Home

Advanced CUBIC protocols for whole-brain and whole

Contents

1. Pancreas Butterfly needle Ma Spleen ee 1 2 reagent 1 4 PBS PBS heparin Kidney C Day 1 in reagent 1 Heart Lung Liver Pancreas d After CB perfusion Kidney Spleen ed After 2 weeks of reagent 1 treatment My A Pelvis o Head Dorsum 2015 Nature America Inc All rights reserved Figure 4 Whole organ imaging with LSFM a a The microscope setup and the customized sample holder inset used in this manuscript b Left a raw TIFF image 2 560 x 2 160 from a cleared Thy1 YFP H Tg mouse brain gt 4 male 23 weeks old imaging conditions z 10 um step x 749 planes zoom 1 6x expose 50 ms x two illuminations from each side total acquisition time 30 min The sample was cleared according to the immersion protocol in this manuscript Right a magnified image of the indicated area on the left These images were minimally processed C sharpness brightness and contrast with ImageJ c The reconstituted 3D image of the acquired data in b A view from the dorsal side is shown d A magnified image of the indicated area in c e A magnified image of the right hippocampus viewed from the midline to lateral as indicated in c f A magnified image of the reconstituted x z image of the right hippocampus as indicated in c g The reconstituted 3D whole organ images from B actin nuc 3xmKate2 KI mouse male 8 weeks old imaging condition z 20 um step x
2. numerical aperture NA 0 15 working distance 87 mm Imaging reservoir 100 quartz LaVision BioTec e Sample holder LaVision BioTec or customized Fig 4a e Green fluorescent signal filter Chroma ET525 50 Red fluorescent signal filter Chroma ET650 60 e Coherent sapphire laser 488LP 100 e Coherent sapphire laser 588LP 50 e Andor sCMOS charge coupled device CCD camera Neo 5 5 The camera and the MVX microscope are connected to a camera adapter Olympus MVX TV1X tube lens Olympus MVX TLU and the Ultramicroscope filter wheel unit LaVision BioTec with adapters LaVision BioTec LV AD MVX 1 and LV AD MVX 2 8 Fig 4a e Customized sample holder e Glass plate for specimen mounting stage Image analyzing software e General image analysis tool e ImageJ freeware from the US National Institutes of Health NIH e Visualization tool e Imaris Bitplane http www bitplane com imaris imaris for 3D reconstitution of TIFF image stacks e ITK SNAP freeware from Paul Yushkevich PhD at the University of Pennsylvania and Guido Gerig PhD at the University of Utah http www itksnap org pmwiki pmwiki php for NIfTl 1 3D images Analysis tools e Python and a C compiler e Code provided as Supplementary Data e ImageMagick http imagemagick org installed with TIFF support e Convert3D http www itksnap org download snap process php link 707 4 amp root nitrc e ANTs 1 9 v4 ANTs freeware from stnava DL URL h
3. 3DISCO for this purpose gt Some of these methods achieved high transparency within a few days by removing lipids and homogenizing refractive indices RIs of the tissue and they were shown to be compatible with whole mount immunohis tochemical analysis However concerns about the quenching of fluorescent proteins and safety issues led to further method development A recent publication addressed this issue reporting that pH control and temperature during clearing are crucial to stabilizing fluorescent proteins Alternative techniques such as Scale use a hydrophilic chemical urea and more recently devel oped tissue clearing methods use other hydrophilic reagents including SeeDB Clear ref 11 or 2 2 thiodiethanol 2 3 and FRUIT 4 These methods are easy and safe and they allow fluorescent signals to be retained however they have a relatively low clearing capability The introduction of CLARITY enabled both fluorescence retention and high transparency by embedding a tissue into hydrogel polymer and removing most of the lipids by electrophoresis gt Possible drawbacks of CLARITY included its technical difficulty and limited scalability because of the need to use a specific device However these difficulties have been addressed by the development of passive clearing protocols that increased the scalability 7 In this protocol we describe how to perform CUBIC CUBIC offers a high performance and device free tissu
4. CB perfusion protocol for whole body clearing A CRITICAL Here we describe clearing of whole adult mouse body only using reagent 1 This overcomes the difficulties of handling whole body samples in the viscous reagent 2 particularly because of bubble formation For infant mouse whole body imaging perfusion and immersion of reagent 1 was sufficient Table 1 19 Adult whole body imaging is not applicable to the microscope setup introduced in this manuscript because of stage size limitations 1 Perform CB perfusion as described in Step 2C i iv i1 Detach the skin from the body Carefully remove as much pelage as possible Make sure that the body is partially transparent after the CB perfusion Typically glands such as the pancreas and submaxillary gland are almost transparent Fig 3d Spleen is also as a good indicator of successful perfusion Immerse the body in 200 ml of reagent 1 Place the container on an orbital shaker set at 60 r p m or a seesaw shaker set at 30 r p m in the incubator at 37 C overnight Use the same concentration of the nuclear staining dye used at Step 2B iv and add to reagent 1 if desired A CRITICAL STEP Inefficient mixing of the reagent and samples during clearing may affect the final clearing performance i11 Replace the same volume of reagent 1 and continue gentle shaking at 37 C Refresh the nuclear staining dye if necessary Replace the reagent and any nuclear staining dye every day in the initial week
5. Dissected organs just after CB perfusion Organs such as the pancreas spleen or kidney are macro scopically cleared and decolored at this point The clearance of these organs is indicative of how well researchers succeed at CB perfusion Scale bars 5 mm c Clearing performance of CB perfused dissected organs at day 1 in reagent 1 and at day 10 in reagent 2 CB perfused organs from a successful procedure are markedly transparent after 1 d of reagent 1 treatment Some of the organs such as pancreas are more transparent in reagent 1 rather than in reagent 2 Scale bars 5 mm d Clearing performance of a CB perfused whole body just after CB perfusion and after 2 weeks of reagent 1 treatment Organs such as pancreas submaxillary gland and spleen quickly tend to become transparent by CB perfusion Major abdominal organs except bones and gastrointestinal content become sufficiently transparent after 2 weeks of reagent 1 treatment C57BL 6 male mice 8 weeks old were used in b d All animal experiments were approved by the Animal Care and Use Committee of the RIKEN Kobe Institute and The University of Tokyo and all of the animals were cared for in accordance with institutional guidelines 1712 VOL 10 NO 11 2015 NATURE PROTOCOLS XLSLPLN25XGMP 25x 1 0 WD 8 mm n 1 41 1 52 Zeiss LD Plan Aphochromat 20X1 0 WD 5 6 mm n 1 43 1 47 Leica HC FLUOTAR L25x 1 00 WD 6 mm n 1 457 b Af B Peristaltic pump
6. Protoc 7 1983 1995 2012 6 Becker K Jahrling N Saghafi S Weiler R amp Dodt H U Chemical clearing and dehydration of GFP expressing mouse brains PLoS ONE 7 e33916 2012 7 Renier N et al iDISCO a simple rapid method to immunolabel large tissue samples for volume imaging Cell 159 896 910 2014 8 Schwarz M K et al Fluorescent protein stabilization and high resolution imaging of cleared intact mouse brains PLoS ONE 10 e0124650 2015 9 Hama H et al Scale a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain Nat Neurosci 14 1481 1488 2011 10 Ke M T Fujimoto S amp Imai T SeeDB a simple and morphology preserving optical clearing agent for neuronal circuit reconstruction Nat Neurosci 16 1154 1161 2013 11 Kuwajima T et al Clear a detergent and solvent free clearing method for neuronal and non neuronal tissue Development 140 1364 1368 2013 12 Aoyagi Y Kawakami R Osanai H Hibi T amp Nemoto T A rapid optical clearing protocol using 2 2 thiodiethanol for microscopic observation of fixed mouse brain PLoS ONE 10 e0116280 2015 13 Costantini I et al A versatile clearing agent for multi modal brain imaging Sci Rep 5 9808 2015 14 15 16 17 18 19 20 21 22 23 24 25 26 af 28 29 30 31 32 33 34 35 36 37 38 Hou B
7. 4c h We use a Windows PC with Intel R Core TM i7 3970X CPU at 3 50 GHz 64 GB of RAM and NVIDIA GeForce GTX 690 and with Imaris software installed CUBIC informatics Here we show the step by step procedures for data processing Steps 7 15 For preprocessing of raw data each TIFF stack Fig 5 Collect raw images is first converted to a 3D image in the NIfTI 1 data format nii extension introduced by the Neuroimaging Informatics Technology Initiative NIfTI http nifti nimh nih gov nifti 1 NIfTI 1 files are visualized using soft ware such as ITK SNAP Because of the memory limitations of the current software tools files need to be downscaled to 25 by discarding three of every four images of the TIFF z stack series and SYTO 16 Raw TIFF image z 3 5 mm 200 u mKate2 SYTO 16 mKate2 8 E op Intestine by changing the resolution of these images from 2 560 x 2 160 to 640 x 540 Fig 5 This limitation should be overcome with the future development of image informatics tools The downscaling procedure is done using ImageMagick http imagemagick org to create a temporary stack of 16 bit PNG files for each original TIFF stack Next each PNG stack is converted to a NIfTI 1 file using the Convert3D tool from ITK SNAP Fig 5 In this step specification of the correct spacing given the pixel number of raw image data and downscaling parameters and the orientation which depends
8. NIfTI 1 files which permits subtraction of signal images to be calculated We show an example of Arc dVenus Tg mouse brains with or without light stimuli which express a destabilized version of yellow fluorescence protein Venus under control of the Arc gene promoter Fig 6a First the raw data are preprocessed as in Figure 5 Then the composite NIfTI 1 images from different samples are aligned Fig 6b As before images are processed in pairs structural and signal images and the process relies on ANTS and WarpImageMultiTransform We first align all brain data sets from the same experiment to an internal reference 1 e one of the brain images among the samples of that experiment Next the internal reference is registered to a brain atlas such as the Allen Brain Atlas These calculations thus provide the aligned 3D images to an atlas Fig 6c Finally the npg 2015 Nature America Inc All rights reserved Figure 6 Calculation of signal subtraction a Here we use the data set of the Arc dVenus Tg mouse brains with or without light stimuli acquired in ref 18 as an example z 10 um step x 625 675 planes zoom 2x expose 3 s x two illuminations for Venus and 300 ms x two illuminations for PI respectively The reconstituted 3D images from raw TIFF stacks are shown as views from dorsal and ventral sides in D V and V D images respectively Yellow Venus blue PI b We first preprocess these data
9. Solution Reagent Setup Possible degradation of Too much heating which Milder and shorter heating during preparation preparation chemicals in CUBIC Causes ammonia odor reagents Precipitate in reagent 2 2A i 2B iii fixation Poor PFA perfusion Step 2A iv viii 2B v ix 2C ii1 clearing Poor organ clearing during reagent 1 treatment Excess shrinkage or deformation of cleared organ after reagent 2 Poor organ clearing during reagent 2 steps Lower room temperature particularly in winter Insufficient cooling of PBS and PFA Wrong position of the tip of the needle Insufficient pressure for perfusion Alkaline pH of PFA Too much fixation time Insufficient incubation time reagent amount or mixing Use of an aged animal Organ dependent differences in clearing Inappropriate salt concentration during treatment with 1 2 reagent 2 Incubation at 37 C Insufficient replacement in 1 2 reagent 2 Use of an infant or juvenile animal Use of samples prepared with CB perfusion Incomplete dissolution or precipitate in reagent 2 Insufficient incubation in reagent 1 Insufficient replacement into reagent 2 Prepare the reagent before use heat it in a microwave for 5 10 s avoid boiling Keep PBS and PFA on ice just before perfusion Make sure that the tip of the needle is in the left ventricle of the heart Make sure that the perfusion outlet only occurs at the cut in the liver Adjust
