uDISCO for Whole Organs and Organisms - A New Method of Large Sample Imaging
In this webinar Dr. Ali Ertürk presents his outreaching uDISCO results as they have been published in Nature Methods. The provision of this paramount technologys will be the basis for a new large sample imaging category providing a high density of information on intact organs and organisms.
Download Insights Tissue Clearing.pdf
Insights tissue clearing
Published on Imaging & Microscopy (http://www.imaging-git.com)
Mar. 16, 2017
Today, imaging entire cleared samples is a vital necessity to understand biological systems and processes. Several protocols have been published describing different clearing protocols. The article will address some of the frequently used techniques.
Frequently asked questions
Can I use DAPI? - DAPI is excited with 405 nm. This is a pretty short wavelength which is scattered and absorbed by the tissue. It is absolutely not suitable for deep imaging. One should try DRAQ5™ or TO-PRO®.
My sample contains GFP, which clearing should I try? - You may try uDISCO, ethyl cinnamate, CUBIC, CLARITY, FluoClearBABB or iDISCO. iDISCO maybe of advantage due to the fact that you can label it with a far red dye for a better penetration of the excitation wavelength.
The tissue I am working with is very dense. What clearing is capable to get it transparent? - Users working with dense samples often use BABB, 3DISCO or iDISCO+.
The sample I would like to image is colorized (e.g.: liver). How can I cope with colorization? - You may try iDISCO+ or CUBIC-perfusion protocol. These protocols were designed to decolorize sample tissue.
The sample I would like to image is very small. How can I mount it? - If you work with organic solvents, you may prepare an empty but cleared cube with 1% low melting agarose. After clearing the cube you make a small cut on top of it where you insert your cleared and stained sample. One can also directly embed sample into agarose and do clearing of the sample within the agarose. In that case extend dehydration (100% over night) so that the agarose is completely dry. If there is only a small amount of water remaining in the cube it will start shrinking and it will get milky as soon as it is transferred to the clearing solution. If you work with aqueous buffers you may insert sample into a FEP tube or a cube of Phytagel™ (Sigma-Aldrich Co. LLC.).
I cleared my sample but it is still not transparent and shows a strong background. -If you have applied an aqueous buffer based protocol you may want to try an organic solvent protocol. Methanol treatment might reduce the background. Please consider to dehydrate the sample completely. If used dehydration solution (tetrahydrofuran, ethanol or methanol) is stored in a bottle which is opened frequently, there already will be a remarkable amount of water in that solution due to hygroscopic characteristic of the solvent.
I cleared my sample using the CLARITY protocol. What kind of solution should I use for imaging? - Some CLARITY imaging solutions are very expensive. To reduce costs you may try sRIMS, 63% thiodieethanol or 80% glycerol. The iDISCO protocol resulted in a sample with a strong labelling only on the surface. What should I do? - Increase the dilution of the antibody and find further information at: http://idisco.info/
My sample is very soft. How can I mount it for the image acquisition? - You may try some glue. Loctite Professional®, krazy glue® or picodent® are even stable in organic solvents.
I would like to label my sample with fluorophores, which are suitable? - Every fluorophore with an excitation maximum between 500 nm and 785 nm is suitable. The extended wavelength can penetrate tissue better. DiI or DiAsp are not compatible with most clearing protocols. Membranes as binding site for these dyes are often altered or affected by the clearing. For example users have good results with using Alexa Fluor® 647, Alexa Fluor® 750 (Thermo Fisher Scientific Inc.), ATTO 647 (ATTO-TEC GmbH), VivoTag® 680, VivoTag® 750 (PerkinElmer Inc.), Cy7, or IRDye ®(LI-COR, Inc.).
 (Chung & Deisseroth, 2013) Chung K, Deisseroth K: CLARITY for mapping the nervous system, Nat Methods (2013) doi: 10.1038/nmeth.2481
 (Yang et al., 2014) Yang B, Treweek JB, Kulkarni RP, Deverman BE, Chen CK, Lubeck E, Shah S, Cai L, Gradinaru V: Single-cell phenotyping within transparent intact tissue through whole-body clearing, Cell (2014) doi: 10.1016/j.cell.2014.07.017
 (Kim et al., 2015) Kim SY, Cho JH, Murray E, Bakh N, Choi H, Ohn K, Ruelas L, Hubbert A, McCue M, Vassallo SL, Keller PJ, Chung K: Stochastic electrotransport selectively enhances the transport of highly electromobile molecules, Proc Natl Acad Sci U S A (2015) doi: 10.1073/pnas.1510133112
 (Murray et al., 2015) Murray E, Cho JH, Goodwin D, Ku T, Swaney J, Kim SY, Choi H, Park YG, Park JY, Hubbert A, McCue M, Vassallo S, Bakh N, Frosch MP, Wedeen VJ, Seung HS, Chung K: Simple, scalable proteomic imaging for high-dimensional profiling of intact systems, Cell (2015) doi: 10.1016/j.cell.2015.11.025.
 (Sylwestrak et al., 2016) Sylwestrak EL, Rajasethupathy P, Wright MA, Jaffe A, Deisseroth K: Multiplexed Intact-Tissue Transcriptional Analysis at Cellular Resolution, Cell (2016) doi: 10.1016/j.cell.2016.01.038
 (Lee et al., 2016) Lee E, Choi J,Jo Y,Kim JY, Jang YJ, Lee HM, Kim SY, Lee HJ, Cho K,Jung N, Hur EM, Jeong SJ, Moon C, Choe Y, Rhyu IJ, Kim H, Sun W: ACT-PRESTO: Rapid and consistent tissue clearing and labeling method for 3-dimensional (3D) imaging, Sci Rep. (2016) doi: 10.1038/srep18631
 (Susaki et al., 2014) Susaki EA, Tainaka K, Perrin, Kishino F, Tawara T, Watanabe TM, Yokoyama C, Onoe H, Eguchi M, Yamaguchi S, Abe T, Kiyonari H, Shimizu Y, Miyawaki A, Yokota H, Ueda HR: Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis, Cell (2014) doi: 10.1016/j.cell.2014.03.042
 (Susaki et al., 2015) Susaki EA, Tainaka K, Perrin D, Yukinaga H, Kuno A, Ueda HR.: Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging, Nat Protoc (11):1709-27 (2015) doi: 10.1038/nprot.2015.085
 (Hama et al., 2011) Hama H1, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A: Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain, Nature neuroscience (2011) doi: 10.1038/nn.2928
 (Hama et al., 2015) Hama H, Hioki H, Namiki K, Hoshida T, Kurokawa H, Ishidate F, Kaneko T, Akagi T, Saito T, Saido T, Miyawaki A: ScaleS: an optical clearing palette for biological imaging, Nat Neurosci (2015) doi: 10.1038/nn.
 (Ke et al., 2013) Ke MT, Fujimoto S, Imai T: SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nature neuroscience (2013) doi: 10.1038/nn.3447
 (Hou et al., 2015) Hou B, Zhang D, Zhao S, Wei M, Yang Z, Wang S, Wang J, Zhang X, Liu B, Fan L, Li Y, Qiu Z, Zhang C, Jiang T: Scalable and DiI-compatible optical clearance of the mammalian brain, Frontiers in neuroanatomy (2015) doi: 10.3389/fnana.2015.00019
 (Ke et al. 2016) Ke MT, Nakai Y, Fujimoto S, Takayama R, Yoshida S, Kitajima TS, Sato M, Imai T: Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent, Cell Rep. (2016) doi: 10.1016/j.celrep.2016.02.057
 (Kuwajima et al., 2013) Kuwajima T, Sitko AA, Bhansali P, Jurgens C, Guido W, Mason C: ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue, Development. (2013) doi: 10.1242/dev.091844
 (Ertürk et al., 2012) Ali Ertürk, Klaus Becker, Nina Jährling, Christoph P Mauch, Caroline D Hojer, Jackson G Egen, Farida Hellal, Frank Bradke, Morgan Sheng & Hans-Ulrich Dodt: Three-dimensional imaging of solvent-cleared organs using 3DISCO, Nature protocols (2012) doi: 10.1038/nprot.2012.119
 (Renier et al., 2014) Renier N, Wu Z, Simon D.J, Yang J, Ariel P, Tessier-Lavigne M: iDISCO: A Simple, Rapid Method to Immunolabel Large Tissue Samples for Volume Imaging, Cell (2014) doi: 10.1016/j.cell.2014.10.010
 (Schwarz et al., 2015) Schwarz MK, Scherbarth A, Sprengel R, Engelhardt J, Theer P, Giese G: Fluorescent-Protein Stabilization and High-Resolution Imaging of Cleared, Intact Mouse Brains, PLoS One (2015) doi: 10.1371/journal.pone.0124650
 (Pan, Ertürk et al, 2016) Pan C, Cai R, Quacquarelli FP, Ghasemigharagoz A, Lourbopoulos A, Matryba P, Plesnila N, Dichgans M, Hellal F, Ertürk A: Shrinkage-mediated imaging of entire organs and organisms using uDISCO, Nat Methods (2016) doi:10.1038/nmeth.3964
 (Klingenberg et al., 2016) Klingberg A, Hasenberg A, Ludwig-Portugall I, Medyukhina A, Männ L, Brenzel A, Engel DR, Figge MT, Kurts C, Gunzer M.: Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy, J Am Soc Nephrol. (2016) doi:10.1681/ASN.2016020232