Skip to main content
SpringerLink
Log in

Three-Dimensional Super-Resolution Imaging of the Cytoskeleton in Hippocampal Neurons Using Selective Plane Illumination

  • Protocol
  • First Online:
Single Molecule Microscopy in Neurobiology

Part of the book series: Neuromethods ((NM,volume 154))

  • 657 Accesses

Abstract

Recent advances in single-molecule-based super-resolution imaging have been in the forefront of biological research for the visualization of the detail structures in cellular and molecular biology. A number of super-resolution optical microscopy techniques have been reported; however, several challenges such as the use of high-activation sources resulting in photobleaching and photodamage effects, restrictions in three-dimensional imaging, long data acquisition, and limited field of view remain unresolved. To address these concerns, a rapid, large-scale, and three-dimensional super-resolution fluorescence microscopy has been developed through the introduction of selective plane illumination microscopy based on scanning Bessel beam and a spontaneously blinking dye HMSiR as a reporter. This localization-based super-resolution microscope offers several advantages. namely minuscule levels of photodamage and phototoxicity effects due to low activation source, good optical sectioning suitable for three-dimensional imaging, large field of view, and fast data acquisition. In this chapter, protocols on the three-dimensional super-resolution imaging of neurons through the application of selective plane illumination technique are discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ok Kyu P, Jina K, Yoo Jung J, Young Ho K, Hyun-Seok H, Byung Joon H, Seung-Hae K, Yun K (2015) 3D light-sheet fluorescence microscopy of cranial neurons and vasculature during zebrafish embryogenesis. Mol Cells 38(11):975–981

    Article  Google Scholar 

  2. Pulvermüller F, Garagnani M (2014) From sensorimotor learning to memory cells in prefrontal and temporal association cortex: a neurocomputational study of disembodiment. Cortex 57:1–21. https://doi.org/10.1016/j.cortex.2014.02.015

    Article  PubMed  Google Scholar 

  3. Peyrache A, Schieferstein N, Buzsáki G (2017) Transformation of the head-direction signal into a spatial code. Nat Commun 8(1):1752. https://doi.org/10.1038/s41467-017-01908-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Giepmans BNG, Adams SR, Ellisman MH, Tsien RY (2006) The fluorescent toolbox for assessing protein location and function. Science 312(5771):217–224. https://doi.org/10.1126/science.1124618

    Article  CAS  PubMed  Google Scholar 

  5. Lippincott-Schwartz J, Patterson GH (2009) Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol 19(11):555–565. https://doi.org/10.1016/j.tcb.2009.09.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Noji H, Yasuda R, Yoshida M, Kinosita K Jr (1997) Direct observation of the rotation of F1-ATPase. Nature 386:299. https://doi.org/10.1038/386299a0

    Article  CAS  PubMed  Google Scholar 

  7. Rief M, Rock RS, Mehta AD, Mooseker MS, Cheney RE, Spudich JA (2000) Myosin-V stepping kinetics: a molecular model for processivity. Proc Natl Acad Sci U S A 97(17):9482–9486. https://doi.org/10.1073/pnas.97.17.9482

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19(11):780–782. https://doi.org/10.1364/OL.19.000780

    Article  CAS  PubMed  Google Scholar 

  9. Gustafsson MGL (2000) Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc 198(2):82–87. https://doi.org/10.1046/j.1365-2818.2000.00710.x

    Article  CAS  PubMed  Google Scholar 

  10. Betzig E (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645

    Article  CAS  Google Scholar 

  11. Huang B, Jones SA, Brandenburg B, Zhuang X (2008) Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat Methods 5:1047–1052

    Article  CAS  Google Scholar 

  12. Chiu S-W, Leake MC (2011) Functioning nanomachines seen in real-time in living bacteria using single-molecule and super-resolution fluorescence imaging. Int J Mol Sci 12(4):2518–2542. https://doi.org/10.3390/ijms12042518

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Amat F, Lemon W, Mossing DP, McDole K, Wan Y, Branson K, Myers EW, Keller PJ (2014) Fast, accurate reconstruction of cell lineages from large-scale fluorescence microscopy data. Nat Methods 11:951. https://doi.org/10.1038/nmeth.3036. https://www.nature.com/articles/nmeth.3036#supplementary-information