10. To stop the clearing procedure wash the sample with 20 ml of PBS 0 01 wt vol sodium azide with gentle shaking or rotation at room temperature three times for at least 2 h each time We typically wash the sample once for 2 h once overnight and again once for 2 h When a sample is stained with PI further staining during this step is needed thus incubate the washed sample in 5 ml of PBS 0 01 wt vol sodium azide containing 5 10 ug ml of PI for an additional 3 d or more if needed at 37 C with rotation 8 A CRITICAL STEP Complete removal of reagent 1 during the washing step is crucial for final clearing efficiency A CRITICAL STEP For cryoprotection at this step we recommend using 30 wt vol sucrose in PBS rather than 20 wt vol sucrose solution to avoid any damage to the sample TROUBLESHOOTING PAUSE POINT Organs can be stored First immerse them in 10 ml of 30 wt vol sucrose in PBS 0 01 wt vol sodium azide per organ with shaking at room temperature overnight When the samples sink to the bottom put them into 0 C T compound and immediately store them at 80 C Thaw the samples as described in the PAUSE POINT callout at Step 2A ii vii Days 8 11 Degas the sample in a limited volume of PBS with a vacuum desiccator Fig 2b To do this immerse the sample in 5 ml of 1 2 PBS diluted reagent 2 and shake it in a 5 ml tube for 6 24 h at 37 C or at room temperature Fig 2e Check whether the sample sinks to the bottom a sign
11. a similar scalability and it may be considered as an alternative Whether a tissue clearing method can be combined with par ticular dyes or stains is an important consideration when selecting the clearing method Whole organ nuclei staining for anatomi cal annotation registration and image analyses has been achieved using the CUBIC clearing procedure 9 Although we did not test other variations of dyes possible limitations on some labeling methods particularly lipophilic dye labeling e g Dil and related dyes may exist given that the CUBIC clearing reagents massively remove lipids Dyes or proteins should be fixed by PFA before clear ing In this sense fluorescent proteins fused to a membrane protein can be observed in the cleared tissue whereas lipophilic dyes such as Dil may not be readily fixed by PFA because of their chemical structures and thus they may be removed during clearing This may be a drawback to CUBIC in which case other clearing methods e g Clear ref 11 SeeDB 9 FRUIT should be considered Structural distortion has been carefully addressed in some clearing methods such as SeeDB and it may need to be con sidered when clearing tissues with the other methods Although we did not observe obvious changes in brain tissue even in the detailed subcellular structures including the axon and spine 8 such structural distortion may happen given that CUBIC reagents remove a large proportion of the lipids and cause t
12. and every 2 or 3 d in the second week Continue the clearing with reagent 1 for at least 2 weeks Typically major abdominal organs except bones and intestinal contents become sufficiently transparent after 2 weeks of treatment with reagent 1 Fig 3d A CRITICAL STEP An ammonia smell indicates the degradation of urea and the reagent should be replaced with fresh medium if this is smell is present TROUBLESHOOTING E PAUSE POINT The whole body can be kept in reagent 1 for up to several months at room temperature Imaging cleared tissues with macrozoom LSFM TIMING 1 3 h per sample A CRITICAL To perform a rapid image acquisition of whole organs a light sheet illumination unit combined with a macrozoom microscope is suitable Here we describe our setup using the Ultramicroscope combined with MVX ZB10 LaVision BioTec and Olympus A confocal or a multiphoton microscope can also be used but for more limited regions 3 Before imaging wipe reagent 2 treated samples softly with a Kimwipe to remove excess reagent 2 on the surface and then immerse the sample into the oil mix for 10 min to 1 h This process also helps remove bubbles around the tissue If bubbles attach on the surface of the sample carefully remove them with a needle or tapered forceps 4 Set the imaging reservoir filled with the immersion oil mix and then set the sample holder Put a glass slide on the sample holder Fig 4a 5 Put the sample on the glass slide Fig 4a Acqui
13. and that the color of the supernatant has turned olive green Replace reagent 1 with the same volume of fresh reagent 1 and continue clearing by shaking at 37 C If appropriate also refresh the nuclear staining dye Replace reagent 1 and any nuclear staining dye again at days 2 and 4 The total incubation time for the complete clearing depends on the organ 1 day of reagent 1 treatment is usually sufficient for pancreas spleen and intestine However note that we treated all indicated organs in Figure 3c with reagent 1 for 5 d A CRITICAL STEP Typically successfully CB perfused organs are turned almost transparent with the exception of liver and lung by day 1 Fig 3c Opacity in the lung occurs mainly from bubbles Note that the color change of the Supernatant indicates decolorization of tissues as a result of heme elution TROUBLESHOOTING vii Days 2 6 To stop the clearing procedure wash the samples with the same volume of PBS with gentle shaking or rotation at room temperature three times for 2 h each time After the PBS wash move to the next step immediately A CRITICAL STEP CB perfused samples are prone to overshrinking in the washing step Do not wash the samples in PBS more than three times for 2 h each time TROUBLESHOOTING viii Immerse the sample in the same volume of 1 2 PBS diluted glycerol and shake it for 6 h to 24 h at room temperature Check whether the sample sinks to the bottom a sign of complete immersion TROUBLESHO
14. containing 10 U ml heparin at 10 ml min to remove the blood from the tissues as much as possible A CRITICAL STEP Insufficient removal of blood inside the tissue prolongs the clearing period and it may cause low clearing performance i11 Perfuse the mice with 150 ml of cold 4 wt vol PFA in PBS pH 7 4 at 15 ml min using a peristaltic pump CAUTION PFA is a very toxic reagent Perform all procedures in a fume hood with a safety glass to avoid inhalation or contact with skin and eyes 1720 VOL 10 NO 11 2015 NATURE PROTOCOLS npg 2015 Nature America Inc All rights reserved PROTOCOL A CRITICAL STEP If the signal from a target reporter protein is weak a prolonged perfusion period may be more effective or clearing as described in Step 2A A CRITICAL STEP Cooling of PBS and 4 wt vol PFA on ice is important for successful perfusion TROUBLESHOOTING iv Perfuse the mice with 20 ml of PBS pH 7 4 at 10 ml min to wash out PFA followed by perfusion of 20 30 ml of 1 2 diluted reagent 1 at the same injection rate Make sure that the organs become translucent by the end of the perfusion Fig 3b Note that a nuclear staining dye such as SYTO 16 1 2 uM and PI 5 10 ug ml can also be added to 1 2 diluted reagent 1 at this CB perfusion step A CRITICAL STEP Perfusion efficiency is crucial to the final clearing efficiency Some organs such as the pancreas and spleen are good indicators to evaluate perfusion efficiency Fi
15. et al Scalable and Dil compatible optical clearance of the mammalian brain Front Neuroanat 9 19 2015 Chung K et al Structural and molecular interrogation of intact biological systems Nature 497 332 337 2013 Tomer R Ye L Hsueh B amp Deisseroth K Advanced CLARITY for rapid and high resolution imaging of intact tissues Nat Protoc 9 1682 1697 2014 Yang B et al Single cell phenotyping within transparent intact tissue through whole body clearing Cell 158 945 958 2014 Susaki E A et al Whole brain imaging with single cell resolution using chemical cocktails and computational analysis Cell 157 726 739 2014 Tainaka K et al Whole body imaging with single cell resolution by tissue decolorization Cell 159 911 924 2014 Keller P J Schmidt A D Wittbrodt J amp Stelzer E H K Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy Science 322 1065 1069 2008 Keller P J et al Fast high contrast imaging of animal development with scanned light sheet based structured illumination microscopy Nat Methods 7 637 642 2010 Ahrens M B Orger M B Robson D N Li J M amp Keller P J Whole brain functional imaging at cellular resolution using light sheet microscopy Nat Methods 10 413 420 2013 Panier T et al Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy F
16. of CAUTION PFA is a very toxic reagent Avoid inhalation or contact with skin cold 4 wt vol PFA 20 ml of PBS and 20 30 ml of 1 2 diluted reagent 1 and eyes Use a draft chamber proper gloves and a mask to handle PFA Great We recommend that PFA be perfused by peristaltic pump for successful and care in handling the injection needle is needed to avoid accidental needlesticks PROCEDURE Anesthesia TIMING 5 min 1 At day 0 deeply anesthetize the mice using pentobarbital 150 mg kg of body weight administer intraperitoneally i p with a 1 ml syringe and a 26 G 1 2 inch injection needle CAUTION Every experiment must follow all relevant governmental and institutional guidelines for the use of experimental animals Transcardial perfusion and tissue clearing TIMING 4 14 d A CRITICAL In this step we particularly focus on the full clearing protocol for the purpose of LSFM imaging However the immersion period and the final transparency of samples can be varied according to the user s experimental purpose 2 Start clearing organs by the simple immersion protocol option A or the CB perfusion and immersion protocol option B CB perfusion clears better than the immersion protocol particularly in heme rich organs but it tends to cause decreased signal intensity because of the shorter fixation time Alternatively start whole body clearing by using the CB perfusion protocol option C A CRITICAL STEP Option A is for a single whole m
17. of complete immersion A CRITICAL STEP Degassing of the sample prevents air bubbles from remaining in the ventricle TROUBLESHOOTING viii Days 8 11 Immerse the sample in 5 ml of reagent 2 in a 5 ml tube and gently shake it at 37 C overnight The next day replace the reagent with fresh reagent and further incubate for 24 h Fig 2e CAUTION Do not rotate the tube to avoid making bubbles Samples do not sink in the highly viscous reagent 2 and it is difficult to take images in the reagent The reagent 2 treated samples should be immersed in the low viscosity immersion oil mix at imaging steps When structural distortion is apparent after reagent 2 treatment at 37 C try incubation at room temperature for a longer time Adjustment of PBS content in 1 2 diluted reagent 2 may also mitigate shrinkage or swelling TROUBLESHOOTING WE PAUSE POINT Organs can be left in reagent 2 for up to 1 week at room temperature Further immersion increases the final transparency but it also causes swelling of the sample After imaging the sample can be washed with PBS 0 01 wt vol sodium azide completely immersed in 30 wt vol sucrose in PBS 0 01 wt vol sodium azide and stored in 0 C T compound at 80 C as described in the PAUSE POINT callout in Step 2A vi B CB perfusion and immersion protocol for faster clearing of whole organs 1 Prepare the surgical setup as shown in Figure 3a 11 Day 0 Perfuse the mice with 20 30 ml of cold PBS pH 7 4
