Skip to main content

Optimized Protocol for Imaging Cleared Neural Tissues Using Light Microscopy

Part of the Methods in Molecular Biology book series (MIMB,volume 1538)

Abstract

Understanding physical and chemical processes at an organismal scale is a fundamental goal in biology. While science is adept at explaining biological phenomena at both molecular and cellular levels, understanding how these processes translate to organismal functions remains a challenging problem. This issue is particularly significant for the nervous system where cell signaling and synaptic activities function in the context of broad neural networks. Recent progress in tissue clearing technologies lessens the barriers that previously prevented the study of large tissue samples while maintaining molecular and cellular resolution. While these new methods open vast opportunities and exciting new questions, the logistics of analyzing cellular processes in intact tissue have to be carefully considered. In this protocol, we outline a procedure to rapidly image intact brain tissue up to thousands of cubic millimeters. This experimental pipeline involves three steps: tissue clearing, tissue imaging, and data analysis. In an attempt to streamline the process for researchers entering this field, we address important considerations for each of these stages and describe an integrated solution to image intact biological tissues. Hopefully, this optimized protocol will lower the barrier of implementing high-resolution tissue imaging and facilitate the investigations of mesoscale questions at molecular and cellular resolution.

Key words

  • Lightsheet
  • Confocal
  • CLARITY
  • OptiView
  • Tissue clearing
  • Whole brain imaging

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-6688-2_11
  • Chapter length: 17 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   109.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-6688-2
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   139.00
Price excludes VAT (USA)
Hardcover Book
USD   169.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Petrovich GD, Canteras NS, Swanson LW (2001) Combinatorial amygdalar inputs to hippocampal domains and hypothalamic behavior systems. Brain Res Brain Res Rev 38(1–2):247–289

    CAS  CrossRef  PubMed  Google Scholar 

  2. Swanson LW (2000) Cerebral hemisphere regulation of motivated behavior. Brain Res 886(1–2):113–164

    CAS  CrossRef  PubMed  Google Scholar 

  3. Graybiel AM, Ragsdale CW Jr (1979) Fiber connections of the basal ganglia. Prog Brain Res 51:237–283

    CAS  PubMed  Google Scholar 

  4. Watabe-Uchida M, Zhu L, Ogawa SK, Vamanrao A, Uchida N (2012) Whole-brain mapping of direct inputs to midbrain dopamine neurons. Neuron 74(5):858–873. doi:10.1016/j.neuron.2012.03.017

    CAS  CrossRef  PubMed  Google Scholar 

  5. Wickersham IR, Lyon DC, Barnard RJ, Mori T, Finke S, Conzelmann KK, Young JA, Callaway EM (2007) Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53(5):639–647. doi:10.1016/j.neuron.2007.01.033

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A, Boe AF, Boguski MS, Brockway KS, Byrnes EJ, Chen L, Chen L, Chen TM, Chin MC, Chong J, Crook BE, Czaplinska A, Dang CN, Datta S, Dee NR, Desaki AL, Desta T, Diep E, Dolbeare TA, Donelan MJ, Dong HW, Dougherty JG, Duncan BJ, Ebbert AJ, Eichele G, Estin LK, Faber C, Facer BA, Fields R, Fischer SR, Fliss TP, Frensley C, Gates SN, Glattfelder KJ, Halverson KR, Hart MR, Hohmann JG, Howell MP, Jeung DP, Johnson RA, Karr PT, Kawal R, Kidney JM, Knapik RH, Kuan CL, Lake JH, Laramee AR, Larsen KD, Lau C, Lemon TA, Liang AJ, Liu Y, Luong LT, Michaels J, Morgan JJ, Morgan RJ, Mortrud MT, Mosqueda NF, Ng LL, Ng R, Orta GJ, Overly CC, Pak TH, Parry SE, Pathak SD, Pearson OC, Puchalski RB, Riley ZL, Rockett HR, Rowland SA, Royall JJ, Ruiz MJ, Sarno NR, Schaffnit K, Shapovalova NV, Sivisay T, Slaughterbeck CR, Smith SC, Smith KA, Smith BI, Sodt AJ, Stewart NN, Stumpf KR, Sunkin SM, Sutram M, Tam A, Teemer CD, Thaller C, Thompson CL, Varnam LR, Visel A, Whitlock RM, Wohnoutka PE, Wolkey CK, Wong VY, Wood M, Yaylaoglu MB, Young RC, Youngstrom BL, Yuan XF, Zhang B, Zwingman TA, Jones AR (2007) Genome-wide atlas of gene expression in the adult mouse brain. Nature 445(7124):168–176. doi:10.1038/nature05453

    CAS  CrossRef  PubMed  Google Scholar 

  7. Oh SW, Harris JA, Ng L, Winslow B, Cain N, Mihalas S, Wang Q, Lau C, Kuan L, Henry AM, Mortrud MT, Ouellette B, Nguyen TN, Sorensen SA, Slaughterbeck CR, Wakeman W, Li Y, Feng D, Ho A, Nicholas E, Hirokawa KE, Bohn P, Joines KM, Peng H, Hawrylycz MJ, Phillips JW, Hohmann JG, Wohnoutka P, Gerfen CR, Koch C, Bernard A, Dang C, Jones AR, Zeng H (2014) A mesoscale connectome of the mouse brain. Nature 508(7495):207–214. doi:10.1038/nature13186

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  8. 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. doi:10.1038/nature12107

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  9. 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 Methods 4(4):331–336. doi:10.1038/nmeth1036

    CAS  CrossRef  PubMed  Google Scholar 

  10. 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. doi:10.1038/nprot.2012.119

    CAS  CrossRef  PubMed  Google Scholar 

  11. 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. doi:10.1038/nn.2928

    CAS  CrossRef  PubMed  Google Scholar 

  12. 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. doi:10.1038/nn.3447

    CAS  CrossRef  PubMed  Google Scholar 

  13. 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. doi:10.1242/dev.091844

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  14. 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. doi:10.1016/j.cell.2014.03.042

    CAS  CrossRef  PubMed  Google Scholar 

  15. Tainaka K, Kubota SI, Suyama TQ, Susaki EA, Perrin D, Ukai-Tadenuma M, Ukai H, Ueda HR (2014) Whole-body imaging with single-cell resolution by tissue decolorization. Cell 159(4):911–924

    CAS  CrossRef  PubMed  Google Scholar 

  16. 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. doi:10.1016/j.cell.2014.07.017

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  17. 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

    CAS  CrossRef  PubMed  Google Scholar 

  18. Schultze O (1897) Über Herstellung and conservirung durchsichtigen embryonen zum stadium der skeletbildung. Anat Anz 13:3–5

    Google Scholar 

  19. Spalteholz W (1914) Über das Durchsichtigmachen von menschlichen und tierischen Präparaten und seine theoretischen Bedingungen. Leipzig, S. Hirzel

    Google Scholar 

  20. Chung K, Deisseroth K (2013) CLARITY for mapping the nervous system. Nat Methods 10(6):508–513. doi:10.1038/nmeth.2481

    CAS  CrossRef  PubMed  Google Scholar 

  21. Kim SY, Chung K, Deisseroth K (2013) Light microscopy mapping of connections in the intact brain. Trends Cogn Sci 17(12):596–599. doi:10.1016/j.tics.2013.10.005

    CrossRef  PubMed  Google Scholar 

  22. 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. doi:10.1038/nprot.2014.123

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  23. Wang K, Milkie DE, Saxena A, Engerer P, Misgeld T, Bronner ME, Mumm J, Betzig E (2014) Rapid adaptive optical recovery of optimal resolution over large volumes. Nat Methods 11(6):625–628. doi:10.1038/nmeth.2925

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  24. Huisken J, Swoger J, Del Bene F, Wittbrodt J, Stelzer EH (2004) Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305(5686):1007–1009. doi:10.1126/science.1100035

    CAS  CrossRef  PubMed  Google Scholar 

  25. Huisken J, Stainier DY (2007) Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM). Opt Lett 32(17):2608–2610

    CrossRef  PubMed  Google Scholar 

  26. Preibisch S, Saalfeld S, Tomancak P (2009) Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25(11):1463–1465. doi:10.1093/bioinformatics/btp184

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  27. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. doi:10.1038/nmeth.2019

    CAS  CrossRef  PubMed  Google Scholar 

  28. Peng H, Bria A, Zhou Z, Iannello G, Long F (2014) Extensible visualization and analysis for multidimensional images using Vaa3D. Nat Protoc 9(1):193–208. doi:10.1038/nprot.2014.011

    CAS  CrossRef  PubMed  Google Scholar 

  29. Bria A, Iannello G (2012) TeraStitcher - a tool for fast automatic 3D-stitching of teravoxel-sized microscopy images. BMC Bioinformatics 13:316. doi:10.1186/1471-2105-13-316

    CrossRef  PubMed  PubMed Central  Google Scholar 

  30. Zhu D, Larin KV, Luo Q, Tuchin VV (2013) Recent progress in tissue optical clearing. Laser Photon Rev 7(5):732–757. doi:10.1002/lpor.201200056

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  31. Efimova OI, Anokhin KV (2009) Enhancement of optical transmission capacity of isolated structures in the brain of mature mice. Bull Exp Biol Med 147(1):3–6

    CAS  CrossRef  PubMed  Google Scholar 

  32. Oldham M, Sakhalkar H, Oliver T, Allan Johnson G, Dewhirst M (2008) Optical clearing of unsectioned specimens for three-dimensional imaging via optical transmission and emission tomography. J Biomed Opt 13(2):021113. doi:10.1117/1.2907968

    CrossRef  PubMed  PubMed Central  Google Scholar 

  33. Genina EA, Bashkatov AN, Tuchin VV (2010) Tissue optical immersion clearing. Expert Rev Med Devices 7(6):825–842. doi:10.1586/erd.10.50

    CrossRef  PubMed  Google Scholar 

  34. Denk W, Strickler JH, Webb WW (1990) Two-photon laser scanning fluorescence microscopy. Science 248(4951):73–76

    CAS  CrossRef  PubMed  Google Scholar 

  35. Haddad R, Lanjuin A, Madisen L, Zeng H, Murthy VN, Uchida N (2013) Olfactory cortical neurons read out a relative time code in the olfactory bulb. Nat Neurosci 16(7):949–957. doi:10.1038/nn.3407

    CAS  CrossRef  PubMed  PubMed Central  Google Scholar 

  36. Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13(1):133–140. doi:10.1038/nn.2467

    CAS  CrossRef  PubMed  Google Scholar 

Download references

Acknowledgments

We would like to thank the Howard Hughes Medical Institute and the National Institute of Health (NIH/NIDCD RO1 5R01DC013087-02; F32 DC10089) for funding. We also thank the Harvard Center for Biological Imaging for providing infrastructure and support.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Joseph Bergan .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Isogai, Y., Richardson, D.S., Dulac, C., Bergan, J. (2017). Optimized Protocol for Imaging Cleared Neural Tissues Using Light Microscopy. In: Poulopoulos, A. (eds) Synapse Development. Methods in Molecular Biology, vol 1538. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6688-2_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6688-2_11

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6686-8

  • Online ISBN: 978-1-4939-6688-2

  • eBook Packages: Springer Protocols