    Article  CAS  PubMed  Google Scholar 

  14. Tomer R, Khairy K, Amat F, Keller PJ (2012) Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy. Nat Methods 9:755. https://doi.org/10.1038/nmeth.2062. https://www.nature.com/articles/nmeth.2062#supplementary-information

    Article  CAS  PubMed  Google Scholar 

  15. Zhang J, Liu X, Xu W, Luo W, Li M, Chu F, Xu L, Cao A, Guan J, Tang S, Duan X (2018) Stretchable transparent electrode arrays for simultaneous electrical and optical interrogation of neural circuits in vivo. Nano Lett 18:2903. https://doi.org/10.1021/acs.nanolett.8b00087

    Article  CAS  PubMed  Google Scholar 

  16. Szymborska A, de Marco A, Daigle N, Cordes VC, Briggs JAG, Ellenberg J (2013) Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 341(6146):655–658. https://doi.org/10.1126/science.1240672

    Article  CAS  PubMed  Google Scholar 

  17. Shim S-H, Xia C, Zhong G, Babcock HP, Vaughan JC, Huang B, Wang X, Xu C, Bi G-Q, Zhuang X (2012) Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proc Natl Acad Sci U S A 109(35):13978–13983. https://doi.org/10.1073/pnas.1201882109

    Article  PubMed  PubMed Central  Google Scholar 

  18. Fernández-Suárez M, Ting AY (2008) Fluorescent probes for super-resolution imaging in living cells. Nat Rev Mol Cell Biol 9:929. https://doi.org/10.1038/nrm2531

    Article  CAS  PubMed  Google Scholar 

  19. Jones SA, Shim S-H, He J, Zhuang X (2011) Fast, three-dimensional super-resolution imaging of live cells. Nat Methods 8:499. https://doi.org/10.1038/nmeth.1605. https://www.nature.com/articles/nmeth.1605#supplementary-information

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. French JB, Jones SA, Deng H, Pedley AM, Kim D, Chan CY, Hu H, Pugh RJ, Zhao H, Zhang Y, Huang TJ, Fang Y, Zhuang X, Benkovic SJ (2016) Spatial colocalization and functional link of purinosomes with mitochondria. Science 351(6274):733–737. https://doi.org/10.1126/science.aac6054

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Huang B, Wang W, Bates M, Zhuang X (2008) Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319(5864):810–813. https://doi.org/10.1126/science.1153529

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Vaziri A, Tang J, Shroff H, Shank CV (2008) Multilayer three-dimensional super resolution imaging of thick biological samples. Proc Natl Acad Sci U S A 105:20221–20226

    Article  CAS  Google Scholar 

  23. Abrahamsson S, Chen J, Hajj B, Stallinga S, Katsov AY, Wisniewski J, Mizuguchi G, Soule P, Mueller F, Darzacq CD, Darzacq X, Wu C, Bargmann CI, Agard DA, Dahan M, Gustafsson MGL (2013) Fast multicolor 3D imaging using aberration-corrected multifocus microscopy. Nat Methods 10(1):60–63. https://doi.org/10.1038/nmeth.2277. http://www.nature.com/nmeth/journal/v10/n1/abs/nmeth.2277.html#supplementary-information

    Article  CAS  PubMed  Google Scholar 

  24. Cella Zanacchi F (2011) Live-cell 3D super-resolution imaging in thick biological samples. Nat Methods 8:1047–1049

    Article  Google Scholar 

  25. Legant WR, Shao L, Grimm JB, Brown TA, Milkie DE, Avants BB, Lavis LD, Betzig E (2016) High-density three-dimensional localization microscopy across large volumes. Nat Methods 13(4):359–365. https://doi.org/10.1038/nmeth.3797. http://www.nature.com/nmeth/journal/v13/n4/abs/nmeth.3797.html#supplementary-information

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Cella Zanacchi F, Lavagnino Z, Perrone Donnorso M, Del Bue A, Furia L, Faretta M, Diaspro A (2011) Live-cell 3D super-resolution imaging in thick biological samples. Nat Methods 8(12):1047–1049. http://www.nature.com/nmeth/journal/v8/n12/abs/nmeth.1744.html#supplementary-information

    Article  Google Scholar 

  27. Schmid B, Shah G, Scherf N, Weber M, Thierbach K, Campos CP, Roeder I, Aanstad P, Huisken J (2013) High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics. Nat Commun 4:2207. https://doi.org/10.1038/ncomms3207. https://www.nature.com/articles/ncomms3207#supplementary-information

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Silvestri L, Paciscopi M, Soda P, Biamonte F, Iannello G, Frasconi P, Pavone FS (2015) Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis. Front Neuroanat 9:68. https://doi.org/10.3389/fnana.2015.00068

    Article  PubMed  PubMed Central  Google Scholar 

  29. Planchon TA, Gao L, Milkie DE, Davidson MW, Galbraith JA, Galbraith CG, Betzig E (2011) Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination. Nat Methods 8:417. https://doi.org/10.1038/nmeth.1586. https://www.nature.com/articles/nmeth.1586#supplementary-information

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chen B-C, Legant WR, Wang K, Shao L, Milkie DE, Davidson MW, Janetopoulos C, Wu XS, Hammer JA, Liu Z, English BP, Mimori-Kiyosue Y, Romero DP, Ritter AT, Lippincott-Schwartz J, Fritz-Laylin L, Mullins RD, Mitchell DM, Bembenek JN, Reymann A-C, Böhme R, Grill SW, Wang JT, Seydoux G, Tulu US, Kiehart DP, Betzig E (2014) Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346(6208):1257998. https://doi.org/10.1126/science.1257998

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Uno S-N, Kamiya M, Yoshihara T, Sugawara K, Okabe K, Tarhan MC, Fujita H, Funatsu T, Okada Y, Tobita S, Urano Y (2014) A spontaneously blinking fluorophore based on intramolecular spirocyclization for live-cell super-resolution imaging. Nat Chem 6(8):681–689. https://doi.org/10.1038/nchem.2002. http://www.nature.com/nchem/journal/v6/n8/abs/nchem.2002.html#supplementary-information

    Article  CAS  PubMed  Google Scholar 

  32. van de Linde S, Löschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, Sauer M (2011) Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6(7):991–1009

    Article  Google Scholar 

  33. Folling J, Bossi M, Bock H, Medda R, Wurm CA, Hein B, Jakobs S, Eggeling C, Hell SW (2008) Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods 5(11):943–945. http://www.nature.com/nmeth/journal/v5/n11/suppinfo/nmeth.1257_S1.html

    Article  Google Scholar 

  34. Ha T, Tinnefeld P (2012) Photophysics of fluorescence probes for single molecule biophysics and super-resolution imaging. Annu Rev Phys Chem 63:595–617. https://doi.org/10.1146/annurev-physchem-032210-103340

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Dempsey GT, Vaughan JC, Chen KH, Bates M, Zhuang X (2011) Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods 8(12):1027–1036. https://doi.org/10.1038/nmeth.1768

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bates M, Huang B, Dempsey GT, Zhuang X (2007) Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317(5845):1749–1753. https://doi.org/10.1126/science.1146598

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Fölling J, Bossi M, Bock H, Medda R, Wurm CA, Hein B, Jakobs S, Eggeling C, Hell SW (2008) Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods 5:943. https://doi.org/10.1038/nmeth.1257. https://www.nature.com/articles/nmeth.1257#supplementary-information

    Article  CAS  PubMed  Google Scholar 

  38. Takakura H, Zhang Y, Erdmann RS, Thompson AD, Lin Y, McNellis B, Rivera-Molina F, Uno S-N, Kamiya M, Urano Y, Rothman JE, Bewersdorf J, Schepartz A, Toomre D (2017) Long time-lapse nanoscopy with spontaneously blinking membrane probes. Nat Biotechnol 35:773. https://doi.org/10.1038/nbt.3876. https://www.nature.com/articles/nbt.3876#supplementary-information

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Gao L, Shao L, Chen B-C, Betzig E (2014) 3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy. Nat Protoc 9:1083. https://doi.org/10.1038/nprot.2014.087. https://www.nature.com/articles/nprot.2014.087#supplementary-information

    Article  CAS  PubMed  Google Scholar 

  40. Huisken J, Stainier DYR (2009) Selective plane illumination microscopy techniques in developmental biology. Development 136(12):1963–1975. https://doi.org/10.1242/dev.022426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Fath T, Ke YD, Gunning P, Götz J, Ittner LM (2008) Primary support cultures of hippocampal and substantia nigra neurons. Nat Protoc 4:78. https://doi.org/10.1038/nprot.2008.199

