Skip to main content

Studying Neuronal Biology Using Spinning Disc Confocal Microscopy

  • Protocol
  • First Online:
Confocal Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2304))


Cytoskeletal integrity is essential for neuronal complexity and functionality. Certain inherited neurological diseases are associated with mutated genes that directly or indirectly compromise cytoskeletal stability. While the large size and complexity of the neurons grown in culture poses certain challenges for imaging, live-cell imaging is an excellent approach to determine the morphological consequences of such mutants. This protocol details the use of spinning disk confocal microscopy and image analysis tools to evaluate branching and neurite length of healthy iPSC-derived glutamatergic neurons that express specific fluorescent proteins. The protocols can be adapted to neuronal cell lines of choice by the investigator.

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

Access this chapter

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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

Similar content being viewed by others


  1. Rao MV, Campbell J, Yuan A, Kumar A, Gotow T, Uchiyama Y et al (2003) The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate. J Cell Biol 163(5):1021–1031.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. de Waegh SM, Lee VM, Brady ST (1992) Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells. Cell 68(3):451–463.

    Article  PubMed  Google Scholar 

  3. Sakaguchi T, Okada M, Kitamura T, Kawasaki K (1993) Reduced diameter and conduction velocity of myelinated fibers in the sciatic nerve of a neurofilament-deficient mutant quail. Neurosci Lett 153(1):65–68.

    Article  CAS  PubMed  Google Scholar 

  4. Kriz J, Zhu Q, Julien JP, Padjen AL (2000) Electrophysiological properties of axons in mice lacking neurofilament subunit genes: disparity between conduction velocity and axon diameter in absence of NF-H. Brain Res 885(1):32–44.

    Article  CAS  PubMed  Google Scholar 

  5. Katsuno H, Toriyama M, Hosokawa Y, Mizuno K, Ikeda K, Sakumura Y et al (2015) Actin migration driven by directional assembly and disassembly of membrane-anchored actin filaments. Cell Rep 12(4):648–660.

    Article  CAS  PubMed  Google Scholar 

  6. Flynn KC, Pak CW, Shaw AE, Bradke F, Bamburg JR (2009) Growth cone-like waves transport actin and promote axonogenesis and neurite branching. Dev Neurobiol 69(12):761–779.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hotulainen P, Hoogenraad CC (2010) Actin in dendritic spines: connecting dynamics to function. J Cell Biol 189(4):619–629.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Cingolani LA, Goda Y (2008) Actin in action: the interplay between the actin cytoskeleton and synaptic efficacy. Nat Rev Neurosci 9(5):344–356.

    Article  CAS  PubMed  Google Scholar 

  9. Kneussel M, Wagner W (2013) Myosin motors at neuronal synapses: drivers of membrane transport and actin dynamics. Nat Rev Neurosci 14(4):233–247.

    Article  CAS  PubMed  Google Scholar 

  10. Kapitein LC, Hoogenraad CC (2011) Which way to go? Cytoskeletal organization and polarized transport in neurons. Mol Cell Neurosci 46(1):9–20.

    Article  CAS  PubMed  Google Scholar 

  11. Munoz-Lasso DC, Roma-Mateo C, Pallardo FV, Gonzalez-Cabo P (2020) Much more than a scaffold: cytoskeletal proteins in neurological disorders. Cell 9(2).

  12. Fernandopulle MS, Prestil R, Grunseich C, Wang C, Gan L, Ward ME (2018) Transcription factor-mediated differentiation of human iPSCs into neurons. Curr Protoc Cell Biol 79(1):e51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wang C, Ward ME, Chen R, Liu K, Tracy TE, Chen X et al (2017) Scalable production of iPSC-derived human neurons to identify Tau-lowering compounds by high-content screening. Stem Cell Rep 9(4):1221–1233.

    Article  CAS  Google Scholar 

  14. Enoki R, Ono D, Hasan MT, Honma S, Honma K (2012) Single-cell resolution fluorescence imaging of circadian rhythms detected with a Nipkow spinning disk confocal system. J Neurosci Methods 207(1):72–79.

    Article  PubMed  Google Scholar 

  15. Miyaoka Y, Chan AH, Judge LM, Yoo J, Huang M, Nguyen TD et al (2014) Isolation of single-base genome-edited human iPS cells without antibiotic selection. Nat Methods 11(3):291–293.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


We would like to thank Eric Balzer from Nikon and Matthew Gastinger from Bitplane for their help with Elements and Imaris and Michael E. Ward (NINDS, NIH) for provide us the i3PSC and the both lentivirus plasmids used in this protocol, the cytosolic mApple and the mNeonGreen-NLS. This work is supported by intramural funding from the National Institute of Allergy and Infectious Diseases at the National Institutes of Health.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Jenny Serra-Vinardell .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2021 This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply and Springer Nature US

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Manzella-Lapeira, J., Brzostowski, J., Serra-Vinardell, J. (2021). Studying Neuronal Biology Using Spinning Disc Confocal Microscopy. In: Brzostowski, J., Sohn, H. (eds) Confocal Microscopy. Methods in Molecular Biology, vol 2304. Humana, New York, NY.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1401-3

  • Online ISBN: 978-1-0716-1402-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics