Advertisement

Light-Sheet Fluorescence Microscopy: Chemical Clearing and Labeling Protocols for Ultramicroscopy

  • Nina JährlingEmail author
  • Klaus Becker
  • Saiedeh Saghafi
  • Hans-Ulrich Dodt
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1563)

Abstract

Light-sheet microscopy is an effective technique in neuroscience, developmental biology, and cancer research for visualizing and analyzing cellular networks and whole organs in three dimensions. Because this technique requires specimens to be translucent they commonly have to be cleared before microscopy inspection. Here, we provide 3DISCO based protocols for preparing cleared samples of immuno-stained neural networks, lectin-labeled vascular networks, and Methoxy-X04 labeled beta-amyloid plaques in mice. 3DISCO utilizes the lipophilic solvents tetrahydrofuran (THF) and dibenzylether (DBE) for dehydration and successive clearing. Crucial steps for obtaining transparent tissues and preserving the fragile endogenous GFP are the transcardial perfusion, as well as the proper implementation of the 3DISCO clearing process using peroxide free chemicals. We further provide a protocol for resin embedding of 3DISCO cleared specimens that allows long term archiving of samples for years with virtually no loss in signal quality.

Key words

Chemical clearing Whole-mount immune-labeling Vascular networks Light-sheet microscopy 3D reconstruction 3DISCO Peroxide elimination Alzheimer disease 

Notes

Acknowledgment

We thank Massih Foroughipour for preparing the technical drawings of the custom-made specimen clamps.

References

  1. 1.
    Keller PJ, Dodt HU (2012) Light sheet microscopy of living or cleared specimens. Curr Opin Neurobiol 22:138–143CrossRefPubMedGoogle Scholar
  2. 2.
    Dodt H-U, Leischner U, Schierloh A et al (2007) Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain. Nat Methods 4:331–336. doi: 10.1038/nmeth1036 CrossRefPubMedGoogle Scholar
  3. 3.
    Ertürk A, Mauch CP, Hellal F et al (2011) Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury. Nat Med 18:166–171. doi: 10.1038/nm.2600 CrossRefPubMedGoogle Scholar
  4. 4.
    Spalteholz W (1911) Über das Durchsichtigmachen von menschlichen und tierischen Präparaten 48Google Scholar
  5. 5.
    Dent JA, Polson AG, Klymkowsky MW (1989) A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus. Development 105:61–74PubMedGoogle Scholar
  6. 6.
    Klymkowsky MW, Hanken J (1991) Whole-mount staining of Xenopus and other vertebrates. Methods Cell Biol 36:419–441CrossRefPubMedGoogle Scholar
  7. 7.
    Becker K, Jährling N, Saghafi S, et al. (2012) Chemical clearing and dehydration of GFP expressing mouse brains. PLoSOne 7:e33916–. doi: 10.1371/journal.pone.0033916Google Scholar
  8. 8.
    Ertürk A, Becker K, Jährling N et al (2012) Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat Protoc 7:1983–1995. doi: 10.1038/nprot.2012.119 CrossRefPubMedGoogle Scholar
  9. 9.
    Hama H, Kurokawa H, Kawano H, Ryoko A et al (2011) Sca/e: a chemical approach for fluorescene imaging and reconstruction of transparent mouse brain. Nat Neurosci 14:1481–1488CrossRefPubMedGoogle Scholar
  10. 10.
    Chung K, Wallace J, Kim S-Y et al (2013) Structural and molecular interrogation of intact biological systems. Nature 497:332–337. doi: 10.1038/nature12107 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Susaki EA, Tainaka K, Perrin D et al (2014) Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157:726–739. doi: 10.1016/j.cell.2014.03.042 CrossRefPubMedGoogle Scholar
  12. 12.
    Alnuami AA, Zeedi B, Qadri SM, Ashraf SS (2008) Oxyradical-induced GFP damage and loss of fluorescence. Int J Biol Macromol 43:182–186CrossRefPubMedGoogle Scholar
  13. 13.
    Eichel FG, Othmer DF (1949) Benzaldehyde by Autoxidation by Dibenzyl Ether. Ind Eng Chem 41:2623–2626. doi: 10.1021/ie50479a054 CrossRefGoogle Scholar
  14. 14.
    Brady OL, v.Elsmie G (1926) The use of 2 : P-Dinitrophenylhydrazine as a reagent for aldehydes and ketones. Analyst 51:77–78.Google Scholar
  15. 15.
    Becker K, Jahrling N, Kramer ER et al (2008) Ultramicroscopy: 3D reconstruction of large microscopical specimens. J Biophotonics 1:36–42CrossRefPubMedGoogle Scholar
  16. 16.
    Jährling N, Becker K, Dodt H (2009) 3D–reconstruction of blood vessels by ultramicroscopy. Organogenesis 5(4):227–230Google Scholar
  17. 17.
    Jährling N, Becker K, Wegenast-braun BM, Grathwohl SA (2015) Cerebral β -Amyloidosis in Mice Investigated by Ultramicroscopy. pp 1–13. doi: 10.1371/journal.pone.0125418Google Scholar
  18. 18.
    Becker K, Hahn CM, Saghafi S, Ja N (2014) Reduction of photo bleaching and long term archiving of chemically cleared GFP-expressing mouse brains. pp 1–11. doi: 10.1371/journal.pone.0114149Google Scholar
  19. 19.
    Klunk WE, Bacskai BJ, Mathis CA et al (2002) Imaging Abeta plaques in living transgenic mice with multiphoton microscopy and methoxy-X04, a systemically administered Congo red derivative. J Neuropathol Exp Neurol 61:797–805CrossRefPubMedGoogle Scholar
  20. 20.
    Jährling N, Becker K, Schönbauer C et al (2010) Three-dimensional reconstruction and segmentation of intact Drosophila by ultramicroscopy. Front Syst Neurosci 4:1. doi: 10.3389/neuro.06.001.2010 PubMedPubMedCentralGoogle Scholar
  21. 21.
    Jährling N, Becker K, Kramer ER, Dodt H-U (2008) 3D-Visualization of nerve fiber bundles by ultramicroscopy. Med Laser Appl 23:209–215. doi: 10.1016/j.mla.2008.06.001 CrossRefGoogle Scholar
  22. 22.
    Cavey MJ, Wong GK-S (1993) Custom silicone rubber molds for epoxy resin embedding. Trans Am Microsc Soc 112:81–84CrossRefGoogle Scholar
  23. 23.
    Enoki S, Saeki K, Maki K, Kuwajima K (2004) Acid denaturation and refolding of green fluorescent protein. Biochemistry 43:14238–14248. doi: 10.1021/bi048733+ CrossRefPubMedGoogle Scholar
  24. 24.
    Schwarz MK, Scherbarth A, Sprengel R et al (2015) Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains. PLoS One 10:e0124650. doi: 10.1371/journal.pone.0124650 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Nina Jährling
    • 1
    • 2
    Email author
  • Klaus Becker
    • 1
    • 2
  • Saiedeh Saghafi
    • 1
    • 2
  • Hans-Ulrich Dodt
    • 1
    • 2
  1. 1.Department of Bioelectronics, FKEVienna University of TechnologyViennaAustria
  2. 2.Center of Brain ResearchMedical University of ViennaViennaAustria

Personalised recommendations