18. one direction is saved to a different folder For further signal comparison steps Steps 7 15 signal and structural images of both D V and V D directions are needed We recommend a simple naming rule for these folders with four fields separated by an underscore information about the experiment including imaging date a unique ID for this brain information about the imaging direction VD or DV and information about the channel nuclear for the nuclear counterstaining and geneExp for the 1722 VOL 10 NO 11 2015 NATURE PROTOCOLS npg 2015 Nature America Inc All rights reserved PROTOCOL signal channel For instance 20131118LAdV_001_nuclear_DV The code provided for the informatics section assumes that the naming convention is respected It is also important to note that white spaces and special characters must be avoided TROUBLESHOOTING Informatics for signal comparison TIMING 1 9 h for a two brain data set depending on the registration method A CRITICAL We provide our source code and an additional user manual as Supplementary Data as well as the NIfTI 1 converted Allen Brain Atlas data and Arc dVenus Tg mouse brain images used in Figure 6 on our website http cubic riken jp Note that timing is roughly proportional to the number of brains and it can vary according to different computer specifications 7 If this is the first time the pipeline is being run install all required software and c
19. organ and whole body scale Overview of the CUBIC pipeline CUBIC provides a platform for a comprehensive analysis of cells in a whole organ or body Here we focus on describing the fol lowing i the advanced CUBIC clearing protocols by simple immersion and CB perfusion Steps 1 2 ii whole brain and whole organ imaging with a LSFM Steps 3 6 and iii CUBIC informatics for preprocessing and comparison of different brain samples Steps 7 15 see also Fig 1 Although CUBIC is also applicable to staining with small chemicals or antibodies over days to weeks as described previously 9 we focus here on imaging of fluorescent proteins together with nuclear counterstaining Tissue clearing Here we provide three clearing procedures Step 2A a simple immersion protocol for dissected whole organs Step 2B the CB perfusion and immersion protocol for faster clearing of whole organs and Step 2C the CB perfusion pro tocol for whole body clearing The immersion protocol in our first CUBIC report has been improved to an advanced version Fig 2 in which the clearing speed and efficiency are increased The CB perfusion protocol Fig 3 is almost identical to that in our second CUBIC report but more detail is given here npg 2015 Nature America Inc All rights reserved PROTOCOL Two photon imaging etc and 1 2 diluted reagent 1 E o o E oo oo o EEN oe ET Reagent 1 refresh 1 d Fixat
20. sets as in Figure 5 Next we align one data set Light to the other Light by registering the first structural data to the second internal reference The internal reference is also registered to a brain atlas such as the Allen Brain Atlas All images are then aligned to the atlas and normalized The 3D reconstituted images from NIfTI 1 data for structure and signal of Light or samples after alignment to the internal reference are shown in dorsal view c Reconstituted 3D images of the aligned and normalized Venus channel yellow and aligned PI channel blue images from the corresponding NIfTI 1 data Views from the dorsal side are shown d Results of subtraction shown as 3D reconstituted images Views from the dorsal side left and top right and the dorsolateral side lower right are shown Signals observed in Light or conditions are shown in magenta and light blue respectively Standardized PI signals of the Light sample are merged and indicated in blue As seen in the magnified panel top right single cells in the sparsely labeled regions can be detected even in the downscaled images The reconstituted 3D images in b d were prepared by using exported TIFF images from the corresponding NIfTI 1 data and with Imaris software as in Figures 4 and 5 All animal experiments here were approved by the Animal Care and Use Committee of the RIKEN Kobe Institute and The University of Tokyo and all animals were car
21. 1 14 d with the exact time required dependent on the sample type and the experimental purposes A single imaging set can be completed in 30 60 min Image processing and analysis can take lt 1 d but it is dependent on the number of samples in the data set The CUBIC clearing protocol can process multiple samples simultaneously We previously used CUBIC to image whole brain neural activities at single cell resolution using Arc dVenus transgenic Tg mice CUBIC informatics calculated the Venus signal subtraction comparing different brains at a whole organ scale These protocols provide a platform for organism level systems biology by comprehensively detecting cells in a whole organ or body INTRODUCTION Since the discovery of the cell as the basic unit of living organisms people have been seeking a way to observe all cells inside the body Comprehensive analysis of cells in organs and whole organisms is expected to provide information about type position number and activity of cells and cellular networks Tissue clearing fol lowed by 3D imaging is one approach that enables the analysis of multiple cells simultaneously in organs Thus the development of this and related technologies has become a recent trend Development of tissue clearing methods Early tissue clearing methods used organic chemicals e g benzyl alcohol methyl salicylate benzyl alcohol benzyl benzoate BABB and solvents used in 3D imaging of solvent cleared organs
22. 2 5 um in a half diameter sphere of a stained nucleus is detected as 2 5 3 7 um in the half diameter actual half diameter optical blurring of lens accord ing to the Rayleigh criterion when 2X zoom 5 2 Um x 5 2 um per pixel is used To distinguish two neighboring cells two param eters need to be considered exclusion volume EV the average voxel volume per cell calculated from cell density and s the voxel 1716 VOL 10 NO 11 2015 NATURE PROTOCOLS volume of nuclei acquired by the camera again considering the cell or nuclei diameter plus the optical blurring as above Each nucleus is detectable and separable if EV is sufficiently larger than s By our calculations s 3 x 3 x 2 voxels 15 6 x 15 6 x 20 um 4 867 2 um gt enough to include a sphere with 2 5 3 7 um in its half diameter The EV of mouse cerebral cortex neuron is 11 000 um per cell ref 32 22 2 um 5 x 5 x 3 voxels which is sufficiently larger than the voxel volume used in this study whereas EV of mouse hippocampal CA1 is 3 900 um per cell ref 33 15 7 um 3 x 3 x 2 to 4 X 4 X 2 voxels which is at the limit of resolution of the examples in the current manuscript Thus the acquired image at its best has single cell resolution in the cortex and regions with similar cell density whereas the voxel size is not sufficient for single cell resolution in denser regions such as the hippocampus or cerebellar granule layer A further consideration
23. 350 500 planes zoom 0 8x 1 6x expose 100 ms to 2 s x two illuminations from each side total acquisition time 45 min The samples were cleared and stained with SYTO 16 according to the CB perfusion protocol The magnified images of SYTO 16 mKate2 and merged signals at the 1 mm depth of each organ zoom 5x h The reconstituted 3D whole organ images from a CAG EGFP Tg GFP green stained with PI male 8 weeks old imaging condition z 20 um step x 350 500 planes zoom 0 8x 1 6x expose 100 ms to 2 s x two illuminations from each side total acquisition time 45 min The samples were cleared and stained with PI red according to the CB perfusion protocol Brightness contrast and minimal gamma value of images in c h were adjusted with Imaris All animal experiments here were approved by the Animal Care and Use Committee of the RIKEN Kobe Institute and The University of Tokyo and all of the animals were cared for in accordance with institutional guidelines Holder Sample jijumi Cover glass nation All raw image data collected in an uncompressed TIFF format 16 bit images for LSFM Fig 4b are typically 7 GB in total per brain color and direction and thus 25 30 GB for a single brain data set which contains structural and signal images acquired in two directions D V and V D Therefore a high spec PC with a large memory size and a good graphics board should be used for 3D reconstitution Fig
24. Foundation for Applied Enzymology by the Brain Sciences Project of the Center for Novel Science Initiatives of the National Institutes of Natural Sciences grant nos BS261004 and BS271005 by the Tokyo Society of Medical Science and by the Shimabara Science Promotion Foundation AUTHOR CONTRIBUTIONS H R U E A S K T and D P designed the study E A S H Y and A K performed most of the immersion protocol K T performed most of the CB perfusion protocol D P performed most of the computational image analysis A K developed the improved immersion protocol All authors discussed the results and commented on the manuscript text COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests Reprints and permissions information is available online at http www nature com reprints index html 1 Keller P J amp Ahrens M B Visualizing whole brain activity and development at the single cell level using light sheet microscopy Neuron 85 462 483 2015 2 Osten P amp Margrie T W Mapping brain circuitry with a light microscope Nat Methods 10 515 523 2013 3 Spalteholz W Uber das Durchsichtigmachen von menschlichen und tierischen Pr paraten S Hirzel 1914 4 Dodt H U et al Ultramicroscopy three dimensional visualization of neuronal networks in the whole mouse brain Nat Methods 4 331 336 2007 5 Ert rk A et al Three dimensional imaging of solvent cleared organs using 3DISCO Nat
25. OTING ix Days 3 7 Immerse the samples in the same volume of reagent 2 and gently shake them at 37 C overnight The next day replace the reagent with fresh reagent and further incubate the samples for several days Typically an apparent transparency plateau is reached after 2 3 d of reagent 2 treatment At day 10 almost all organs should be transpar ent as shown in Figure 3c The gastrointestinal contents of the stomach and intestine should be removed as much as possible before the following imaging step CAUTION To avoid making bubbles do not rotate the tube Samples do not sink in the highly viscous reagent 2 and it is difficult to take images in the reagent The reagent 2 treated samples should be immersed in the low viscosity immersion oil mix at imaging steps When structural distortion is apparent after reagent 2 treatment at 37 C try incubating room temperature for a longer time or testing the simple immersion protocol Step 2A TROUBLESHOOTING E PAUSE POINT Tissues can be left in reagent 2 for up to 1 week at room temperature Further immersion increases the final transparency but it also causes swelling of the sample After imaging the sample can be washed with PBS completely immersed in 30 wt vol sucrose in PBS and stored in 0 C T compound at 80 C as in the PAUSE POINT callout in Step 2A vi NATURE PROTOCOLS VOL 10 NO 11 2015 1721 npg 2015 Nature America Inc All rights reserved PROTOCOL C
26. ane n m Ventral Use V D First e First plane First n 1 Figure 5 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 18 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 here only YFP channel shown in the panel are z 2 25 mm sharp and 5 5 mm blurred for top and bottom respectively The raw data sets are downscaled to 25 and converted to NIfTI 1 files shown as capture images of ITK SNAP Next structural D V data shown as reconstituted 3D images via nuclei counterstaining are registered to the corresponding V D data This step is to calculate transformation parameters which is applied to the signal D V data in the following step alignment We then merge the aligned images in order to ensure sharpness throughout the resulting 3D image YFP channel is shown 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 images This is used to define two threshold values at the z slice position n and m and to create the merged composite NIfTI 1 image shown as capture images of ITK SNAP according to these values The reconstituted 3D images and plane images in the Align and Combine panels were prepared by using exported TIFF images from the corresponding NIfTI 1 data 3D recon
27. calable observation of tissue to subcellular structures in a single cleared organ The method will therefore support organism level systems biology and facilitate our understanding of complicated biological phenomena in multicellular organisms Note Any Supplementary Information and Source Data files are available in the online version of the paper ACKNOWLEDGMENTS We thank the lab members at RIKEN QBiC and The University of Tokyo in particular S I Kubota for his kind help in preparing the materials A Millius for his critical reading and editing of the manuscript and T Mano for his kind contributions and suggestions to discuss image resolution This work was supported by the Program for Innovative Cell Biology by Innovative Technology and the Brain Mapping by Integrated Neurotechnologies for Disease Studies Brain MINDS from the Ministry of Education Culture Sports Science and Technology MEXT of Japan a Grant in Aid for Scientific Research S grant No 25221004 for Scientific Research on Innovative Areas grant no 23115006 and for Young Scientists A grant no 15H05650 from MEXT Japan Society for the Promotion of Science JSPS by the strategic programs for R amp D President s discretionary fund of RIKEN by an intramural Grant in Aid from the RIKEN QBiC by a grant from AMED CREST by the RIKEN Special Postdoctoral Research Program by the RIKEN Foreign Postdoctoral Researcher Program by a Grant in Aid from the Japan
28. copy confocal microscopy Two photon microscopy Optical sectioning micro scopy stereomicroscopy Two photon microscopy confocal microscopy two photon serial sectioning tomography Confocal microscopy two photon microscopy COLM and other custom LSFM setups Whole brain signal comparison analysis extraction of anatomical and histological structures quantitative analysis of pancreatic Langerhans islets 3D reconstruction visualization of intensities axon tracing cell number quantification tumor volume calculation 3D reconstruction distance measurement 3D reconstruction axon and dendrite tracing cell distribution analysis 3D reconstruction 3D reconstruction neurite tracing aUse acrylamide embedded specimens MATERIALS REAGENTS Animal samples used for imaging e Animals expressing fluorescent proteins can be used Strong expression of a bright fluorescent protein gives the best imaging results A bright red fluorescent protein such as mKate 2 is best So far we have confirmed good imaging performance with Thyl YFP H Tg The Jackson Laboratory R26 H2B EGFP KI RIKEN Center for Developmental Biology CDB R26 H2B mCherry KI RIKEN CDB R26 CAG nuc 3xmKate2 KI RIKEN CDB amp QBiC B actin CAG nuc 3xXmKate2 KI RIKEN CDB amp QBiC CAG EGFP Tg Japan SLC and Arc dVenus Tg Gifu University We also usually use C57BL 6 CLEA Japan to prepare cleared organs and bodies CAUTION Animal exp
29. d it is scalable from subcellular structures e g neuronal axons or spines to marmoset brains to whole animal bodies 9 Furthermore whole organ counterstaining with a nucleic dye enables precise positioning of genetically labeled cells in the whole organ structure extraction of specific anatomical struc tures and alignment of different samples for comparing signal intensities 8 9 CUBIC for whole organ or whole body imaging and image informatics CUBIC cleared samples can be used in LSFM We use an optimized Ultramicroscope LaVision BioTec for this purpose In rapid whole organ imaging a single cleared whole mouse 1710 VOL 10 NO 11 2015 NATURE PROTOCOLS brain can be imaged within 30 60 min per color and orientation Fluorescence wavelength affects the quality of imaging results in general red wavelengths can penetrate deeper in tissue and thus better imaging results are obtained with red fluorescence than green fluorescence particularly in deeper regions To ensure that weaker signals are detected in deeper regions the sample is imaged in two orientations in the case of whole brain imaging we took z stack images of the dorsal side up D V and ventral side up V D directions Image visualization software such as Imaris can be used for depicting the reconstituted 3D image Imaris implements numer ous image analysis functions including spot counting and surface extraction We performed extractions of anatomical st
30. ds further investigation Not cleared with the current reagents aWe performed imaging of postnatal day 1 samples19 Imaging of cleared adult mouse was not tested because of size limitation of current LSFM setup The cleared body specimen can be stocked in the reagent cSo far we tested a brain sample of postnatal day 3 Adult brain will be tested in future studies 4E A S unpublished result Needs hair removal fBone clearing of infant mice was sufficient for imaging 9 clearing methods also and thus we are unable to suggest a suit able alternative in this scenario Thus this issue needs further investigation in future studies As discussed earlier imaging resolution is also a point to be considered We have detected and counted signals from a single cell and in this manuscript we therefore define this as providing single cell resolution This criterion is roughly evaluated by using spot analysis of Imaris software in the data set in Figure 4 hippocampal cells of Thy1 YFP H Tg Supplementary Video 1 In our opinion this criterion is supported overall by the calculated optical resolution According to the vendor s specifications the optical resolution of microscopy that we use in this manuscript has 4 2 um and 3 7 um in x y images with 1 6x and 2X zoom respectively The thickness of the light sheet is lt 10 um at the thinnest region which is smaller than the typical step size 10 um In a typical nuclei stained image a
31. e clearing method based on hydrophilic reagents which preserves fluorescence It enables reproducible whole organ and whole body clearing We have used CUBIC for clearing and rapid 3D imaging of whole mouse brains a whole marmoset hemisphere whole mouse organs e g lung and heart and whole mouse body These images were used for image analyses for extracting biological information 9 Methods for imaging cleared tissues Tissues cleared using the above methods can be imaged in 3D with optical microscopies Because some of the above clearing methods render tissues highly transparent light sheet fluores cence microscopy LSFM has also been used for imaging 2 This type of microscopy can collect z stack images in a rapid manner and it has been applied to 3D and 4D imaging such as a time lapse imaging of developing embryos or whole brain calcium dynamics 0 23 One of the earliest cases of whole mouse brain imaging was rapid whole brain imaging of a BABB cleared brain using a macrozoom compatible light sheet unit ultra microscopy More recently COLM CLARITY optimized light sheet microscopy has been used for whole brain scale imaging of CLARITY processed brains Thus the use of LSFM after an efficient tissue clearing method facilitates a high throughput collection of multiple 3D images Rapid 3D imaging with LSFM can be used after whole organ and whole body clearing by CUBIC CUBIC also provides processing and analysis of 3D ima
32. ed for in accordance with institutional guidelines Adapted with permission from ref 18 signal channel images of different brains are compared To do so we normalize these aligned images so that the median intensity inside the brain is the same across all brains Next the fslmaths function is used to compare pairs of brains by subtracting one image from the other Fig 6d We provide the set of scripts for all steps and a brief user guide as Supplementary Data with up to date versions also available on a GitHub repository https github com SystemsResearch CUBIC_nprot On our website http cubic riken jp we also share example raw TIFF data of Arc dVenus Tg mice shown in Figure 6 and NIfTI 1 converted Allen Brain Atlas data Limitations of the current version of CUBIC CUBIC was developed and optimized for whole organ and whole body imag ing and the informatics analysis was developed to enable a com prehensive pipeline Thus there are several important advantages to CUBIC compared with other clearing methods The first is the active tissue decoloring ability This method is milder for pro teins as opposed to the simple flushing or harsh decolorization methods with peroxidase or acetone used previously gt gt This enables a wide range of applications for not only the brain but also other organs inside Table 1 Because aqueous reagents are used to clear tissue and fluores cence signals are preserved tissues can be
33. er clearing methods 9 and it can be used for two photon imaging and possibly as part of the sample preparation for single photon imaging NATURE PROTOCOLS VOL 10 NO 11 2015 1719 npg 2015 Nature America Inc All rights reserved PROTOCOL iv Discard 1 2 diluted reagent 1 Immediately add 8 10 ml of reagent 1 and gently shake it or rotate the sample at 37 C overnight If desired the same concentration of nuclear staining dye used in the previous step should be added to reagent 1 CAUTION Reagent 1 can erase oily pen marks easily Make sure that the tube is sealed by wrapping Parafilm around the lid and the top of it Sample labels should be written on both the body and lid of the tube to avoid loss of information An ammonia smell indicates degradation of urea and that the reagent should be replaced by fresh medium TROUBLESHOOTING v Day 2 Replace 8 10 ml of reagent 1 and continue gentle shaking or rotation at 37 C Replace the reagent every 2 d days 4 and 6 Also refresh any nuclear staining dye on day 4 and day 6 Typically the brain will be sufficiently cleared by days 7 8 Fig 2e CAUTION As CUBIC treated organs soften we recommend using spoons instead of forceps for handling them in order to avoid damage A CRITICAL STEP If the white matter has not substantially cleared by 8 d of immersion try further immersion by placing it into fresh reagent 1 for an additional 1 2 d TROUBLESHOOTING vi Days 7 10
34. eriments must be performed in accordance with governmental and institutional regulations regarding the use of animals for research purposes All animal experiments and housing conditions in this manuscript were approved by the Animal Care and Use Committee of the RIKEN Kobe Institute The University of Tokyo and the Gifu University and all animals were cared for and treated humanely in accordance with the institutional guidelines for experiments using animals Fixative perfusion and storage reagents e PBS tablets Takara cat no T9181 e Heparin sodium Mochida Pharmaceutical 10 000 U 10 ml e PFA Nacalai Tesque cat no 02890 45 1 CAUTION PFA is toxic Perform all procedures in a fume hood e HCI Nacalai Tesque cat no 18320 15 or 18321 05 CAUTION HCl is toxic Perform all procedures in a fume hood e NaOH Nacalai Tesque cat no 31511 05 1 CAUTION NaOH is toxic Perform all procedures in a fume hood e Sucrose Nacalai Tesque cat no 30403 55 or 30404 45 e TISSUE TEK O C T compound Sakura Finetek cat no 4583 1 2 diluted ScaleCUBIC 1 1 2 reagent 1 ScaleCUBIC 1 is mixed with an equivalent volume of dH O A CRITICAL ScaleCUBIC 1 should not be diluted with PBS because contamination with salt decreases the clearing performance Clearing nuclei staining and imaging reagents e N N N N Tetrakis 2 hydroxypropyl ethylenediamine Quadrol Tokyo Chemical Industry cat no T0781 2 2 2 Nitrilotrie
35. ersion of sample in reagent 1 clearing is obvious within several hours Fig 2e and such partially cleared samples are even applicable to deep region imaging with two photon microscopy The CUBIC reagents also decolorize organs and the whole body by removing heme which is also apparent just after CB perfusion Fig 3a b and this ability enables whole body clearing within 2 weeks Fig 3c d We summarize the organs we have used CUBIC on in Table 1 Removal of the CUBIC reagents by PBS washing reverses the cleared state Fig 2e but the tissue is clear again if it is re immersed into CUBIC reagents Figure 4a f shows typical imaging results for the Thy1 YFP H Tg mouse brain at 1 6x zoom Overall soma and other subcellular structures such as neurites in the mouse brain can be captured provided that they are sparsely labeled Fig 4b d e and Supplementary Video 1 Other organs were also subjected to rapid multicolor 3D imaging in the same platform Fig 4g h Of note these data were collected for 30 60 min for each direction color According to the user s experimental purpose high or low resolution images may be acquired We use relatively low resolution because our primary purpose is to analyze cells within the context of a whole organ or body in a high throughput manner However subcellular structures can be observed in the cleared tissue Fig 4b f 18 and thus CUBIC permits more detailed observations by using a higher zoom on the LSFM inst
36. fused with diluted reagent 1 Samples are subsequently treated with reagent 1 and reagent 2 Counterstaining is performed during and after reagent 1 treatment 9 Samples can be stored at various points in the PROCEDURE which are indicated as PAUSE POINTS Whole organ or whole body imaging We use a commercially available LSFM instrument Ultramicroscope LaVision BioTec supplied with an optimized macrozoom microscope MVX ZB10 Olympus and a scientific complementary metal oxide semicon ductor sCMOS camera Andor NEO 5 5 2 560 x 2 160 pixels A customized sample holder is also used for larger brain and body samples and soft abdominal organs Fig 4 A suitable pair of excitation laser and emission filters is also installed We typically use 100 mW of 488 nm laser ET525 50 emission filter for green fluorescence and 50 mW of 588 nm laser ET650 60 emission NATURE PROTOCOLS VOL 10 NO 11 2015 1711 npg 2015 Nature America Inc All rights reserved PROTOCOL pe Before dissolution Complete dissolution PBS washed day 8 and 9 fiz cee A E maa ae Er F Fixed brain 1 2 reagent 1 day 1 6h Reagent 1 day 7 H E jae a J Sam T Figure 2 Procedure of the simple immersion protocol a Preparation of reagent 2 This reagent contains a high concentration of urea 25 wt and sucrose 50 wt top These can be completely dissolved by heat and stirring with a microwave and a hot st
37. g YFP Venus with a strong expression promoter e g CAG avoid green channel and use a red fluorescent protein e g mKate2 Adjust the pH of PFA to 7 4 Use a younger animal Select a bright fluorescent protein with a strong expression promoter as above Use more PFA for perfusion and pause the perfusion procedure after PFA perfusion to increase fixation reaction 3 h try the immersion protocol select an animal strain with a bright fluorescent signal Higher temperature during clearing may decrease fluorescence signals Try to incub ate at room temperature rather than 37 C Note that lower temperature also decreases clearing efficiency Check the setting of laser power filter and exposure time Adjust the light sheet focus to the region of interest for the LaVision Ultramicroscope the width of focused sheet is 1 3 1 4 of an adult mouse hemisphere and thus it is impossible to take images with adjusted focus throughout the brain continued NATURE PROTOCOLS VOL 10 NO 11 2015 1725 npg 2015 Nature America Inc All rights reserved PROTOCOL TABLE 3 Troubleshooting table continued Step Problem Possible reason Solution 8 15 analysis Brain appears deformed Incorrect scaling Check the parameter file and ensure that the in the NIfTI 1 file correct voxel dimensions are given for the Step 8 raw TIFF image in mm Incorrect brain Different brain position during imaging Reorie
38. g 3b v Dissect the organs of interest and immerse these in reagent 1 Several organs can be processed in a single tube but the stomach and intestine should be separated into different tubes Gastrointestinal content in these organs should be removed as much as possible in this step Thus immerse several organs such as heart lung kidney spleen pancreas and a piece of liver in 40 ml of reagent 1 or immerse each small organ such as heart lung kidney spleen and pancreas in 5 ml of reagent 1 Incubate the samples with shaking 60 r p m if you are using an orbital shaker in a hybridization oven as shown in Fig 2c or 30 r p m if you are using a seesaw shaker as shown in Fig 2d or rotation 5 r p m at 37 C overnight Add the same concentration of any nuclear staining dye used at Step 2B iv to reagent 1 CAUTION Reagent 1 erases oily pen marks easily Make sure that the tube is sealed by wrapping Parafilm around the lid Sample labels should be written on both the body and the lid of the tube to avoid loss of information An ammonia smell indicates degradation of urea and in this scenario the reagent should be replaced with fresh medium A CRITICAL STEP Efficient mixing of the reagent and samples during clearing may affect the final clearing performance For efficient clearing the samples of interest should be immersed in at least a fivefold volume of reagent 1 TROUBLESHOOTING vi Day 1 Make sure that the organs are transparent
39. ges for extracting biological NATURE PROTOCOLS VOL 10 NO 11 2015 1709 npg 2015 Nature America Inc All rights reserved PROTOCOL information Therefore CUBIC presents a platform of whole organ or whole body imaging and image informatics which enables a wide range of users to perform experiments targeting cellular and organ layers with multiple samples Development of CUBIC for efficient and reproducible whole organ or whole body clearing In developing a clearing technique for whole brain and whole body imaging we considered two main criteria one efficiency and transparency with the preservation of fluorescence for a rapid whole brain body imaging with LSFM and two reproducibility for comparative analysis of multiple samples Because a clearing method with hydrophilic reagents had the potential to fulfill these criteria we started by modifying the Scale recipe For this pur pose we constructed a new chemical screening method in which reduction of turbidity of a fixed brain suspension was measured before and after mixing with a candidate chemical solution This screening enables nonbiased discovery of brain clearing chemicals We screened 40 Scale related chemicals and found that aminoalcohols in addition to urea and Triton X 100 in Scale clear tissue with minimal fluorescence quenching In the CUBIC clearing protocol we prepared two reagents ScaleCUBIC 1 reagent 1 and ScaleCUBIC 2 reagent 2 whic
40. h also mini mize light scattering inside the tissue The first reagent works as a potential lipid remover Lipid is thought to be the main light scattering material inside tissue and its removal is correlated with the degree of transparency A fixed brain was treated with the first reagent for 1 week washed with buffer and then immersed into the second reagent which has an RI close to 1 49 which is similar to that of the SeeDB reagent 9 Moving from the buffer to the second reagent matched the RIs between the sample and the reagent which further reduced light scattering within the tissue Thus a whole mouse brain became transparent within 14 d ref 18 However some chemicals seem to have additional or different roles during the procedure and thus further studies are needed to elucidate tissue clearing mechanisms In addition to light scattering light absorption is another chal lenge in tissue clearing We accidentally found that aminoalcohols can remove heme in blood and tissues 9 thus CUBIC is able to decolorize tissue We used perfusion of the CUBIC reagent CB perfusion to efficiently penetrate the mouse body and to accelerate the clearing and decoloring procedure The CB perfusion protocol enabled not only faster clearing of dissected tissues but also whole body clearing of infant and adult mice Because of its efficiency and reproducibility the CUBIC pro tocol can be applied to multiple samples in a single experiment an
41. imaged by fluorescence microscopy To achieve efficient clearing for LSFM application the optimal CUBIC reagent comprises five chemicals Table 2 and takes several days to 2 weeks to process However the proce dure can be modified for the user s purpose for example if users plan to image by two photon microscopy a one step immersion in reagent 1 for 1 3 d is sufficient 8 The actual scalability of CUBIC has not yet been fully inves tigated So far we have tested a hemisphere of infant marmoset and infant and adult whole mice Table 1 Clearing of these samples was efficient for example in the case of cleared infant PROTOCOL D V V D Venus oO V D image O Register and align to internal reference Light D V image V D image Light C D V V D d Aligned and normalized Venus high in Light Venus high in Light PI Magnified Rotated Subtraction light Light mouse internal structures of the brain could be imaged directly even through the skull However we have not yet tested adult primate brains e g marmoset although we plan to investigate this in future studies Although clearing of larger adult primate brains might be more difficult longer incubation times and CB perfusion may address this issue The passive clarity technique PACT perfusion assisted agent release in situ PARS protocol of CLARITY has apparently achieved
42. into 0 C T compound and immediately transfer them to 80 C To continue the clearing protocol thaw the samples gradually at room temperature and wash them with PBS at least twice with each wash for 1 h to remove sucrose and 0 C T compound The sample will now be ready for the next step however we find that the clearing efficiency is reduced in samples that have been stored iii Immerse the sample in 8 10 ml of 1 2 water diluted reagent 1 with shaking 60 r p m if you are using the orbital shaker of a hybridization oven shown in Fig 2c and 30 r p m if you are using a seesaw shaker as shown in Fig 2d or rotation 5 r p m at 37 C for 3 6 h We recommend using a 30 ml conical tube for clearing a single brain rather than a 15 ml tube in this and further clearing Step 2A iv v because of sample swelling Clearing effects can be observed during this step Fig 2e Note that a nuclear staining dye such as SYTO 16 1 2 uM and PI 5 10 ug ml can also be added to 1 2 diluted reagent 1 at this step A CRITICAL STEP Inefficient mixing of the reagent and samples during clearing may affect the final clearing performance Pretreatment with 1 2 diluted reagent 1 gives a more effective final clearing efficiency than direct immersion in reagent 1 However this step can be skipped for other purposes such as two photon imaging with a partially cleared sample for example This direct immersion procedure gives better clearing results than some oth
43. ion Lipid removal RI matching c 5 rr incre eaiieseather Imaging E 2 Step 1 2A ii Step 2A iii vi i Step 2A ii iii with LSFM T g Step 2B v vii Step 2B viii ix z z 1 Steps 3 6 XD O 1 3 h per sample K 5 10 min 1d 1 7 d Ad 2d 10 min Perfused with PBS Postfixation Reagent 1 Wash Reagent 2 Immersion and 4 PFA in 4 PFA refresh 2 3 d refresh 1 d oil T eoe ooo o o eo EZ o o o EE Fixation CB perfusion Lipid removal decolorization 2s Steps 1 2B iv re Step 2C i iii 25 Steps 1 2C i i ala SO ane EA gi 7 min a 7d 7d ta Perfused with PBS 4 PFA Whole _______ Immersion body oil Reagent 1 refresh 2 3 d Preprocessing Steps 7 and 8 Steps 9 and 10 Raw TIFF Acquision e E dorsal x gt side D wu o Create E lt composite NifTl 1 data Acquision V D images S viewed in ITK SNAP Use z 2 E l ventral i fi N side son sh b a bix 3 5 h Create D V V D composite Convert to NIfTI 1 downscaling Structure Signal subtraction Steps 11 and 12 Steps 13 15 Sample A Sample B Reference m oe ia Signal 3h 0 5 1 5 h Alignment Normalization and subtraction between samples Figure 1 Overview of the CUBIC pipeline CUBIC is composed of three major stages clearing imaging and analysis For efficient and reproducible clearing we provide three protocols a simple immersion protocol Step 2A which takes 11 d for a whole brain from an adu
44. ion or 3 5 min if you are using affine transformation only and lt 1 min for the alignment of both channels TROUBLESHOOTING 12 Use atlasAlignment py to register the internal reference to the brain atlas 1 h 30 min and align all brains to the atlas 1 min per brain TROUBLESHOOTING 13 Calculate the normalization factors 7 min plus 7 min per brain with median_brainOnly py TROUBLESHOOTING 14 Normalize and compare brains e g for subtraction with normalisation_comparison py exact timing is dependent on comparison but it typically takes 10 60 min TROUBLESHOOTING 15 Export a TIFF stack for the resulting files with exportTiffStack py about 4 min per NIfTI 1 file A CRITICAL STEP Although registration with Symmetric Normalization SyN is effective in some cases it may cause structural deformation or distortion Users can choose whether to use SyN or affine only registration We recommend checking the quality of final aligned data and if necessary trying SyN or affine only instead In this manuscript we show data aligned with SyN in Figure 5 and with affine only transformation in Figure 6 for illustrative purposes TROUBLESHOOTING TROUBLESHOOTING Troubleshooting advice can be found in Table 3 NATURE PROTOCOLS VOL 10 NO 11 2015 1723 npg 2015 Nature America Inc All rights reserved PROTOCOL TABLE 3 Troubleshooting table Step Problem Possible reason
45. irrer bottom b A vacuum desiccator for the degassing steps c An incubator with a shaker a hybridization incubator that we use for the clearing procedure Inset five brain samples treated with reagent 1 in a single tube day 5 d A table shaker used for the PBS washing step at room temperature e Appearance of a brain sample in each step A brain from C57BL 6 male mouse 8 weeks old was used Reagent 1 treated sample is temporally swollen but the size is recovered after immersion in reagent 2 Scale bar 5 mm All animal experiments were approved by the Animal Care and Use Committee of the RIKEN Kobe Institute and The University of Tokyo and all of the animals were cared for in accordance with institutional guidelines 1 2 reagent 2 day 9 6 5 h waa i fae wl fa ae IN ka E structural images via whole organ coun terstaining should be collected To ensure sharpness of signals throughout the 3D image data of the same sample from dif ferent directions D V and V D in the case of whole brain imaging are also collected This is recommended because the ventral horizontal slices are sharper in V D images and dorsal horizontal slices are sharper in D V images Fig 5 8 If a proper LSFM instrument is not available widely used confocal or two photon microscopes can also be used Partially cleared samples by one step immersion in reagent 1 for 1 3 d are even applicable to deep region imaging with two pho
46. is that the thinnest area of the sheet is limited and does not cover the entire imaged field In addition the analysis software used in this manuscript does not support large image data and collected data must be downscaled Although CUBIC has the potential to detect all signals with single cell resolution these issues will need to be further addressed in future studies npg 2015 Nature America Inc All rights reserved TABLE 2 Other clearing methods and their applications to imaging and analysis PROTOCOL Methods Chemical contents Applied imaging methods Applied computational analysis methods CUBIC18 19 Quadrol Triton X 100 and urea for reagent 1 triethanolamine urea and sucrose for reagent 2 3DISCO4 9 7 36 38 Ethanol or methanol xylene benzyl alcohol benzyl benzoate for BABB method Tetrahydrofuran dichloromethane dibenzyl ether for THF DBE method Scale Urea glycerol Triton X 100 SeeDB10 D Fructose a thioglycerol FRUIT 4 p Fructose urea Formamide for Clear formamide and PEG for Clear ref 2 Clear ref 11 2 2 thiodiethanol 2 13 2 2 Thiodiethanol SDS in borate buffer and one of FocusClear RIMS Histodenz or Sorbitol or 2 2 thiodiethanol CLARITY2 13 15 17 Ultramicroscope LSFM two photon microscopy confocal microscopy Ultramicroscope LSFM two photon microscopy confocal microscopy Two photon microscopy confocal microscopy Two photon micros
47. logy 80 1240 1246 2013 Feng G P et al Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP Neuron 28 41 51 2000 Okabe M Ikawa M Kominami K Nakanishi T amp Nishimune Y Green mice as a source of ubiquitous green cells FEBS Lett 407 313 319 1997 Ert rk A et al Three dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury Nat Med 18 166 171 2012 Soderblom C et al 3D imaging of axons in transparent spinal cords from rodents and nonhuman primates eNeuro 2 doi 10 1523 ENEURO 0001 15 2015 2015 Weber T G et al Apoptosis imaging for monitoring DR5 antibody accumulation and pharmacodynamics in brain tumors noninvasively Cancer Res 74 1913 1923 2014 NATURE PROTOCOLS VOL 10 NO 11 2015 1727
48. lt mouse but varies according to the experimental purpose and organs a CB perfusion protocol for the whole adult mouse Step 2C which takes 14 d and the CB perfusion and immersion hybrid protocol Step 2B in which dissected organs after CB perfusion can be continuously cleared according to the simple immersion protocol Rapid 3D imaging can be performed with an LSFM The collected data are processed and analyzed such as by signal comparison between samples as described in this manuscript Images of actual samples correspond to the samples in Figures 2 and 3 All animal experiments here were approved by the Animal Care and Use Committee of the RIKEN Kobe Institute and The University of Tokyo and all animals were cared for in accordance with institutional guidelines The clearing performance of CB perfusion surpasses the immer sion protocol particularly in heme rich organs heart muscle kidney or liver but it tends to cause decreased signal intensity because of a short fixation time Incubation period can be varied and it is dependent on the organ and imaging methods to be used Figs 1 3 Users may select either of these options and determine the desired final transparency for their experimental purposes In either option a paraformaldehyde PFA fixation is needed Thus animals are transcardially perfused with 4 wt vol PFA and then organs are dissected for postfixation Step 1 Alternatively the fixed body can be further per
49. min cool the mixture at room temperature add 5 g of triethanolamine and stir it further The 0 1 vol vol of Triton X 100 included in the original recipe is not necessary Finally degas the reagent with a vacuum desiccator 0 1 MPa 30 min Fig 2b Prepare 1 2 reagent 2 by mixing 1 1 of reagent 2 and PBS Reagent 2 can be stored at room temperature for up to 2 weeks CAUTION The acrid ammonia smell in these reagents indicates degradation of urea Generation of ammonia itself is not apparently a problem because the reagents are buffered with aminoalcohol in an alkaline pH range pH 11 ref 19 We recommend that users avoid excess heating during preparation If users experience the smell during clearing change to fresh medium see also TROUBLESHOOTING section A CRITICAL Reagent 2 becomes highly viscous and therefore we use wt rather than wt vol or vol vol for convenience Because water evaporation will make it difficult for highly concentrated chemicals to dissolve the weight should be monitored for the addition of evaporated water after completely dissolving urea and sucrose Avoid boiling during the preparation A CRITICAL Reagent 2 should not be prepared with PBS because salt contamination decreases the clearing performance We use PBS only in preparing 1 2 reagent 2 PBS because tissues after reagent 1 treatment tend to be easily swollen in 1 2 reagent 2 water which might cause distortion of the overall struc
50. npg 2015 Nature America Inc All rights reserved PROTOCOL Advanced CUBIC protocols for whole brain and whole body clearing and imaging Etsuo A Susaki 3 Kazuki Tainaka 37 Dimitri Perrin47 Hiroko Yukinaga gt Akihiro Kuno 2 gt 6 amp Hiroki R Ueda 3 1Department of Systems Pharmacology The University of Tokyo Tokyo Japan Japan Agency for Medical Research and Development AMED Core Research for Evolutionary Science and Technology CREST AMED Tokyo Japan Laboratory for Synthetic Biology RIKEN Quantitative Biology Center QBiC Osaka Japan 4School of Electrical Engineering and Computer Science Science and Engineering Faculty Queensland University of Technology QUT Brisbane Queensland Australia gt Department of Anatomy and Embryology Faculty of Medicine University of Tsukuba Ibaraki Japan PhD Program in Human Biology School of Integrative and Global Majors University of Tsukuba Ibaraki Japan 7These authors contributed equally to this work Correspondence should be addressed to H R U uedah tky umin ac jp Published online 8 October 2015 doi 10 1038 nprot 2015 085 Here we describe a protocol for advanced CUBIC Clear Unobstructed Brain Body Imaging Cocktails and Computational analysis The CUBIC protocol enables simple and efficient organ clearing rapid imaging by light sheet microscopy and quantitative imaging analysis of multiple samples The organ or body is cleared by immersion for
51. nt the NIfTI 1 file in ITK SNAP until orientation Step 8 it matches your raw TIFF stack Note the correct orientation e g RPS and edit the convertTiffFiles py script accordingly line 148 for DV acquired files line 150 for VD acquired files Error message No such Wrong folder name or brain ID in the Check the parameter file for that step and file or directory parameter file ensure that all brain IDs are valid and that all folders exist Error message Command Missing software Make sure that all the required tools are not found installed that they are accessible from the command line and that our two C programs are compiled TIMING Step 1 anesthesia 5 min Step 2 transcardial perfusion and tissue clearing 4 14 d Steps 3 6 imaging of the cleared tissues with the macrozoom LSFM 1 3 h per sample depending on the number of samples color and direction and the required exposure time 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 registration instead of symmetric normalization ANTICIPATED RESULTS The CUBIC pipeline can be used for whole organ or whole body clearing It is simple efficient and reproducible and thus it can be applied to simultaneous multisample clearing in a single tube Fig 2c or a plastic container The procedure can be performed using equipment usually used in a typical biology laboratory Fig 2a d By simple imm
52. on the acquisition direction is needed In preparation for merging NIfTI 1 data in the same color channel of the same brain acquired from opposite directions D V and V D the files need to be aligned Fig 5 In this step a pair of NIfTI 1 data images the structural and signal images is processed The structural image is used to calculate the trans formation parameter which is needed to align the D V image to the V D image This is calculated by the ANTS function of the ANTS software The transformation can allow deformation for example Symmetric Normalization or it can be restricted to aff ine operations only Then using the WarpImageMultiTransform VOL 10 NO 11 2015 1713 npg 2015 Nature America Inc All rights reserved PROTOCOL D V image D V image Structure Dorsal Horizontal Sagittal Coronal 3 Apply to uen 5 p3 signal and A TIFF PNG 5 D gt structural z ue voxel resolution a A E D n Q Register Calculate Ventral 2 160 structural transformation 2 560 7 V D image image e parameter eb Horizontal Sagittal Coronal nite D a 21 Ventral io 2L amp N 5s S II Z T n o a I z N E a gt n Dorsal Structure Aligned NIfTI 1 files signal D V V D NIfTI 1 file signal D V image V D image Last plane Last plane Horizontal Sagittal Coronal Dorsal Use D V m 1 last Use weighed th pl m th plane ee j J D v V D n th plane n th pl
53. opy the provided code to the computer used for the analysis Compile the C files for edge detection g 03 edge_detection_Prewitt cpp o edge_detection_Prewitt and file merging g 03 file_merging cpp o file_merging 8 Construct a 3D NIfTI 1 file for each TIFF stack with the convertTiffFiles py script This takes 2 min per stack and each brain sample corresponds to four stacks two channels two acquisition directions TROUBLESHOOTING 9 Align images of the same brain acquired from opposite directions with the sameBrainAlignment py script For each brain this includes the following registration of the V D acquired nuclear counterstaining image to the D V acquired one alignment of the V D acquired nuclear counterstaining image and alignment of the V D acquired signal channel image The registration takes 1 h 30 min using symmetric normalization or 3 5 min using affine transformations only and both alignments take under 1 min TROUBLESHOOTING 10 Merge the V D acquired and D V acquired images Use edgeDetection py to calculate the n and m thresholds for each brain and channel 4 min per brain and use these results in fileMerging py to merge the files 7 8 min per brain TROUBLESHOOTING 11 Choose one brain to be used as internal reference and align all the other brains to this reference with internalAlignment py for each brain 1 h 30 min for the registration if you are using symmetric normalizat
54. or 227261 Peristaltic pump EYELA model no MP 2000 Fig 3a Intravenous i v injection needle 23 G butterfly type Terumo cat no SV 23CLK e 26 G 1 2 inch injection needle Terumo cat no NN 2613S T shape stopcock Terumo cat no TS TL2K e Disposable syringes 1 10 and 20 ml Terumo cat no SS 01T SS 1010SZ SS 20ESZ e Vacuum desiccator AS ONE cat no VXS 1 5943 01 with vacuum pump ULVAC model no DA 15D Fig 2b e Incubation devices We use hybridization incubator TAITEC model no HB 80 Fig 2c or incubator EYELA model no FMS 1000 or MHS 2000 with a rotator TAITEC model no RT 5 Shaker TAITEC model no Wave PR or MixerXR 36 Fig 2d e Magnetic stirrer for preparing highly viscous reagents ASH model no AMG S or IKEDA Scientific model no IS 20PC Hot stirrer for preparing highly viscous reagents IKA model no C MAG HS10 or Advantec model no SRS710HA e pH meter HORIBA scientific model no LAQUA twin e Positive displacement pipettor Gilson model no Microman M 1000 A CRITICAL We highly recommend this pipettor for measuring the weight of the viscous materials such as Triton X 100 and aminoalcohols e Microwave e Fume hood Imaging microscopy for whole mouse organs Light sheet illumination device with a macrozoom microscope 8 In this study we used the Ultramicroscopes from LaVision BioTec and the MVX ZB10 from Olympus equipped with Olympus MVPLAPO0 63x lens
55. ouse brain Fig 2 and it may need some modifications when other organs are cleared Because handling of whole body samples in the viscous reagent 2 become difficult particularly by causing bubbles the cleared whole body with option C is kept in reagent 1 but not in reagent 2 A Simple immersion protocol for dissected whole brain i Day 0 Perfuse the mice with 10 ml of cold PBS pH 7 4 containing 10 U ml of heparin at 10 ml min to remove the blood from the organ as much as possible Next perfuse 25 ml of cold 4 wt vol PFA pH 7 4 at 10 ml min Dissect the brain and postfix it in 10 ml of 4 wt vol PFA with shaking at 4 C for 18 24 h A CRITICAL STEP Cooling of PBS and 4 wt vol PFA on ice is important for successful perfusion Muscle stiffness after perfusion is a good indicator of successful perfusion Residual blood in the mouse brain increases auto fluorescence especially in the green laser excitation The pH value of PFA is also crucial for efficient clearing and lower autofluorescence Overfixation causes both lower clearing efficiency and autofluorescence TROUBLESHOOTING 11 Day 1 Wash the tissue sample with 10 ml of PBS 0 01 wt vol sodium azide for at least 2 h twice at room temperature to remove the remaining PFA Fig 2e PAUSE POINT The fixed organs can be stored First immerse them in 10 ml of 20 30 wt vol sucrose in PBS per organ with shaking at 4 C for 1 2 d When the samples sink to the bottom put them
56. ransient swelling during the procedure In addition CUBIC has not yet been optimized to fully clear bone and melanin pigments Table 1 However this issue remains unaddressed by other NATURE PROTOCOLS VOL 10 NO 11 2015 1715 npg 2015 Nature America Inc All rights reserved PROTOCOL TABLE 1 Tissues to which CUBIC has been successfully applied Tissue Clearing protocol Reagent before imaging Clearing efficiency Mouse whole body 19 CB perfusion Mouse brain18 19 Simple immersion or CB perfusion Marmoset hemisphere 48 Simple immersion Mouse heart 9 Simple immersion or CB perfusion Mouse lung 9 Simple immersion or CB perfusion Mouse spleen 9 Simple immersion or CB perfusion Mouse liver Simple immersion or CB perfusion Mouse stomach 9 Simple immersion or CB perfusion Mouse intestine Simple immersion or CB perfusion Mouse kidney 9 Simple immersion or CB perfusion Mouse pancreas Simple immersion or CB perfusion Mouse lymph node Simple immersion or CB perfusion Mouse muscle Simple immersion or CB perfusion Mouse skin 19 Simple immersion or CB perfusion Mouse bone CB perfusion Tissues with melanin eye and hair 19 CB perfusion Reagent 15 Good Reagent 2 Good Reagent 2 Good Reagent 2 Good Reagent 2 Good Reagent 2 Good Reagent 2 Good Reagent 2 Good Reagent 2 Good Reagent 2 Good Reagent 2 or reagent 1 Good Reagent 2 Good Reagent 2 Good Reagent 2 Good Reagent 1 Partially cleared but nee
57. re a live image with an appropriate laser and filter pair to adjust the focus and the sample position to the center A whole mouse brain image can be captured using 1 6x to 2x zoom on the MVX ZB10 A CRITICAL STEP To avoid fluorescence quenching laser power should be weakened during the adjustment of position and focus 6 Set the focus position of the illumination sheet z range in the case of whole brain typically 7 mm in total Z step size typically 10 um per step laser power typically 70 100 and exposure time typically 50 ms 1 s for each side Each plane should be illuminated from both the right and left sides and a merged image with maximum intensity should be saved The exposure times should be adjusted according to the fluorescent signal intensities of each sample After all parameters are adjusted start image acquisition When multicolor images are needed repeat the image acquisition procedure with the same z range and readjust the laser power and exposure time To collect both D V and V D data sets manually flip the sample upside down and acquire the image again for the opposite orientation A CRITICAL STEP To take images with a high signal to noise ratio it is important to use bright fluorescent proteins and chemicals Because 700 images 11 MB per image total 7 GB are acquired per color and direction each image should be saved to a secondary storage location e g a hard drive during acquisition Each stack one color
58. ront Neural Circuits 7 65 2013 Murphy K et al Evaluation of registration methods on thoracic CT the EMPIRE10 challenge IEEE Trans Med Imaging 30 1901 1920 2011 Yushkevich P A et al User guided 3D active contour segmentation of anatomical structures significantly improved efficiency and reliability Neuroimage 31 1116 1128 2006 Eguchi M amp Yamaguchi S In vivo and in vitro visualization of gene expression dynamics over extensive areas of the brain Neuroimage 44 1274 1283 2009 Prewitt J M S Object enhancement and extraction In Picture Processing and Psychopictorics eds Lipkin B S amp Rosenfeld A 75 149 Academic Press 1970 Jenkinson M Beckmann C F Behrens T E Woolrich M W amp Smith S M Fsl Neuroimage 62 782 790 2012 Lein E S et al Genome wide atlas of gene expression in the adult mouse brain Nature 445 168 176 2007 Alnuami A A Zeedi B Qadri S M amp Ashraf S S Oxyradical induced GFP damage and loss of fluorescence Int J Biol Macromol 43 182 186 2008 Steinke H amp Wolff W A modified Spalteholz technique with preservation of the histology Ann Anat 183 91 95 2001 Faisal A A White J A amp Laughlin S B Ion channel noise places limits on the miniaturization of the brain s wiring Curr Biol 15 1143 1149 2005 Richards K L et al Hippocampal volume and cell density changes in a mouse model of human genetic epilepsy Neuro
59. ructures in the 3D images for comparison of Langerhans islets in normal and diabetic pancreases For more complicated analyses we implemented image informatics often used in functional MRI fMRI analysis 8 First structural images via counterstaining were registered to a reference brain to calculate transformation parameters Next the transformation parameters were applied to the corresponding signal images transgenes etc for alignment These aligned images could be merged with each other to calculate signal subtraction between samples This analysis was performed with open source software such as Advanced Normalization Tools ANTS 24 and ITK SNAP but it requires advanced informat ics and computer science skills For the user s convenience we provide an easier analysis pipeline with prepared scripts in this manuscript As an example of the comparative analysis we demonstrate 3D image analysis of Arc dVenus Tg mouse brains with or without light stimulation and calculate the signal subtrac tion 8 26 The final subtraction data clearly depict regions and cells in the whole brain in which neurons responded to the light stimuli Such direct comparative analysis by using whole brain fluorescent 3D images was first reported using CUBIC informat ics 8 CUBIC informatics enables quantitative identification of stimulus or timing dependent neural activities and it will help delineate structural abnormalities in disease samples at the whole
60. rument or by using higher NA objective lenses on other confocal and two photon microscopes We implemented an image informatics method originally used in fMRI analysis to compare different brains In this pipeline the acquired data sets are preprocessed by merging D V and V D to make sure that the resulting images are clear throughout the z stack Fig 5 Then signal subtraction between samples is calculated Fig 6 Here we show an example of Arc dVenus Tg mouse brains with or without light stimuli4 2 Fig 6a The raw images were preprocessed aligned and normalized and then subtraction of Venus signal was calculated to visualize light responsive regions at the whole brain scale Fig 6b d These calculations could be achieved by whole organ counterstaining in the CUBIC clearing protocol Note that the final resolution of images in this process is downscaled and it is more difficult to achieve single cell resolution throughout the entire imaging field because of the current software limitations This will be addressed in future studies 1726 VOL 10 NO 11 2015 NATURE PROTOCOLS npg 2015 Nature America Inc All rights reserved PROTOCOL In summary CUBIC provides a platform for comprehensive cell detection and analysis across whole organs and the body Possible applications of CUBIC will be for whole organ samples from multiple conditions or time points detection of aberrant 3D morphological changes in diseased tissues or a s
61. s e g brain ventricles after reagent 2 Tissue damage when freezing Floating sample during imaging 6 imaging Bubble on the surface High autofluorescence Weak or nondetectable fluorescence Poor z resolution Degradation of urea Insufficient degassing Rotation during reagent 2 Keeping the sample at 20 C to 30 C which possibly causes growth of water crystals inside the tissue Insufficient replacement by sucrose solution Use of reagent 2 rather than the immersion oil mix Insufficient removal of the reagent 2 Insufficient fluorescent signal Alkaline pH of PFA Use of an aged animal Insufficient fluorescence signals Signal decrease in CB perfusion Temperature during clearing Inappropriate setting of microscopy Inappropriate setting of the light sheet focus Replace with fresh medium and avoid too much heating during preparation of reagents Degas the reagent 2 after preparation degas the sample in a limited volume of PBS before the reagent 2 incubation Incubate in the reagent 2 with gentle Shaking rather than rotation Stock the sample in 0 C T compound at 80 C thaw the sample gradually at room temperature Increase incubation time in the sucrose solution i e until organs sink Immerse the sample in oil Remove excess reagent 2 before immersion into the oil mix remove bubbles in the oil with a needle or a tapered forceps Select a bright fluorescent protein e
62. stitutions were performed with Imaris software as in Figure 4 and they are shown as views from the dorsal and ventral side in D V and 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 Tokyo and all animals were cared for in accordance with institutional guidelines Adapted with permission from ref 18 function in ANTS we apply the transformation parameters to align both structural and signal images of the D V image to the V D image Next we merge the aligned images in order to ensure sharp ness throughout the resulting 3D image Fig 5 To do so we use the Prewitt operator to calculate the edge content of the two images as a proxy for image sharpness This is used to define two thresholds n and m so that slices below n only come from the V D image slices above m only come from the D V image and slices in between the two are a linear combination of the two images We then take the image pairs and the thresholds and we create the merged NIfTI 1 image In order to access the values of indi vidual pixels in the images we use the fsl2ascii and fslascii2img functions of FSL28 The steps above produce a pair of D V V D composite NIfTI 1 data for both structural and signal images 1714 VOL 10 NO 11 2015 NATURE PROTOCOLS Next to facilitate analysis across different brain data sets we align these merged
63. thanol triethanolamine Wako cat no 145 05605 e Urea Nacalai Tesque cat no 35904 45 or 35907 15 Polyethylene glycol PEG mono p isooctylphenyl ether Triton X 100 A CRITICAL In our first CUBIC paper we mentioned that the quality of Triton X 100 product seems crucial for preserving fluorescence signals and we recommended a product from Nacalai Tesque cat no 25987 85 However that product has been discontinued We further checked the same chemical from Nacalai Tesque cat no 12967 45 Sigma Aldrich cat no X100 T9284 T8787 T8532 or Tokyo Chemical Industry cat no P0873 and none of them caused fluorescence quenching in the final reagent 1 recipe at least in the short term and thus they can be used as substitutes e Sucrose Nacalai Tesque cat no 30403 55 or 30404 45 e Sodium azide Nacalai Tesque cat no 31208 82 CAUTION Sodium azide is highly toxic e SYTO 16 Life Technologies cat no 7578 e Propidium iodide PI Life Techologies cat no P21493 e Silicon oil TSF4300 Momentive RI 1 498 NATURE PROTOCOLS VOL 10 NO 11 2015 1717 npg 2015 Nature America Inc All rights reserved PROTOCOL e Mineral oil RI 1 467 Sigma Aldrich cat no M8410 EQUIPMENT Tubes 5 ml Eppendorf cat no 0030119 401SG e Conical tubes 15 ml Corning cat no 352096 or 188271 e Conical tubes 30 ml Sarstedt cat no 60 544 e Conical tubes 50 ml Corning cat no 352070
64. the pH of PFA to 7 4 Stop postfixation within 24 h Incubate it longer in reagent 1 a few days more exchange reagent 1 more frequently every day rather than every 2 d shake or rotate appropriately Use a younger animal Use reagent 1 rather than reagent 2 for the final clearing reagent e g pancreas becomes clearer in reagent 1 see Fig 3 If the organ has shrunk too much decrease the concentration of PBS in the 1 2 reagent 2 e g mix 1 1 2 of dH 0 PBS reagent 2 rather than 1 1 mix of PBS reagent 2 use of 1 2 reagent 2 water instead of PBS causes swelling Try to incubate samples at room temperature during reagent 2 steps takes more time Sufficiently incubate the organ in the reagent for complete replacement 24 h Organs from an infant or juvenile animal tend to shrink more in reagent 2 and need less incubation time during reagent 1 treatment Try the simple immersion protocol Make sure that no precipitate exists in the prepared stocked reagent 2 Increase reagent 1 treatment time Use more reagent 2 and exchange the reagent one or two more times 1724 VOL 10 NO 11 2015 NATURE PROTOCOLS continued npg 2015 Nature America Inc All rights reserved TABLE 3 Troubleshooting table continued PROTOCOL Step Problem Possible reason Solution Step 2A iv viii 2B v ix 2C ii1 clearing Ammonia smell during clearing Bubbles on inside structures of the organ
65. ton microscopy 8 because the clearing performance surpasses that of some of the other clearing methods developed for these imaging purposes 9 Microscope vendors have released objective lenses for deep tissue imaging e g Olympus XLPLNIOXSVMP 10x 0 6 working distance WD 8 mm n adjustable refractive index range 1 33 1 52 and Reagent 2 filter for red to far red fluorescence The size of the acquired image per pixel is dependent on the zoom of the microscope such that one pixel is 5 2 x 5 2 um at 2X zoom and one pixel is 6 5 X 6 5 um at 1 6X zoom These pixel sizes are sufficient for detect ing signal from single cells in regions such as the cerebral cortex or even from subcellular structures when they were sparsely labeled as discussed below Fig 4b f and Supplementary Video 1 Users can also use higher magnified zoom values 6 3x if finer resolution is needed Z step size is selected according to the thickness of the laser sheet We typically select the thinnest sheet of the used LSFM and set 10 um as the z step size For further image analysis both signal images e g transgenes and Figure 3 Procedure of the CB perfusion protocol a Surgical setup for CB perfusion The transcardiac perfusion line is connected to heparin PBS for flushing the blood 4 wt vol PFA on ice with peristaltic pump for fixation PBS for flushing PFA and 1 2 diluted reagent 1 for accelerative clearing through the vascular system b
66. ttp sourceforge net projects advants files ANTS ANTS_Latest FSL http fsl fmrib ox ac uk fsl fslwiki FslInstallation REAGENT SETUP PBS Prepare PBS according to the vendor s manual When using PBS tablets Takara cat no T9181 the tablet is dissolved in 1 liter of dH O When PBS 0 01 wt vol sodium azide is prepared directly dissolve 0 1 g of sodium azide in 1 liter of PBS This solution can be stored at room temperature 18 25 C for several months 1718 VOL 10 NO 11 2015 NATURE PROTOCOLS PFA solution To prepare 4 wt vol PFA in PBS dissolve 40 g of PFA in 1 liter total of PBS Heat the PBS solution avoid boiling and add PFA pow der and a 1 500 1 1 000 volume of 1 N NaOH to help dissolving PFA faster After complete dissolution adjust the pH to 7 4 using HCI PFA can be stored at 20 C until use for several months CAUTION PFA is a very toxic reagent Avoid inhalation or contact with skin and eyes Use a draft chamber proper gloves and a mask to handle PFA HCl and NaOH A CRITICAL The pH value of PFA is a crucial factor for an efficient clearing with lower autofluorescence 80 wt Quadrol Quadrol is a highly viscous liquid and it can be used as an 80 wt working solution In this case add 125 g of dH O to 500 g of Quadrol reagent bottle and stir it for at least 30 min Store the solution at room temperature for up to 1 month A CRITICAL Quadrol is highly viscous and we use wt rather than
67. ture and because the gradual exchange from PBS through 1 2 reagent 2 PBS to salt free reagent 2 does not affect the final transparency Before clearing 1 2 reagent 1 water is not a problem A CRITICAL Make sure that there is no precipitation in the reagent 2 solution stock before use The precipitation in the stock can be dissolved again by mild heating in a microwave Insufficient degassing may cause bubbles around and inside the tissue during reagent 2 treatment Immersion oil mix Mix 1 1 of TSF4300 and mineral oil completely with a stirrer and degas it with a vacuum desiccator 0 1 MPa 30 min Fig 2b before use The oil mix can be repeatedly used for imaging by filtering contaminants clearing reagents etc Its RI is 1 48 1 49 a compa rable RI of reagent 2 The oil can be wiped out with 70 vol vol EtOH A CRITICAL The mix ratio can be optionally changed because the best RI matching may be different between organs npg 2015 Nature America Inc All rights reserved PROTOCOL EQUIPMENT SETUP reproducible surgeries Thus we devised a surgical instrument with a combi Surgical setup for the CB perfusion protocol Typical surgical setup for nation of T shape stopcocks a peristaltic pump connected with silicon tube an CB perfusion is depicted in Figure 3a In the protocol the adult mouse is i v injection needle and a disposable syringe as shown in Figure 3a perfused with four solutions 20 30 ml of cold heparin PBS 150 ml
68. wt vol or vol vol for convenience ScaleCUBIC 1 reagent 1 Reagent 1 is a mixture of urea 25 wt final concentration Quadrol 25 wt final concentration Triton X 100 15 wt final concentration and dH O For example to prepare 500 g of reagent 1 solution mix 125 g of urea and 156 g of 80 wt Quadrol in 144 g of dH O with a hot stirrer After complete dissolution further stir the mixture at room temperature and add 75 g of Triton X 100 Finally degas the reagent with a vacuum desiccator 0 1 MPa 30 min Fig 2b The reagent can be stored at room temperature for up to 1 month Prepare 1 2 reagent 1 by mixing 1 1 of reagent 1 and dH O A CRITICAL Quadrol and Triton X 100 are viscous and therefore we use wt rather than wt vol or vol vol for convenience Reagent 1 should not be prepared with PBS because salt contamination decreases the clearing performance We usually prepare the stock of reagent 1 for convenience However longer storage may cause quenching of fluorescent signals Avoid boiling during the preparation see also TROUBLESHOOTING section ScaleCUBIC 2 reagent 2 Reagent 2 is a mixture of urea 25 wt final concentration sucrose 50 wt final concentration triethanolamine 10 wt final concentration and dH O To prepare 50 g of reagent 2 solution dissolve 12 5 g of urea and 25 g of sucrose in 7 5 g of dH O in a microwave using a hot stirrer Fig 2a After complete dissolution typically it takes 10 15

Download Pdf Manuals

Related Search

Related Contents

Comersus Sophisticated Cart User's Guide  Craftsman 917.28955 Lawn Mower User Manual  Feux de route – avril 2010  König CSGCGALS4MWH mobile phone case    Miele DA 422-4 Ext  User Manual Draco KVM Extender Model: K477 Series  Casio Watch 3232 User's Manual  Contour for Windows User Guide  - Radiocrafts AS  

Copyright © All rights reserved. Failed to retrieve file