Abstract
Plant histology and imaging traditionally involve the transformation of tissues into thin sections to minimize light scatter in opaque material, allowing optical clarity and high-resolution microscopy. Recently, new techniques in 3D tissue clearing, including PEA-CLARITY, have been developed to minimize light scatter within intact, whole samples. These techniques can achieve equivalent microscopic resolution to that of thin section imaging with the added benefit of maintaining the original 3D structure and position of biomolecules of interest. Furthermore, PEA-CLARITY is compatible with standard stains and immunohistochemistry, allowing molecular interrogation of intact, 3D tissues. This chapter outlines the current methods available for 3D histology in plants and details the materials, equipment, reagents, and procedure for the PEA-CLARITY technique.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Furbank RT (2016) Walking the C4 pathway: past, present, and future. J Exp Bot 67(14):4057–4066. https://doi.org/10.1093/jxb/erw161
Palmer WM, Martin AP, Flynn JR, Reed SL, White RG, Furbank RT, Grof CPL (2015) PEA-CLARITY: 3D molecular imaging of whole plant organs. Sci Rep 5:13492. https://doi.org/10.1038/srep13492
Becker K, Jahrling N, Saghafi S, Weiler R, Dodt HU (2012) Chemical clearing and dehydration of GFP expressing mouse brains. PLoS One 7(3):e33916. https://doi.org/10.1371/journal.pone.0033916
Hama H, Kurokawa H, Kawano H, Ando R, Shimogori T, Noda H, Fukami K, Sakaue-Sawano A, Miyawaki A (2011) Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci 14(11):1481–1488. https://doi.org/10.1038/nn.2928
Hama H, Hioki H, Namiki K, Hoshida T, Kurokawa H, Ishidate F, Kaneko T, Akagi T, Saito T, Saido T, Miyawaki A (2015) ScaleS: an optical clearing palette for biological imaging. Nat Neurosci 18(10):1518–1529. https://doi.org/10.1038/nn.4107
Kuwajima T, Sitko AA, Bhansali P, Jurgens C, Guido W, Mason C (2013) ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. Development 140(6):1364–1368. https://doi.org/10.1242/dev.091844
Erturk A, Becker K, Jahrling N, Mauch CP, Hojer CD, Egen JG, Hellal F, Bradke F, Sheng M, Dodt HU (2012) Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat Protoc 7(11):1983–1995. https://doi.org/10.1038/nprot.2012.119
Erturk A, Bradke F (2013) High-resolution imaging of entire organs by 3-dimensional imaging of solvent cleared organs (3DISCO). Exp Neurol 242:57–64. https://doi.org/10.1016/j.expneurol.2012.10.018
Renier N, Wu Z, Simon DJ, Yang J, Ariel P, Tessier-Lavigne M (2014) iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159(4):896–910. https://doi.org/10.1016/j.cell.2014.10.010
Ke MT, Fujimoto S, Imai T (2013) SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat Neurosci 16(8):1154–1161. https://doi.org/10.1038/nn.3447
Ke MT, Imai T (2014) Optical clearing of fixed brain samples using SeeDB. Curr Protoc Neurosci 66:Unit 2.22. https://doi.org/10.1002/0471142301.ns0222s66
Susaki EA, Tainaka K, Perrin D, 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 (2014) Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157(3):726–739. https://doi.org/10.1016/j.cell.2014.03.042
Susaki EA, Tainaka K, Perrin D, Yukinaga H, Kuno A, Ueda HR (2015) Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nat Protoc 10(11):1709–1727. https://doi.org/10.1038/nprot.2015.085
Chung K, Wallace J, Kim SY, Kalyanasundaram S, Andalman AS, Davidson TJ, Mirzabekov JJ, Zalocusky KA, Mattis J, Denisin AK, Pak S, Bernstein H, Ramakrishnan C, Grosenick L, Gradinaru V, Deisseroth K (2013) Structural and molecular interrogation of intact biological systems. Nature 497(7449):332–337. https://doi.org/10.1038/nature12107
Tomer R, Ye L, Hsueh B, Deisseroth K (2014) Advanced CLARITY for rapid and high-resolution imaging of intact tissues. Nat Protoc 9(7):1682–1697. https://doi.org/10.1038/nprot.2014.123
Lee H, Park JH, Seo I, Park SH, Kim S (2014) Improved application of the electrophoretic tissue clearing technology, CLARITY, to intact solid organs including brain, pancreas, liver, kidney, lung, and intestine. BMC Dev Biol 14:48. https://doi.org/10.1186/s12861-014-0048-3