    Article  CAS  Google Scholar 

  42. Chen CY, Chen YT, Wang JY, Huang YS, Tai CY (2017) Postsynaptic Y654 dephosphorylation of β-catenin modulates presynaptic vesicle turnover through increased n-cadherin-mediated transsynaptic signaling. Dev Neurobiol 77(1):61–74. https://doi.org/10.1002/dneu.22411

    Article  CAS  PubMed  Google Scholar 

  43. Brewer GJ (1997) Isolation and culture of adult rat hippocampal neurons. J Neurosci Methods 71(2):143–155. https://doi.org/10.1016/S0165-0270(96)00136-7

    Article  CAS  PubMed  Google Scholar 

  44. Homaei AA, Sajedi RH, Sariri R, Seyfzadeh S, Stevanato R (2010) Cysteine enhances activity and stability of immobilized papain. Amino Acids 38(3):937–942. https://doi.org/10.1007/s00726-009-0302-3

    Article  CAS  PubMed  Google Scholar 

  45. Lautenschläger J, Mosharov EV, Kanter E, Sulzer D, Kaminski Schierle GS (2018) An easy-to-implement protocol for preparing postnatal ventral mesencephalic cultures. Front Cell Neurosci 12:44. https://doi.org/10.3389/fncel.2018.00044

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Lustre. http://lustre.org/

  47. Adaptive Computing Torque Resource Manager. http://www.adaptivecomputing.com/products/open-source/torque/

  48. Oxford Academic ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. https://academic.oup.com/bioinformatics/article/30/16/2389/2748167

  49. LibTIFF – TIFF Library and Utilities. http://www.simplesystems.org/libtiff/

  50. rsync. https://rsync.samba.org/

  51. BMC Bioinformatics ImageJ2: ImageJ for the next generation of scientific image data. https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-017-1934-z

  52. XVFB Xvfb − virtual framebuffer X server for X Version 11. https://www.x.org/releases/X11R7.7/doc/man/man1/Xvfb.1.xhtml

  53. SQLite. https://www.sqlite.org/index.html

  54. Europe PMC Fourier ring correlation as a resolution criterion for super-resolution microscopy. http://europepmc.org/abstract/MED/23684965

  55. Bitbucket Torque and Thunderstorm. https://bitbucket.org/account/user/cbc-group/projects/LOC

Download references

Acknowledgments

The control software of the selective plane illumination microscope was licensed by Howard Hughes Medical Institute, Janelia Farm Research Campus. P.C. and B.-C.C. acknowledge the partial support of the Ministry of Science and Technology of Taiwan under contract numbers 106-2119-M-001-023 and 105-2119-M-001-026-MY2. P.C. is grateful for the support of the Thematic project of Academia Sinica.

Author information

Authors and Affiliations

  1. Research Center for Applied Sciences, Academia Sinica, Taipei, Taiwan

    Frances Camille M. Wu, Feby Wijaya Pratiwi, Chin-Yi Chen, Chieh-Han Lu, Wei-Chun Tang, Yen-Ting Liu, Bi-Chang Chen & Peilin Chen

Authors
  1. Frances Camille M. Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Feby Wijaya Pratiwi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chin-Yi Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chieh-Han Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wei-Chun Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yen-Ting Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Bi-Chang Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Peilin Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Bi-Chang Chen or Peilin Chen .

Editor information

Editors and Affiliations

  1. Laboratory of Cellular and Molecular Neurobiology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan

    Nobuhiko Yamamoto

  2. Laboratory for Cell Polarity Regulation, RIKEN Center for Biosystems Dynamics Research, Suita, Osaka, Japan

    Yasushi Okada

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Wu, F.C.M. et al. (2020). Three-Dimensional Super-Resolution Imaging of the Cytoskeleton in Hippocampal Neurons Using Selective Plane Illumination. In: Yamamoto, N., Okada, Y. (eds) Single Molecule Microscopy in Neurobiology . Neuromethods, vol 154. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0532-5_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0532-5_13

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0531-8

  • Online ISBN: 978-1-0716-0532-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation