬁciency M m) f 3 = 5 × 5 × 3 voxels, M m M 1 M 9 m in in m 3 . m m = per per

s .

© 2015 Nature America, Inc. All rights reserved. • • • • Fixative, andstorage reagents perfusion • Animal for samplesused imaging REAGENTS M a T • CLARITY 2,2 Clear FRUIT SeeDB Sca 3DISCO CUBIC Methods Use acrylamide-embedded specimens. A Perform procedures all inafume hood. NaOH (Nacalai Tesque, cat. no. 31511-05) toxic. Perform procedures all inafumehood. HCl (Nacalai Tesque, cat. no. 18320-15 or 18321-05) procedures inafumehood. PFA (Nacalai Tesque, cat. no. 02890-45) Heparin sodium(Mochida Pharmaceutical, 10,000U/10ml) (Takara,PBS tablets cat. no. T9181) institutional guidelines for usinganimals. experiments animalswereand all cared for andtreated humanely inaccordance the with RIKEN Kobe Institute, University The Tokyo of andtheGifuUniversity, weremanuscript approved by the Animal Care andUse the Committee of research purposes. andhousingconditions animalexperiments All inthis governmental animalsfor andinstitutional regulations regarding theuseof ! usually useC57BL/6(CLEAJapan) to prepare cleared andbodies organs CAG-EGFP SLC) Tg(Japan and Arc-dVenus Tg(GifuUniversity). We also (RIKEN CDB&QBiC), KI(RIKEN CDB),R26-H2B-mCherry R26-CAG-nuc-3 ×mKate2 KI R26-H2B-EGFP KI(RIKENCenter for Developmental (CDB)), Biology Thy1-YFP-H performance with imaging Jackson Tg(The Laboratory), fluorescent protein such asmKate2 isbest. far, So we have confirmed good fluorescent abright proteinof gives results. imaging thebest red A bright Animals expressing fluorescent proteins used. canbe Strong expression ATER

B CAUT ` LE l -Thiodiethanol e T 9 (ref. 11 (ref. 1 18,1 1 2 4 I I 0 4,5,7,36–3 ON ALS a, | 9 13,15,1

Other clearing methods and their applications to imaging and analysis. inaccordanceAnimal must experiments performed be with ) 7 8 12,1 B -actin-CAG-nuc-3×mKate2 KI(RIKENCDB&QBiC), 3 Sorbitol) or 2,2 FocusClear, RIMS (Histodenz or SDS in borate buffer and one of 2,2 and PEG for Formamide for D D Urea/glycerol/Triton X-100 dibenzyl ether for THF-DBE method Tetrahydrofuran/dichloromethane/ method alcohol/benzyl benzoate for BABB Ethanol or methanol/xylene/benzyl urea and sucrose for reagent-2 for reagent-1, triethanolamine, Quadrol, Triton X-100 and urea (-)-Fructose/urea (-)-Fructose/ ` -Thiodiethanol C

hemical contents !

CAUT ! Clear

CAUT A Clear ` I -thioglycerol -thiodiethanol ON T (ref. 2) (ref. I ON PFA istoxic. Perform all T , , formamide ! NaOH istoxic.

CAUT I ON HCl is

A Optical sectioning micro microscopy Two-photon confocal microscopy Two-photon microscopy, confocal microscopy Two-photon microscopy, confocal microscopy two-photon microscopy, Ultramicroscope (LSFM), confocal microscopy two-photon microscopy, Ultramicroscope (LSFM), other custom LSFM setups photon microscopy, COLM and Confocal microscopy, two- tomography two-photon serial sectioning confocal microscopy, Two-photon microscopy, scopy, stereomicroscopy pplied imagingmethods

• • • • • • • • • Clearing, reagents nuclei-staining andimaging • • • Silicon oil TSF4300 (Momentive, RI=1.498) Propidium iodide(PI; Life Techologies, cat. no. P21493) SYTO 16(Life Technologies, cat. no. S7578) toxic.is highly azideSodium (Nacalai Tesque, cat. no. 31208-82) Sucrose (Nacalai Tesque, cat. no. 30403-55or 30404-45) assubstitutes.used the finalreagent-1 recipe, term, atleastintheshort canbe and thus they (cat. no. P0873), them fluorescence caused andnone of quenching in (cat. no. X100, T9284, T8787, T8532)or Tokyo Chemical Industry same chemical from Nacalai Tesque (cat. no. 12967-45), Sigma-Aldrich However, thatproduct discontinued. hasbeen We checked further the and we recommended aproduct from Nacalai Tesque (cat. no. 25987-85). Triton fluorescence X-100product for seemspreserving crucial signals, M Polyethylene glycol (PEG) mono- Urea (Nacalai Tesque, cat. no. 35904-45 or 35907-15) 2,2 Chemical Industry, cat. no. T0781) N,N,N performance. PBS,diluted with contaminationsaltdecreases because theclearing with an equivalent dH volume of 1/2-diluted Sca TISSUE TEKO.C.T. compound Finetek, (Sakura cat. no. 4583) Sucrose (Nacalai Tesque, cat. no. 30403-55or 30404-45)

CR ` ,2 I p T -Nitrilotriethanol (triethanolamine; (triethanolamine; Wako,-Nitrilotriethanol cat. no. 145-05605) ` ,N I CAL ` -Tetrakis(2-hydroxypropyl)ethylenediamine In ourﬁrst CUBICpaper l

eCUBIC-1 (1/2-reagent-1). Sca -

NATUREPROTOCOLS

A tracing, cell distribution analysis 3D reconstruction, axon and dendrite 3D reconstruction, distance measurement quantification, tumor volume calculation intensities, axon tracing, cell number 3D reconstruction, visualization of pancreatic Langerhans islets structures, quantitative analysis of extraction of anatomical and histological Whole-brain signal comparison analysis, 3D reconstruction, neurite tracing 3D reconstruction 2 pplied computationalanalysis methods O M

p CR -isooctylphenyl ether(Triton-isooctylphenyl X-100) I T 1 I 8 CAL , of we mentioned thatthequality Sca

| VOL.10 NO.11VOL.10 l eCUBIC-1 ismixed with l eCUBIC-1 should not be eCUBIC-1 shouldnotbe !

CAUT (Quadrol; Tokyo PROTOCOL I ON Sodium azide Sodium | 2015

1717

© 2015 Nature America, Inc. All rights reserved. 0.1 0.1 g sodium azide of in 1 liter PBS.of This solution can be stored at room When PBS/0.01% (wt/vol) sodium azide is prepared, directly dissolve tablets (Takara, cat. no. T9181), the tablet is dissolved in 1 liter dH of PBS REAGENT SETUP • • • • • • Analysis tools • • • • • Image-analyzing software • • • • • • • • • • • microscopyImaging mouseorgans for whole • • • • • • • • • • • • • • • • • • EQUIPMENT • 1718 months. several for °C) (18–25 temperature MVX-ZB10 from MVPLAPO0.63× lens Olympus Olympus, with equipped In thisstudy, we theUltramicroscopes used from LaVision BioTec andthe amacrozoom with illumination device microscopeLight-sheet Fume hood Microwave materials such theviscous asTritonof X-100andaminoalcohols. M Positive-displacement pipettor (Gilson, no. model Microman M-1000) pH meter (HORIBA scientific, no. model LAQUA twin) HS10, or Advantec, no. model SRS710HA) Hot stirrer forreagents viscous preparing highly (IKA, no. model C-MAG no.model AMG-S, or IKEDA Scientific, no. model IS-20PC) stirrerMagnetic forreagents viscous preparing highly (ASH, Shaker (TAITEC, no. model Wave-PR or MixerXR-36, arotatorMHS-2000) with (TAITEC, no. model RT-5) no.model HB-80, Incubation devices. We usehybridization incubator (TAITEC, (ULVAC, no. model DA-15D; Vacuum desiccator (ASONE, cat. no. pump vacuum VXS 1-5943-01)with SS-20ESZ) syringes,Disposable 1, 10and20ml(Terumo, cat. no. SS-01T, SS-1010SZ, stopcockT shape (Terumo, cat. no. TS-TL2K) 26-G, (Terumo, 1/2-inchneedle injection cat. no. NN-2613S) cat. no. SV-23CLK) Intravenous (i.v.) needle, injection 23-G, (Terumo, butterfly type Peristaltic pump(EYELA, no. model MP-2000; Conical tubes, 50ml(Corning, cat. no. 352070or 227261) Conical tubes, 30ml(Sarstedt, cat. no. 60.544) Conical tubes, 15ml(Corning, cat. no. 352096or 188271) Tubes, 5ml(Eppendorf, cat. no. 0030119.401SG) Mineral oil (RI=1.467; PROTOCOL FSL ( net ANTs 1.9-v4(ANTs, freeware from stnava, DLURL: 4&root=nitr Convert3D ( ImageMagick ( Code provided as andaC++compilerPython http://www.it Pennsylvaniaof andGuido Gerig, PhDattheUniversity Utah): of ITK-SNAP (freeware from Paul Yushkevich, PhDattheUniversity reconstitution TIFFimage stacks of (Bitplane,Imaris Visualization tool ImageJ (freeware from theUSNational Health Institutes (NIH)) of imageGeneral analysis tool Glass plate stage for mounting specimen Customized sampleholder MVX-1 andLV AD MVX-2) filter unit(LaVision wheel BioTec) adapters (LaVision with BioTec, LV AD MVX-TV1X), MVX-TLU) lens tube (Olympus andtheUltramicroscope and theMVXmicroscope are connected to adapter acamera (Olympus Andor sCMOScharge-coupled Neo (CCD)camera device 5.5. camera The Coherent sapphire laser 588LP-50 Coherent sapphire laser 488LP-100 Red fluorescent filter signal (Chroma ET650/60) Green fluorescent filter signal (Chroma ET525/50) holderSample (LaVision BioTec or customized; reservoirImaging LaVision (100%quartz; BioTec) (NA) aperture (numerical =0.15, working distance =87mm)

/projects/advants/ﬁ Prepare PBS according to the vendor’s manual. When using PBS

| I http://fsl.fmrib.o VOL.10 NO.11VOL.10 T I CAL We recommend highly thispipettor theweight for measuring c http://www.i ksnap.org/pmwiki/pmwiki.ph ) http://imagemagi http://www. Supplementary Data Supplementary Fig. 2 | x.ac.uk/fsl/fslwiki/ 2015 les/ANTS/ANTS_Latest c Sigma-Aldrich, cat. no. M8410) ) or incubator (EYELA, no. model FMS-1000or tksnap.org/download/ 1 | 8 bitplane.com/imaris/

Fig. 2 ( NATUREPROTOCOLS Fig. 4 ck.org b ) a ) / ) installed with TIFFsupport with ) installed FslInstallatio p ) for NlfTl-13Dimages / ) Fig. 3 snap/process.php?lin Fig. 4 imari ht a n a ) Fig. 2 tp://sourceforge. ) ) s ) for 3D d

)

4,1 8

2 O. O.

k=707

matching may different be between organs. M reagent-2. 70%(vol/vol) out with RIof rable EtOH. oilwiped The canbe filtering contaminants reagents, (clearing etc.). Its RIis1.48–1.49, acompa Fig. 2 desiccator avacuum itwith astirrer and degas (~0.1MPa,with ~30min; Immersion oil mix treatment. reagent-2 during tissue the inside and around bubbles maycause microwave. a degassing in Insufficient heating mild by again dissolved be can stock the in precipitation use. The before stock solution M transparency. Before clearing, 1/2-reagent-1/water isnotaproblem. 1/2-reagent-2/PBS through to salt-free reagent-2 does not affect the final exchangePBS from gradual the because and structure, overall the of distortion cause 1/2-reagent-2/water, might in which swollen easily be to tend treatment reagent-1 after tissues because 1/2-reagent-2/PBS, preparing performance. We clearing in only the PBS decreases use contamination M ureadissolving andsucrose. Avoid thepreparation. boilingduring completely water monitoredafter be evaporated for of theaddition dissolve,to chemicals should theweight concentrated highly for difficult it make will convenience.evaporation for water (vol/vol) Because % or (wt/vol) % than rather wt% use we therefore and viscous, highly alsoTROUBLESHOOTING(see section). medium. fresh to change clearing, during smell the experience users If ref. amin with buffered are reagents the because problem a apparently not is itself ammonia of Generation urea. inthesereagentsammonia of smell indicates degradation recipe original the in MPa, (~0.1 desiccator min; vacuum ~30 a with reagent the degas included X-100 Triton triethanolamineadd and5 g stirof it further. The 0.1% (vol/vol) of ittakes 10–15min),(typically cool themixture atroom temperature, a microwave usingahotstirrer ( solution, dH sucrose urea dissolve in7.5gof and25gof 12.5gof finalconcentration) anddH (10 wt% triethanolamine concentration), final wt% (50 sucrose concentration), the stock of reagent-1the stock for convenience. of However, longer storage may cause contamination decreases performance. theclearing We usually prepare convenience. Reagent-1 prepared shouldnotbe PBS, with salt because and therefore or than%(wt/vol) (vol/vol) rather we usewt% for reagent-1 anddH of at room temperature for upto 1month. Prepare 1/2-reagent-1 by mixing1:1 desiccatorvacuum (~0.1MPa, ~30min; Tritontemperature 75gof andadd X-100. Finally, thereagent a degas with ahotstirrer.with After complete dissolution, themixture stir atroom further dH Quadrol in144gof 80wt% solution, urea and156gof mix125gof final concentration) anddH concentration), finalconcentration), Quadrol (25wt% Triton X-100(15wt% Sca or than%(wt/vol) (vol/vol) rather and we usewt% for convenience. room temperature for upto 1month. Quadrol reagent itfor andstir bottle atleast30min. Store thesolution at working solution.80 wt% In thiscase, dH 125gof add Quadrol 80 wt% PFAof lower factor for isacrucial with anefficient clearing autofluorescence. gloves andamaskto PFA, handle HCl andNaOH. Avoid skinandeyes. inhalationor contact with Use chamber, adraft proper useforat −20°Cuntil several months. After complete dissolution, adjust thepHto 7.4usingHCl. PFA stored canbe der 1NNaOH anda1/500–1/1,000volume of to PFA dissolving help faster. PBS.liter (total) of Heat thePBSsolution (avoid PFA boiling)andadd pow PFA solution be stored at room temperature for up to 2 weeks. can PBS. Reagent-2 and reagent-2 of 1:1 mixing by Prepare1/2-reagent-2 Sca alsoTROUBLESHOOTING(see section). fluorescentquenching of signals. Avoid thepreparation boilingduring

CR CR CR l l 1 eCUBIC-1 (reagent-1) eCUBIC-2 (reagent-2) 9 I I I b T ). We recommend thatusers avoid excess preparation. during heating T T ) before use. oilrepeatedly The mixcanbe by for used imaging I I I CAL CAL CAL The mix ratio can be optionally RI changed canbe thebest because mixratio The Make sure that there is no precipitation in the reagent-2 reagent-2 the in precipitation no is there Make that sure salt PBS, because with prepared be not should Reagent-2

To prepare PFA 4%(wt/vol) inPBS, PFA dissolve 40gof in1

Quadrol liquid, viscous isahighly asan used anditcanbe

2 Mix 1:1 of TSF4300 oil completely andmineral Mix 1:1of O. O. M

2 Reagent-1 final urea (25wt% isamixture of Reagent-2 is a mixture of urea (25 wt% final final wt% (25 urea of mixture a is Reagent-2 O. For example, to prepare reagent-1 500gof I T I o CAL Fig.2 alcohol (~pH11; inanalkalinepHrange Quadrol andTriton X-100are viscous, 2 M a O.To reagent-2 of g 50 prepare ! ). After complete dissolution ).dissolution complete After

CAUT CR Fig. 2 M I 1 T

8 CR I isnotnecessary. Finally, I b CAL ON ). reagent The stored canbe I T PFA toxic isavery reagent. I Quadrol viscous, ishighly ! M CAL

CAUT

CR 2 Reagent-2 becomes becomes Reagent-2 O to 500 g of O to 500gof I T I I ON CAL The acrid acrid The The pHvalue The

Fig.2 2

O in in O

O ).

( bubbles, the cleared whole body with option C is kept in reagent-1 but not in reagent-2. are cleared. Because handling of whole-body samples in the viscous reagent-2 become difficult, particularly by causing M CB-perfusion protocol (option C). decreased signal intensity becauseof the shorter ﬁxation time). Alternatively, startwhole-body clearing byusing the (option B;CB-perfusion clearsbetterthanthe immersion protocol, particularly in heme-rich organs, butittends tocause 2| the immersion period and the ﬁnal transparency of samplescanbevaried according tothe user’s experimental purpose. M T animals. ! (i.p.) witha1-mlsyringe and a26-G,1/2-inch injection needle). 1| A PROCE We recommend that PFA be byperfused pump for peristaltic successful and cold 4% (wt/vol) PFA, 20 ml PBS,of and 20–30 ml 1/2-diluted of reagent-1. perfused with four solutions: 20–30 ml of cold heparin-PBS, 150 ml of CB-perfusion is depicted in protocol CB-perfusion the for setup Surgical EQUIPMENT SETUP ranscardial perfusion and tissue clearing clearing tissue and perfusion ranscardial

A nesthesia (iii) CAUT

(ii) ) CR CR (i) S Start clearing organs bythe simpleimmersion protocol (option A)orthe CB-perfusion and immersion protocol At day 0,deeply anesthetize the mice using pentobarbital (~150mg/kg of body weight, administer intraperitoneally I I imple immersion protocol for dissectedwholebrain single-photon imaging. clearing methods partially cleared sample, for example immersion inreagent-1. However, this step canbeskippedfor other purposessuch astwo-photon imaging witha performance. Pretreatment with1/2-dilutedreagent-1 givesamore effective final clearing efficiency thandirect M can alsobeadded to1/2-dilutedreagent-1 atthisstep. observed during thisstep( than a15-mltubeinthisand further clearing Step2A(iv,v), becauseof sampleswelling. Clearing effectscanbe or rotation (~5r.p.m.) at37°Cfor 3–6h.We recommend using a30-mlconical tubefor clearing asingle brain rather shaker of ahybridization ovenshown in that havebeenstored. The samplewillnow beready for the next step;however, wefind thatthe clearing efficiency isreduced insamples temperature and washthem withPBSatleasttwice, witheachwashfor 1h,toremove sucrose and O.C.T. compound. and immediately transfer them to−80°C.To continue the clearing protocol, thawthe samplesgradually atroom per organ withshaking at4°Cfor 1–2d. When the samplessinktothe bottom, putthem into O.C.T. compound J temperature toremove the remaining PFA ( after perfusion isagood indicator of successful perfusion. Residual bloodinthe mouse brain increases auto M Dissect the brain and postfixitin10mlof 4%(wt/vol)PFA withshaking at4°Cfor 18–24h. blood from the organ asmuch aspossible. Next, perfuse~25mlof cold4%(wt/vol)PFA (pH7.4)at~10ml/min. ? autofluorescence. Overfixation causesbothlowerclearing efficiency and autofluorescence. fluorescence especially inthe green-laser excitation. The pHvalueof PFA isalsocrucial for efficient clearing and lower T T Immerse the samplein8–10mlof 1/2-water-dilutedreagent-1 withshaking (~60r.p.m. ifyou are using the orbital

Day 0 Day 1 D I I I

ON URE

CAL

CAL TROU CR PAUSE CR Every experiment must follow all relevant governmental and institutional guidelines for the use of experimental I I . Wash the tissuesamplewith10mlof PBS/0.01%(wt/vol)sodiumazide for at least2htwice atroom . Perfuse the mice with10mlof coldPBS(pH7.4)containing 10U/mlof heparin at~10ml/mintoremove the

Inthisstep,weparticularly focus onthe fullclearing protocol for the purposeof LSFMimaging. However, L T T STEP I I B

CAL CAL T LES

IMI PO Option A is for a single whole mouse brain ( brain mouse whole single a for is A Option

H I STEP STEP N NT OOT G Figure Figure 3 The fixed organs canbestored. First,immerse them in10mlof 20–30%(wt/vol)sucrose inPBS 5min 1 Inefficient mixing of the reagent and samplesduring clearing may affectthe final clearing Cooling of PBSand 4%(wt/vol)PFA onice isimportant for successful perfusion. Muscle stiffness 9 I , and itcanbeusedfor two-photon imaging N G a . In the protocol, the adult mouse is Fig. 2

Typical setup surgical for e ). Note thatanuclear staining dye, such asSYTO 16(1–2 1 8 . Thisdirect immersion procedure givesbetterclearing results thansome other L

Fig. 2 T IMI Fig. 2 N c G , and ~30r.p.m. ifyouare using aseesawshaker asshown in 4–14 d 4–14 e

Fig.

care in handling the injection needle is needed to avoid accidental needlesticks. and eyes. Use a draft chamber, proper gloves and a mask to handle PFA. Great ! i.v. injection needle and a disposable syringe, as shown in nation of T shape stopcocks, a peristaltic pump connected with silicon tube, an combi a with instrument surgical a devised we Thus, surgeries. reproducible

CAUT 2 ), ), and it may need some modifications when other organs 1 I 8 ON and possiblyaspartof the samplepreparation for PFA is a very toxic reagent. Avoid inhalation or contact with skin NATUREPROTOCOLS

M M) and PI(5–10

| VOL.10 NO.11VOL.10 Figure Figure 3 PROTOCOL | - 2015

Fig. 2

. M

g/ml),

1719 d

- )

| B-perfusion andimmersion protocol for faster clearing of wholeorgans VOL.10 NO.11VOL.10 or contact withskinand eyes. ! clearing performance. M blood from the tissuesasmuch aspossible. and stored inO.C.T. compound at−80°C,asdescribed inthe PAUSE POINTcalloutinStep2A(vi). PBS/0.01% (wt/vol)sodiumazide, completelyimmersed in30%(wt/vol)sucrose inPBS/0.01%(wt/vol)sodiumazide the final transparency, butitalsocausesswelling of the sample. Afterimaging, the samplecanbewashed with J ? mitigate shrinkage orswelling. incubation atroom temperature for alonger time. Adjustment of PBS content in1/2-dilutedreagent-2 may also immersion oilmixatimaging steps. When structural distortion isapparent afterreagent-2 treatment at37°C,try it isdifficulttotake images inthe reagent. The reagent-2–treated samplesshould beimmersed inthe low-viscosity ! The next day, replace the reagent withfresh reagent and further incubate for ~24h( ? M temperature ( the samplein~5mlof 1/2-PBS-dilutedreagent-2 and shake itina5-mltubefor 6–24hat37°Corroom callout atStep2A(ii). into O.C.T. compound and immediately store them at−80°C.Thawthe samplesasdescribed inthe PAUSE POINT sodium azide perorgan withshaking atroom temperature overnight. When the samplessinktothe bottom,putthem J ? 20% (wt/vol)sucrose solution toavoid any damage tothe sample. M M 5–10 this stepisneeded: thus, incubate the washed samplein~5mlof PBS/0.01%(wt/vol)sodiumazide containing once for 2h,once overnight and againonce for 2h.When asampleisstained withPI,further staining during gentle shaking orrotation atroom temperature three times for atleast2heachtime. We typically washthe sample ? information. Anammonia smell indicates degradation of urea, and thatthe reagent should bereplaced byfresh medium. the lid and the topof it.Samplelabelsshould bewrittenonboththe body and lid of the tubetoavoid lossof ! to reagent-1. 37 °Covernight. Ifdesired, the same concentration of nuclear staining dye usedinthe previous stepshould beadded Discard 1/2-dilutedreagent-1. Immediately add 8–10mlof reagent-1 and gently shake itorrotate the sampleat Perfuse the mice with150 mlof cold4%(wt/vol)PFA inPBS(pH7.4)at~15ml/min using aperistaltic pump. Day 0 Days 8–11 Days 8–11 Days 7–10 Day 2 ? placing itinto fresh reagent-1 for anadditional 1–2d. M to avoid damage. ! cleared bydays 7–8( 2 d(days 4and 6).Alsorefresh any nuclear staining dye onday 4and day 6.Typically, the brain willbesufficiently

CAUT CAUT CAUT CAUT

TROU

TROU TROU TROU

TROU CR PAUSE CR PAUSE CR CR CR I I I I I . Perfuse the mice with20–30mlof coldPBS(pH7.4)containing 10U/mlheparin at~10ml/mintoremove the . Replace8–10mlof reagent-1 and continue gentle shaking orrotation at37°C.Replacethe reagent every M T T T T T I I I g/ml of PIfor anadditional 3d(ormore, ifneeded) at37°Cwithrotation I I I I I I B B B B B ON ON ON ON CAL CAL CAL CAL CAL LES LES LES LES LES

PO PO . Immerse the samplein~5mlof reagent-2 ina5-ml tubeand gently shake itat37°Covernight. . Degasthe sampleinalimitedvolume of PBSwithavacuumdesiccator ( . To stopthe clearing procedure, washthe samplewith20mlof PBS/0.01% (wt/vol)sodiumazide with PFA isaverytoxic reagent. Perform allprocedures inafume hood with asafetyglasstoavoid inhalation Donot rotate the tubetoavoid making bubbles. Samplesdo not sinkinthe highly viscousreagent-2, and Reagent-1 canerase oilypenmarks easily. Make sure thatthe tube issealedbywrapping Paraﬁlm around As CUBIC-treated organs soften, werecommend using spoons instead of forceps for handling them inorder |

2015

H H H H I H I STEP STEP STEP STEP STEP

NT Fig. 2 NT OOT OOT OOT OOT OOT Organs canbeleftinreagent-2 for upto1weekatroom temperature. Further immersion increases Organs canbestored. First,immerse them in10ml of 30%(wt/vol)sucrose inPBS/0.01%(wt/vol)

| Insufficient removal of bloodinside the tissueprolongs the clearing period, and itmay causelow Degassing of the sampleprevents airbubblesfrom remaining inthe ventricle. Forcryoprotection atthisstep,werecommend using 30%(wt/vol)sucrose inPBSrather than Completeremoval of reagent-1 during the washing stepiscrucial for final clearing efficiency. Ifthe whitematter hasnot substantially cleared by8dof immersion, tryfurther immersion by

I I I I I NATUREPROTOCOLS N N N N N e G G G G G ). Check whether the samplesinkstothe bottom(asign of completeimmersion). Fig. 2

e ).

Figure 3

a .

1 8 Fig. 2 . Fig. 2

b e ). To do this, immerse ).

(viii) (vii) (ix) (vi) (iv) (v) ? M effective, orclearing asdescribed inStep2A. M ? Check whether the samplesinkstothe bottom(asign of completeimmersion). ? PBS more thanthree times for 2heachtime. M rotation atroom temperature three times for 2heachtime. Afterthe PBSwash,move tothe next stepimmediately. callout inStep 2A(vi). completely immersed in30%(wt/vol)sucrose inPBS and stored inO.C.T. compound at −80°C,asinthe PAUSE POINT the final transparency, butitalsocauses swelling of the sample. Afterimaging, the samplecanbewashed with PBS, J ? incubating room temperature for alonger time or testing the simpleimmersion protocol (Step2A). immersion oilmixatimaging steps. When structural distortion isapparent afterreagent-2 treatment at37°C,try it isdifficulttotake images inthe reagent. The reagent-2–treated samplesshould beimmersed in the low-viscosity ! possible before the following imaging step. ent, asshown in transparency plateauisreached after2–3dof reagent-2 treatment. At day 10,almost allorgans should betranspar day, replace the reagent withfresh reagent and further incubate the samplesfor several days. Typically, anapparent ? supernatant indicates decolorization of tissuesasaresult of heme elution. and lung, byday 1( M pancreas, spleenand intestine. However, note thatwetreated allindicated organs in incubation time for the complete clearing depends onthe organ: 1day of reagent-1 treatment isusually sufficient for refresh the nuclear staining dye. Replacereagent-1 and any nuclear staining dye againatdays 2and 4.The total reagent-1 withthe same volume of fresh reagent-1 and continue clearing byshaking at37°C. If appropriate, also spleen are good indicators toevaluate perfusion efficiency ( M added to1/2-dilutedreagent-1 atthisCB-perfusion step. perfusion ( 1/2-diluted reagent-1 atthe same injection rate. Make sure thatthe organs become translucent bythe end of the Immerse the sampleinthe same volume of 1/2-PBS-diluted glycerol and shake itfor 6hto24atroom temperature. Dissectthe organs of interest and immerse these inreagent-1. Several organs canbeprocessed inasingle tube, but Perfuse the mice with20mlof PBS(pH7.4)at~10ml/mintowashoutPFA, followed byperfusion of 20–30mlof Day 1 Days 3–7 Days 2–6 ? For efficient clearing, the samplesof interest should beimmersed inatleastafivefold volume of reagent-1. M An ammonia smell indicates degradation of urea, and inthisscenario the reagent should bereplaced withfresh medium. the lid. Samplelabelsshould bewrittenonboththe body and the lid of the tubetoavoid lossof information. ! reagent-1. rotation (~5r.p.m.) at37°Covernight. Add the same concentration of any nuclear staining dye usedatStep2B(iv)to in ahybridization ovenasshown in and pancreas in5mlof reagent-1. Incubate the sampleswithshaking (~60r.p.m. ifyouare using anorbitalshaker pancreas and apiece of liverin40mlof reagent-1 orimmerse eachsmall organ such asheart, lung, kidney, spleen be removed asmuch aspossibleinthisstep.Thus, immerse several organs such asheart, lung, kidney, spleen, the stomach and intestine should beseparated into different tubes. Gastrointestinal content inthese organs should

CAUT CAUT

TROU

TROU TROU TROU

TROU CR CR CR PAUSE CR CR CR I I I I I I . Make sure thatthe organs are transparent and thatthe colorof the supernatant hasturned olivegreen. Replace T T T T T T I I I I I I I I B B B B B B ON ON CAL CAL CAL CAL CAL CAL LES . Immerse the samplesinthe same volume of reagent-2 and gently shake them at37°Covernight. The next . To stopthe clearing procedure, washthe sampleswiththe same volume of PBSwithgentle shaking or LES LES LES LES LES

Fig. 3 To avoid making bubbles, do not rotate the tube. Samplesdo not sinkinthe highly viscousreagent-2, and Reagent-1 erases oilypenmarks easily. Make sure thatthe tubeissealedbywrapping Paraﬁlm around

