Abstract
Optical tissue clearing enables the precise imaging of cellular and subcellular structures in whole organs and tissues without the need for physical tissue sectioning. By combining tissue clearing with confocal or lightsheet microscopy, 3D images can be generated of entire specimens for visualization and large-scale data analysis. Here we demonstrate two different passive tissue clearing techniques that are compatible with immunofluorescent staining and lightsheet microscopy: PACT, an aqueous hydrogel–based clearing method, and iDISCO+, an organic solvent–based clearing method.
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References
Ariel P (2017) A beginner's guide to tissue clearing. Int J Biochem Cell Biol 84:35–39. https://doi.org/10.1016/j.biocel.2016.12.009
Matryba P, Kaczmarek L, Gołąb J (2019) Advances in ex situ tissue optical clearing. Laser Photonics Rev 13(8). https://doi.org/10.1002/lpor.201800292
Ueda HR, Erturk A, Chung K, Gradinaru V, Chedotal A, Tomancak P, Keller PJ (2020) Tissue clearing and its applications in neuroscience. Nat Rev Neurosci 21(2):61–79. https://doi.org/10.1038/s41583-019-0250-1
Richardson DS, Lichtman JW (2015) Clarifying tissue clearing. Cell 162(2):246–257. https://doi.org/10.1016/j.cell.2015.06.067
Susaki EA, Ueda HR (2016) Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals. Cell Chem Biol 23(1):137–157. https://doi.org/10.1016/j.chembiol.2015.11.009
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
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
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
Tainaka K, Murakami TC, Susaki EA, Shimizu C, Saito R, Takahashi K, Hayashi-Takagi A, Sekiya H, Arima Y, Nojima S, Ikemura M, Ushiku T, Shimizu Y, Murakami M, Tanaka KF, Iino M, Kasai H, Sasaoka T, Kobayashi K, Miyazono K, Morii E, Isa T, Fukayama M, Kakita A, Ueda HR (2018) Chemical landscape for tissue clearing based on hydrophilic reagents. Cell Rep 24(8):2196–2210. e2199. https://doi.org/10.1016/j.celrep.2018.07.056
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
Renier N, Adams EL, Kirst C, Wu Z, Azevedo R, Kohl J, Autry AE, Kadiri L, Umadevi Venkataraju K, Zhou Y, Wang VX, Tang CY, Olsen O, Dulac C, Osten P, Tessier-Lavigne M (2016) Mapping of brain activity by automated volume analysis of immediate early genes. Cell 165(7):1789–1802. https://doi.org/10.1016/j.cell.2016.05.007
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
Gradinaru V, Treweek J, Overton K, Deisseroth K (2018) Hydrogel-tissue chemistry: principles and applications. Annu Rev Biophys 47:355–376. https://doi.org/10.1146/annurev-biophys-070317-032905
Klingberg A, Hasenberg A, Ludwig-Portugall I, Medyukhina A, Mann L, Brenzel A, Engel DR, Figge MT, Kurts C, Gunzer M (2017) Fully automated evaluation of Total glomerular number and capillary tuft size in nephritic kidneys using Lightsheet microscopy. J Am Soc Nephrol 28(2):452–459. https://doi.org/10.1681/ASN.2016020232
Masselink W, Reumann D, Murawala P, Pasierbek P, Taniguchi Y, Bonnay F, Meixner K, Knoblich JA, Tanaka EM (2019) Broad applicability of a streamlined ethyl cinnamate-based clearing procedure. Development 146(3). https://doi.org/10.1242/dev.166884
Acknowledgments
This work was funded by Mission Connect, a program of TIRR Foundation. Use of the Texas A&M Microscopy and Imaging Center is acknowledged.
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Jalufka, F.L. et al. (2022). Hydrophobic and Hydrogel-Based Methods for Passive Tissue Clearing. In: Heit, B. (eds) Fluorescent Microscopy. Methods in Molecular Biology, vol 2440. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2051-9_12
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DOI: https://doi.org/10.1007/978-1-0716-2051-9_12
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