Epp JR, Niibori Y, (Liz) Hsiang H-L, Mercaldo V, Deisseroth K, Josselyn SA, Frankland PW (2015) Optimization of CLARITY for clearing whole-brain and other intact organs. eNeuro 2:3. https://doi.org/10.1523/eneuro.0022-15.2015
Zheng H, Rinaman L (2015) Simplified CLARITY for visualizing immunofluorescence labeling in the developing rat brain. Brain Struct Funct 221(4):2375–2383. https://doi.org/10.1007/s00429-015-1020-0
Kim SY, Chung K, Deisseroth K (2013) Light microscopy mapping of connections in the intact brain. Trends Cogn Sci 17(12):596–599. https://doi.org/10.1016/j.tics.2013.10.005
Yang B, Treweek JB, Kulkarni RP, Deverman BE, Chen CK, Lubeck E, Shah S, Cai L, Gradinaru V (2014) Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell 158(4):945–958. https://doi.org/10.1016/j.cell.2014.07.017
Dodt HU, Leischner U, Schierloh A, Jahrling N, Mauch CP, Deininger K, Deussing JM, Eder M, Zieglgansberger W, Becker K (2007) Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat Meth 4(4):331–336. https://doi.org/10.1038/nmeth1036
Warner CA, Biedrzycki M, Jacobs SS, Wisser RJ, Caplan JL, Sherrier DJ (2014) An optical clearing technique for plant tissues allowing deep imaging and compatible with fluorescence microscopy. Plant Physiol 166(4):1684–1687. https://doi.org/10.1104/pp.114.244673
Kurihara D, Mizuta Y, Sato Y, Higashiyama T (2015) ClearSee: a rapid optical clearing reagent for whole-plant fluorescence imaging. Development 142(23):4168–4179. https://doi.org/10.1242/dev.127613
Hasegawa J, Sakamoto Y, Nakagami S, Aida M, Sawa S, Matsunaga S (2016) Three-dimensional imaging of plant organs using a simple and rapid transparency technique. Plant Cell Physiol 57(3):462–472. https://doi.org/10.1093/pcp/pcw027
Truernit E, Bauby H, Dubreucq B, Grandjean O, Runions J, Barthélémy J, Palauqui J-C (2008) High-resolution whole-mount imaging of three-dimensional tissue organization and gene expression enables the study of phloem development and structure in Arabidopsis. Plant Cell 20(6):1494–1503. https://doi.org/10.1105/tpc.107.056069
Richardson DS, Lichtman JW (2015) Clarifying tissue clearing. Cell 162(2):246–257. https://doi.org/10.1016/j.cell.2015.06.067
Timmers AC (2016) Light microscopy of whole plant organs. J Microsc 263(2):165–170. https://doi.org/10.1111/jmi.12394
Treweek JB, Chan KY, Flytzanis NC, Yang B, Deverman BE, Greenbaum A, Lignell A, Xiao C, Cai L, Ladinsky MS, Bjorkman PJ, Fowlkes CC, Gradinaru V (2015) Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping. Nat Protoc 10(11):1860–1896. https://doi.org/10.1038/nprot.2015.122
Danila FR, Quick WP, White RG, Furbank RT, von Caemmerer S (2016) The metabolite pathway between bundle sheath and mesophyll: quantification of plasmodesmata in leaves of C3 and C4 monocots. Plant Cell 28(6):1461–1471. https://doi.org/10.1105/tpc.16.00155
Liu AK, Hurry ME, Ng OT, DeFelice J, Lai HM, Pearce RK, Wong GT, Chang RC, Gentleman SM (2015) Bringing CLARITY to the human brain: visualisation of Lewy pathology in three-dimensions. Neuropathol Appl Neurobiol 42(6):573–587. https://doi.org/10.1111/nan.12293
Kim SY, Cho JH, Murray E, Bakh N, Choi H, Ohn K, Ruelas L, Hubbert A, McCue M, Vassallo SL, Keller PJ, Chung K (2015) Stochastic electrotransport selectively enhances the transport of highly electromobile molecules. Proc Natl Acad Sci USA 112(46):E6274–E6283. https://doi.org/10.1073/pnas.1510133112
Li J, Czajkowsky DM, Li X, Shao Z (2015) Fast immuno-labeling by electrophoretically driven infiltration for intact tissue imaging. Sci Rep 5:10640. https://doi.org/10.1038/srep10640
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Palmer, W.M. et al. (2018). 3D Clearing and Molecular Labeling in Plant Tissues. In: Covshoff, S. (eds) Photosynthesis. Methods in Molecular Biology, vol 1770. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7786-4_17
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7786-4_17
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7785-7
Online ISBN: 978-1-4939-7786-4
eBook Packages: Springer Protocols