H H H H H H I STEP STEP STEP STEP STEP STEP NT OOT OOT OOT OOT OOT OOT Tissuescanbeleftinreagent-2 for upto1weekatroom temperature. Further immersion increases b Figure 3 Efficient mixing of the reagent and samples during clearing may affect the final clearing performance. Cooling of PBSand 4%(wt/vol)PFA onice isimportant for successful perfusion. Ifthe signal from atarget reporter protein isweak,aprolonged perfusion period may bemore CB-perfusedsamplesare prone toovershrinking inthe washing step.Donot wash the samplesin Typically, successfully CB-perfusedorgans are turned almost transparent, withthe exception of liver Perfusion efficiency iscrucial tothe final clearing efficiency. Some organs such asthe pancreas and I I I I I I ). Note thatanuclear staining dye, such asSYTO 16(1–2 N N N N N N Fig. 3 G G G G G G

c c . The gastrointestinal contents of the stomach and intestine should be removed asmuch as ). Opacityinthe lung occursmainly from bubbles. Note thatthe colorchange of the Fig. 2 c

, or~30r.p.m. ifyouare using aseesawshaker asshown in

Fig. 3 b ).

M M) and PI(5–10 NATUREPROTOCOLS

Figure 3

c M | VOL.10 NO.11VOL.10 withreagent-1 for 5d. g/ml), canalsobe

PROTOCOL Fig. 2 |

2015 d

) or

1721 -

© 2015 Nature America, Inc. All rights reserved. direction (‘VD’ or ‘DV’) and information about the channel (‘nuclear’ for the nuclear counterstaining and ‘geneExp’ for the information about the experiment (including imaging date), a unique ID for this brain, information about the imaging directions are needed. We recommend a simple naming rule for these folders with four fields separated by an underscore: to a different folder. For further signal comparison steps (Steps 7–15), signal and structural images of both D-V and V-D saved to a secondary storage location (e.g., a hard drive) during acquisition. Each stack (one color, one direction) is saved chemicals. Because ~700 images (~11 MB per image, total ~7 GB) are acquired per color and direction, each image should be M the sampleupside down and acquire the image againfor the oppositeorientation. with the same parameters are adjusted, startimage acquisition. When multicolor images are needed, repeat the image acquisition procedure be saved. The exposure times should beadjustedaccording tothe ﬂuorescent signal intensities of eachsample. Afterall Each plane should beilluminated from boththe right and leftsides, and amerged image withmaximum intensity should size (typically 10 6| and focus. M MVX-ZB10. focus and the sampleposition tothe center. Awhole mouse brain image canbecaptured using 1.6×to2zoomonthe 5| sample holder ( 4| If bubblesattachonthe surface of the sample, carefully remove them withaneedle ortapered forceps. then immerse the sampleinto the oilmixfor 10min to1h.Thisprocess alsohelps remove bubblesaround the tissue. 3| (LaVision BioTec and Olympus).Aconfocal oramultiphoton microscope canalsobeused, butfor more-limited regions. macrozoom microscope issuitable. Here wedescribe oursetupusing the Ultramicroscope combined withMVX-ZB10 M Imaging cleared tissues with macrozoom to the microscope setupintroduced inthismanuscript becauseof stage sizelimitations. body imaging, perfusion and immersion of reagent-1 wassufﬁcient ( handling whole-body samplesinthe viscousreagent-2, particularly becauseof bubbleformation. Forinfant mouse whole- M ( 1722 PROTOCOL C (iii)

)

(ii) CR CR CR CR (i) Perform CB-perfusion asdescribed inStep2C(i–iv). C

Set the focus position of the illumination sheet, Put the sampleonthe glassslide ( Set the imaging reservoir ﬁlledwiththe immersion oilmix,and then setthe sampleholder. Putaglassslide onthe Before imaging, wipereagent-2–treated samplessoftly withaKimwipe toremove excess reagent-2 onthe surface, and | I I B-perfusion protocol for whole-bodyclearing I I VOL.10 NO.11VOL.10 J ? fresh medium ifthisissmell ispresent. M bones and intestinal contents become sufﬁciently transparent after2weeksof treatment withreagent-1 ( the second week.Continue the clearing withreagent-1 for atleast2weeks. Typically, major abdominal organs except necessary. Replacethe reagent (and any nuclear staining dye) everyday inthe initial week,and every2or3din performance. M reagent-1 ifdesired. incubator at37°Covernight. Use the same concentration of the nuclear staining dye usedatStep2B(iv),and add to reagent-1. Placethe container onanorbitalshaker setat~60r.p.m. oraseesawshaker setat~30r.p.m. inthe transparent ( transparent afterthe CB-perfusion. Typically, glands such asthe pancreas and submaxillary gland are almost T T T T Replacethe same volume of reagent-1 and continue gentle shaking at37°C.Refresh the nuclear staining dye if Detachthe skinfrom the body. Carefully remove asmuch pelage aspossible. Make sure thatthe body ispartially I I I I

CAL CAL TROU CAL CAL PAUSE CR CR I I

Here wedescribe clearing of whole adult mouse body onlyusing reagent-1. Thisovercomes the difﬁculties of To perform arapid image acquisition of whole organs, alight-sheet illumination unitcombined witha T T STEP STEP I I B CAL CAL z LES

Fig. 4 -range and readjust the laserpowerand exposure time. To collectbothD-Vand V-Ddata sets, manually ﬂip PO To take images with a high signal-to-noise ratio, it is important to use bright fluorescent proteins and To avoid fluorescence quenching, laser power should be weakened during the adjustment of position M |

2015 H I Fig. 3 STEP m perstep),laserpower(typically 70–100%)and exposure time (typically 50ms–1 sfor eachside). STEP NT OOT a The whole body canbekept inreagent-1 for uptoseveral months atroom temperature. ). | Anammonia smell indicates the degradation of urea, and the reagent should bereplaced with Inefﬁcient mixing of the reagent and samplesduring clearing may affectthe ﬁnal clearing

I NATUREPROTOCOLS d N ). Spleenisalsoasagood indicator of successful perfusion. Immerse the body in200mlof

Fig. 4 LS a

FM ). Acquire aliveimage withanappropriate laserand ﬁlterpairtoadjustthe L

T IMI z -range (inthe caseof whole brain, typically ~7mm intotal),Z-step N G 1–3 h per sample per h 1–3 T able 1 ) 1 9 . Adult whole-body imaging isnot applicable Fig. 3

© 2015 Nature America, Inc. All rights reserved. Troubleshooting advice canbefound in ? ? data aligned withSyNin checking the quality of final aligned data and, ifnecessary, trying SyNoraffine-only instead. Inthismanuscript, weshow structural deformation ordistortion. Users canchoose whether touseSyNoraffine-only registration. We recommend M 15| ? comparison, butittypically takes 10–60min). 14| ? 13| ? atlas (~1minperbrain). 12| ? affine transformation only, and <1minfor the alignment of bothchannels). (for eachbrain, ~1h30minfor the registration ifyouare using symmetric normalization or3–5minifyouare using 11| ? brain and channel (4minperbrain), and usethese results in‘fileMerging.py’ tomerge the files(7–8minperbrain). 10| ? alignments take under 1min. The registration takes ~1 h30minusing symmetric normalization or3–5minusing affine transformations only, and both alignment of the V-D–acquired nuclear counterstaining image and alignment of the V-D–acquired signal channel image. brain, thisincludes the following: registration of the V-D–acquired nuclear counterstaining image tothe D-V–acquired one, 9| ? brain samplecorresponds tofour stacks(twochannels, twoacquisition directions). 8| edge_detection_Prewitt) and filemerging (g++-O3file_merging.cpp -ofile_merging). computer usedfor the analysis. Compilethe C++filesfor edge detection (g++-O3edge_detection_Prewitt.cpp -o 7| specifications. riken.j converted AllenBrain Atlas data and Arc-dVenus Tgmouse brain images usedin M Informatics for signal comparison ? be avoided. that the naming convention is respected. It is also important to note that white spaces and special characters must signal channel). For instance, 20131118LAdV_001_nuclear_DV. The code provided for the informatics section assumes

TROU TROU TROU TROU TROU TROU TROU TROU TROU TROU

CR CR Choose one brain to be used as internal reference, and align all the other brains to this reference with ‘internalAlignment.py’ ExportaTIFFstackfor the resulting fileswith‘exportTiffStack.py’ (about4min per NIfTI-1file). Normalize and compare brains (e.g., for subtraction) with‘normalisation_comparison.py’ (exact timing isdependent on Calculatethe normalization factors (7min,plus7minperbrain) with‘median_brainOnly.py’. Use ‘atlasAlignment.py’ toregister the internal reference tothe brain atlas(~1h30min),and align allbrains tothe Merge the V-D–acquired and D-V–acquired images. Use ‘edgeDetection.py ‘tocalculatethe Align images of the same brain acquired from oppositedirections, withthe ‘sameBrainAlignment.py’ script.Foreach Construct a3DNIfTI-1filefor eachTIFFstackwiththe If thisisthe firsttime the pipeline isbeing run,install allrequired software and copythe provided code tothe I I T T p I I B B B B B B B B B B ). Note thattiming isroughly proportional tothe number of brains, and itcanvaryaccording todifferent computer CAL CAL LES LES LES LES LES LES LES LES LES LES We provide oursource code and anadditional usermanual as

H H H H H H H H H H STEP OOT OOT OOT OOT OOT OOT OOT OOT OOT OOT Although registration withSymmetric Normalization (SyN)iseffective, insome casesitmay cause I I I I I I I I I I N N N N N N N N N N G G G G G G G G G G Figure 5 and withafﬁne-only transformation in L

T T IMI able N G 3 1–9 h for a two-brain data set, depending on the registration method registration the on depending set, data two-brain a for h 1–9 . ‘convertTiffFiles.py’ Figure S upplementary Data script.Thistakes ~2minper stack,and each Figure 6 , for illustrative purposes. NATUREPROTOCOLS 6 onourwebsite( n , aswellthe NIfTI-1- and

m | VOL.10 NO.11VOL.10 thresholds for each http://cubic. PROTOCOL

| 2015

1723

| VOL.10 NO.11VOL.10 3 |

Troubleshooting table. Troubleshooting | 2015 during reagent-2 steps reagent-2 during Poor organ clearing organ after reagent-2 deformation of cleared Excess shrinkage or treatment during reagent-1 Poor organ clearing perfusion PFA Poor Precipitate in reagent-2 reagents chemicals in CUBIC Possible degradation of P roblem |

NATUREPROTOCOLS

Insufficient replacement into reagent-2 Insufficient incubation in reagent-1 in reagent-2 Incomplete dissolution or precipitate CB-perfusion Use of samples prepared with animal juvenile or infant an of Use reagent-2 Insufficient replacement in 1/2 Incubation at 37 °C treatment with 1/2 reagent-2 Inappropriate salt concentration during Organ-dependent differences in clearing animal aged an of Use mixing or amount reagent Insufficient incubation time, time fixation much Too Alkaline pH of PFA Insufficient pressure for perfusion Wrong position of the tip of the needle Insufficient cooling of PBS and PFA in winter Lower room temperature, particularly causes ammonia odor) Too much heating (which P ossible reason

Stop postfixation within 24 h ~7.4 to PFA of pH the Adjust occurs at the cut in the liver Make sure that the perfusion outlet only left ventricle of the heart Make sure that the tip of the needle is in the perfusion before just ice on PFA and PBS Keep microwave for 5–10 s (avoid boiling) Prepare the reagent before use; heat it in a Milder and shorter heating during preparation S one or two more times Use more reagent-2 and exchange the reagent Increase reagent-1 treatment time prepared/stocked reagent-2 Make sure that no precipitate exists in the protocol immersion simple the Try treatment reagent-1 need less incubation time during tend to shrink more in reagent-2 and Organs from an infant or juvenile animal h) (~24 replacement complete for Sufficiently incubate the organ in the reagent (takes more time) temperature during reagent-2 steps Try to incubate samples at room swelling reagent-2/water instead of PBS causes than 1:1 mix of PBS:reagent-2); use of 1/2 (e.g., mix 1:1:2 of dH the concentration of PBS in the 1/2 reagent-2 If the organ has shrunk too much, decrease clearer in reagent-1; see final clearing reagent (e.g., pancreas becomes Use reagent-1 rather than reagent-2 for the animal younger a Use appropriately rotate (every day rather than every 2 d); shake or more); exchange reagent-1 more frequently Incubate it longer in reagent-1 (a few days olution 2 O:PBS:reagent-2 rather Fig. 3 )

(continued)

Troubleshooting table (continued). table Troubleshooting Poor Poor fluorescence Weak or nondetectable High autofluorescence Bubble on the surface imaging Floating sample during freezing Tissue damage when after reagent-2 (e.g., brain ventricles) structures of the organs Bubbles on inside clearing Ammonia smell during P roblem z -resolution

focus Inappropriate setting of the light-sheet Inappropriate setting of microscopy clearing during Temperature Signal decrease in CB-perfusion Insufficient fluorescence signals animal aged an of Use Alkaline pH of PFA Insufficient fluorescent signal Insufficient removal of the reagent-2 immersion oil mix Use of reagent-2 rather than the solution Insufficient replacement by sucrose crystals inside the tissue) (which possibly causes growth of water Keeping the sample at 20 − °C to 30 − °C Rotation during reagent-2 Insufficient degassing Degradation of urea P ossible reason

Immerse the sample in oil solution (i.e., until organs sink) Increase incubation time in the sucrose temperature room 80 − °C; thaw the sample gradually at Stock the sample in O.C.T. compound at shaking rather than rotation Incubate in the reagent-2 with gentle the reagent-2 incubation the sample in a limited volume of PBS before Degas the reagent-2 after preparation; degas reagents of preparation during heating much Replace with fresh medium, and avoid too S decrease fluorescence signals fluorescence decrease Higher temperature during clearing may with a bright fluorescent signal immersion protocol; select an animal strain increase fixation reaction (~3 h); try the perfusion procedure after PFA perfusion to Use more PFA for perfusion, and pause the strong expression promoter as above Select a bright fluorescent protein with a animal younger a Use ~7.4 to PFA of pH the Adjust mKate2) (e.g., protein fluorescent red (e.g., CAG); avoid green channel and use a Venus) with a strong expression promoter Select a bright fluorescent protein (e.g., YFP, oil with a needle or a tapered forceps into the oil mix; remove bubbles in the Remove excess reagent-2 before immersion focus throughout the brain) the throughout focus impossible to take images with adjusted an adult mouse hemisphere, and thus it is the width of focused sheet is 1/3–1/4 of interest (for the LaVision Ultramicroscope, Adjust the light-sheet focus to the region of time exposure Check the setting of laser power, filter and clearing efficiency) (Note that lower temperature also decreases ate at room temperature rather than 37 °C. NATUREPROTOCOLS olution

| VOL.10 NO.11VOL.10 8 PROTOCOL . Try to incub | 2015 (continued)

1725 - © 2015 Nature America, Inc. All rights reserved. the final resolution of images inthisprocess isdownscaled, and itismore difficulttoachieve single-cell resolution ( normalized, and then subtraction of Venus signal was calculated to visualize light-responsive regions at the whole-brain scale of Arc-dVenus Tgmouse brains withorwithout light stimuli throughout the pipeline, the acquired data setsare preprocessed bymerging D-Vand V-Dtomake sure thatthe resulting images are clear confocal and two-photon microscopes. detailed observations by using ahigher zoomonthe LSFMinstrument, orbyusing higher-NA objectivelenses onother manner. However, subcellularstructures canbeobservedinthe cleared tissue( resolution becauseourprimary purposeistoanalyze cellswithinthe context of awhole organ orbody inahigh-throughput platform ( ( subcellular structures such asneurites inthe mouse brain canbecaptured, provided thatthey are sparselylabeled reverses the cleared state ( ( heme, which isalsoapparent justafterCB-perfusion ( region imaging withtwo-photon microscopy reagent-1, clearing isobvious withinseveral hours ( be performed using equipment usually usedinatypical biology laboratory ( it canbeapplied tosimultaneous multisample clearing inasingle tube( The CUBIC pipeline canbeusedfor whole-organ or whole-body clearing. It issimple, efficient and reproducible, and thus ANT registration instead of symmetric normalization) Steps 7–15, informatics for signal comparison: 8–9 h for a two-brain data set (reduced to 1–2 h if you are using affine color and direction, and the required exposure time) Steps 3–6, imaging of the cleared tissues with the macrozoom LSFM: 1–3 h per sample (depending on the number of samples, Step 2, transcardial perfusion and tissue clearing: 4–14 d Step 1, anesthesia: 5 min L T 1726 throughout the entire imaging field, becauseof the current software limitations. Thiswillbeaddressed infuture studies. A Fig. 4b Fig. 3c Fig. 6b 8–15 (analysis) S PROTOCOL

tep We implemented animage informatics method originally usedinfMRI analysis tocompare different brains. Inthis According tothe user’sexperimental purpose, high- orlow-resolution images may beacquired. We userelatively low Figure 4a B T LE IMI I

C | VOL.10 NO.11VOL.10 I 3 PATE N , | , – d G d

d Troubleshooting table (continued). table Troubleshooting ). We summarize the organs wehaveusedCUBIC onin Fig. 4g , ). These calculations couldbeachieved bywhole-organ counterstaining inthe CUBIC clearing protocol. Note that e – D and f shows typical imaging results for the Thy1-YFP-H Tgmouse brain at1.6×zoom.Overall, soma and other RESULTS z -stack ( , S | h upplementary Video1 2015 not found’ not Error message: ‘Command file or directory’ Error message: ‘No such orientation (Step 8) Incorrect brain (Step 8) in the NIfTI-1 file Brain appears deformed P ). Ofnote, these data were collectedfor ~30–60minfor eachdirection/color. roblem |

Fig. NATUREPROTOCOLS Fig. 2 5 ). Then, signal subtraction betweensamplesiscalculated (

e ), butthe tissueisclearagainifitre-immersed into CUBIC reagents.

). Other organs were alsosubjectedtorapid, multicolor 3Dimaging inthe same Missing software parameter file Wrong folder name or brain ID in the Different brain position during imaging Incorrect scaling P 1 ossible reason 8 . The CUBIC reagents also decolorize organs and the whole body byremoving Fig. 2 Fig. 3a e ), and such partially cleared samplesare evenapplicable todeep 18,2 , b 6 ), and thisabilityenables whole-body clearing within2weeks ( T Fig. 6 able 1 a . Removal of the CUBIC reagents byPBSwashing ). The raw images were preprocessed, aligned and Fig. 2 Fig. 2a c Fig. 4b ) oraplastic container. The procedure can – are compiled command line and that our two C++ programs installed, that they are accessible from the Make sure that all the required tools are exist folders ensure that all brain IDs are valid and that all Check the parameter file for that step and VD-acquired files) (line 148 for DV-acquired files, line 150 for the convertTiffFiles.py script accordingly correct orientation (e.g., RPS) and edit it matches your raw TIFF stack. Note the Reorient the NIfTI-1 file in ITK-SNAP until mm) in image, TIFF raw correct voxel dimensions are given (for the Check the parameter file and ensure that the S d olution ). Bysimpleimmersion of samplein – f ) 1 Fig. 8 , and thus CUBIC permits more 6 ). Here weshow anexample

© 2015 Nature America, Inc. All rights reserved. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. c at online available is informationpermissions and Reprints interests. INTERESTS FINANCIAL COMPETING discussed the results and commented on the manuscript text. image analysis. A.K. developed the improved immersion protocol. All authors most of the CB-perfusion protocol. D.P. performed most of the computational E.A.S., H.Y. and A.K. performed most of the immersion protocol. K.T. performed AUT and by the Shimabara Science Promotion Foundation. (grant nos. BS261004 and BS271005); by the Tokyo Society of Medical Science; for Novel Science Initiatives of the National Institutes of Natural Sciences Foundation for Applied Enzymology; by the Brain Sciences Project of the Center Foreign Postdoctoral Researcher Program; by a Grant-in-Aid from the Japan AMED-CREST; by the RIKEN Special Postdoctoral Research Program; by the RIKEN RIKEN; by an intramural Grant-in-Aid from the RIKEN QBiC; by a grant from (JSPS); by the strategic programs for R&D (President’s discretionary fund) of (grant no. 15H05650) from MEXT/Japan Society for the Promotion of Science Research on Innovative Areas (grant no. 23115006) and for Young Scientists (A) a Grant-in-Aid for Scientific Research (S) (grant No. 25221004), for Scientific of Education, Culture, Sports, Science and Technology (MEXT) of Japan; Neurotechnologies for Disease Studies (Brain/MINDS) from the Ministry Cell Biology by Innovative Technology and the Brain Mapping by Integrated image resolution. This work was supported by the Program for Innovative manuscript; and T. Mano for his kind contributions and suggestions to discuss preparing the materials; A. Millius for his critical reading and editing of the The University of Tokyo, in particular S.I. Kubota for his kind help in A online version of the pape Note: Any and Information Source Data Supplementary files are available in the biological phenomena inmulticellular organisms. organ. The method willtherefore supportorganism-level systems biology and facilitate ourunderstanding of complicated 3D morphological changes indiseasedtissuesorascalableobservation of tissue-to-subcellularstructures inasingle cleared Possible applications of CUBIC willbefor whole-organ samplesfrom multiple conditions ortime points, detection of aberrant om/reprints/index.htm CKNO

In summary, CUBIC provides aplatform for comprehensive celldetection and analysis across whole organs and the body.

H imaging. brain. mouse ﬁxed of observation 2,2 using protocol clearing optical tissue. non-neuronal and neuronal Neurosci. Nat. reconstruction.circuit neuronal for agent clearing optical preserving (2011). brain. mousetransparent of reconstruction brains. mouse intact cleared, of imaging imaging. volume for samples tissue (2012). e33916 brains. mouse expressing GFP dehydrationof and clearing 3DISCO. brain. mouse whole the in networks Präparaten tierischen Methods Nat. Neuron microscopy.light-sheet using level single-cell the at development Costantini, I. Costantini, rapid A T.Nemoto, & T. Hibi, H., Osanai, R., Kawakami,Y., Aoyagi, T. Kuwajima, morphology- and simple a SeeDB: T.Imai, & S. Fujimoto,M.T., Ke, H. Hama, M.K. Schwarz, Renier,N. Chemical H.U. Dodt, & Weiler,R. S., Saghaﬁ, N., Jährling, Becker,K., A. Ertürk, H.U. Dodt, W.Spalteholz, microscope.light a with circuitry Margrie,T.W.brain MappingP.& Osten, and activity whole-brain Visualizing M.B.Ahrens, Keller,P.J.& OR W

LE CONTR DGM

85 Nat. Protoc. Nat. Sci. Rep. Sci. et al. et , 462–483 (2015). 462–483 , et al. et et al. et et al. et IB ENTS

et al. et UT

10 et al. et 16 et al. et Über das Durchsichtigmachen von menschlichen und menschlichen von Durchsichtigmachen das Über Sca Three-dimensional imaging of solvent-cleared organs using organs solvent-cleared of Three-dimensionalimaging iDISCO: a simple, rapid method to immunolabel large immunolabel to method rapid simple, a iDISCO: I Ultramicroscopy: three-dimensional visualization neuronalof

, 515–523 (2013). 515–523 , We thank the lab members at RIKEN QBiC and ONS , 1154–1161 (2013). 1154–1161 ,

5 l r Clear A versatile clearing agent for multi-modal brain multi-modal for agent clearing versatile A . l . Fluorescent-protein stabilization and high-resolution and stabilizationFluorescent-protein

e: a chemical approach for ﬂuorescence imaging and imagingﬂuorescence for approach chemical a e: , 9808 (2015). 9808 , (S. Hirzel, 1914). Hirzel, (S.

7 H.R.U., H.R.U., E.A.S., K.T. and D.P. designed the study. , 1983–1995 (2012). 1983–1995 , T : a detergent-solvent-freea and:clearing method for The authors declare no competing financial declare no The financial authors competing Development PLoS ONE PLoS ` -thiodiethanol for microscopic for -thiodiethanol Nat. Methods Nat. Cell

159 PLoS ONE PLoS Nat. Neurosci. Nat.

, 896–910 (2014). 896–910 , 10

140 , e0116280 (2015). e0116280 ,

4 , 331–336 (2007). 331–336 ,

, 1364–1368 (2013). 1364–1368 , 10 , e0124650 (2015). e0124650 , http://www.nature.

14 PLoS ONE PLoS , 1481–1488 ,

, 21. 20. 19. 18. 17. 16. 15. 14. 38. 37. 36. 35. 34. 33. 32. 31. 30. 29. 28. 27. 26. 25. 24. 23. 22.

Nat. Methods Nat. microscopy.structured-illuminationsheet-based light scanned with microscopy. sheet light scanned by development embryonic early zebrafish of decolorization. tissue by (2014). 726–739 analysis. computational and cocktails chemical using clearing. whole-body through (2014). 1682–1697 tissues. intact of imaginghigh-resolution and rapid systems. brain. mammalian Cancer Res. Cancer noninvasively.tumors brain in pharmacodynamicsand accumulation ENEURO.0001-15.201 primates.nonhuman and rodents from cords Med. Nat. injury. after responses glial regenerationand axon assess to cord spinal (1997). cells. green ubiquitous of source a as mice’ GFP.of variants spectral multiple epilepsy. genetic human of model mouse (2005). wiring. brain’s the of miniaturization the on histology.the of (2008). fluorescence.of loss and damage brain. Fsl. 1970). Press, Psychopictorics and (2009). 1274–1283 brain. the of areas extensive over dynamicsexpression Neuroimage reliability.and efficiency improved significantly structures: anatomical (2011). 1901–1920 challenge. EMPIRE10 the CT: Circuits Neural Front. microscopy.illumination plane selective using larvae zebrafish intact Methods Nat. microscopy.light-sheet using resolution cellular at imagingfunctional Keller,P.J. Reconstruction E.H.K. Stelzer, & J. Wittbrodt, A.D., Keller,P.J.,Schmidt, Tainaka,K. E.A. Susaki, B.Yang, for CLARITY Advanced K. Deisseroth, & B. Hsueh, Ye,L., Tomer,R., K. Chung, B. Hou, Weber,T.G. C. Soderblom, A. Ertürk, ‘GreenY. Nishimune, & T.Nakanishi, K., Kominami, M., Ikawa, M., Okabe, G.P.Feng, K.L. Richards, limits places noise Ion-channel S.B. Laughlin, & J.A. White, A.A., Faisal, preservation with technique Spalteholz modified W.A Wolff, & H. Steinke, Oxyradical-inducedGFP S.S. Ashraf, & S.M. Qadri, B., Zeedi, A.A., Alnuami, E.S. Lein, S.M.C.F.,Beckmann, Behrens,T.E.,Smith,Woolrich,Jenkinson,M., M.W. & In extraction. and enhancement Object J.M.S. Prewitt, Yamaguchi,S. & M. Eguchi, P.A.Yushkevich, Murphy,K. Panier,T. Keller,P.J.Whole-brain & J.M. Li, D.N., Robson, Orger,M.B.,M.B., Ahrens, Neuroimage Nature et al. et et al. et Nature

et al. et et al. et 18 et al. et et al. et et al. et

et al. et et al. et et al. et et al. et 74 31 Science et al. et

, 166–171 (2012). 166–171 , Scalable and DiI-compatible optical clearance of the of clearance optical DiI-compatible and Scalable 7 10 445 et al. et Single-cell phenotyping within transparent intact tissue intacttransparent within phenotyping Single-cell , 1913–1923 (2014). 1913–1923 , et al. et , 1116–1128 (2006). 1116–1128 , , 637–642 (2010). 637–642 , Fast functional imaging of multiple brain regions in regions brain multiple of imagingfunctional Fast

Genome-wide atlas of gene expression in the adult mouse adult the in expressiongene of atlas Genome-wide Three-dimensional imaging of the unsectioned adult unsectioned the of Three-dimensionalimaging Structural and molecular interrogation of intact biological intact of interrogation molecular and Structural Imaging neuronal subsets in transgenic mice expressing micetransgenic in subsets neuronalImaging 62 497 , 413–420 (2013). 413–420 , Fast, high-contrast imaging of animal development animal of imaginghigh-contrast Fast, Evaluation of registration methods on thoracic on methodsregistration of Evaluation et al. et Whole-body imaging with single-cell resolution single-cell with imaging Whole-body Apoptosis imaging for monitoring DR5 antibody DR5 monitoring for imaging Apoptosis Ann. Anat. Ann. Whole-brain imaging with single-cell resolution single-cell with imaging Whole-brain , 168–176 (2007). 168–176 , Front. Neuroanat. Front. (eds. Lipkin, B.S. & Rosenfeld, A.) 75–149 (Academic 75–149 A.) Rosenfeld, & B.S. Lipkin, (eds. , 782–790 (2012). 782–790 ,

Hippocampal volume and cell density changes in a in changesdensity cell and volume Hippocampal 3D imaging of axons in transparent spinaltransparent in axons of imaging 3D 322 , 332–337 (2013). 332–337 ,

5 NATUREPROTOCOLS 7 (2015). User-guided 3D active contour segmentation of segmentation contour active 3D User-guided , 65 (2013). 65 , , 1065–1069 (2008). 1065–1069 , Cell

In vivo In

183 IEEE Trans. Med. Imaging Med. Trans. IEEE 159 Cell , 91–95 (2001). 91–95 ,

, 911–924 (2014). 911–924 , Int. J. Biol. Macromol. Biol. J. Int. Neuron 158 and

9 , 19 (2015). 19 , , 945–958 (2014). 945–958 ,

in vitro in Neurology 28

| VOL.10 NO.11VOL.10 , 41–51 (2000). 41–51 , FEBS Lett. FEBS eNeuro Curr. Biol. Curr. visualization of gene of visualization

80 2 Nat. Protoc. Nat. Cell

, 1240–1246 (2013). 1240–1246 ,

PROTOCOL d

30 407 Picture Processing Picture Neuroimage 15 oi:10.1523/

, 157 , 1143–1149 , , 313–319 ,

, 182–186 , | 2015 ,

44 |

1727