Skip to main content

Viral Injection and Cranial Window Implantation for In Vivo Two-Photon Imaging

  • Protocol
  • First Online:
High-Resolution Imaging of Cellular Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1474))

Abstract

Two-photon (2P) imaging has proven to be a powerful tool for investigating neural structure and function both in brain slices and in intact systems. In vivo 2P imaging presents significant challenges in sample preparation, which are exacerbated in non-murine species. Here, we describe procedures for the effective virally mediated labeling of neurons and for the implantation of cranial windows for imaging. The procedures described here are applicable to a range of species, including mice, and are routinely used in ferrets and tree shrews to provide large-scale labeling of cortical volumes and high-quality imaging data.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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. Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci U S A 100:7319–7324. doi:10.1073/pnas.1232232100

    Article  CAS  PubMed Central  Google Scholar 

  2. Ohki K, Chung S, Ch’ng YH, Kara P, Reid RC (2005) Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433(7026):597–603. doi:10.1038/nature03274

    Article  CAS  Google Scholar 

  3. Peron SP, Freeman J, Iyer V, Guo C, Svoboda K (2015) A cellular resolution map of barrel cortex activity during tactile behavior. Neuron 86(3):783–799. doi:10.1016/j.neuron.2015.03.027

    Article  CAS  Google Scholar 

  4. Dombeck DA, Khabbaz AN, Collman F, Adelman TL, Tank DW (2007) Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron 56(1):43–57. doi:10.1016/j.neuron.2007.08.003

    Article  CAS  PubMed Central  Google Scholar 

  5. Tsai P, Kleinfeld D (2009) In vivo two-photon laser scanning microscopy with concurrent plasma-mediated ablation principles and hardware realization. In: Frostig RD (ed) In vivo optical imaging of brain function, 2nd edn. CRC Press, Boca Raton, FL, pp 59–115

    Chapter  Google Scholar 

  6. Svoboda K, Yasuda R (2006) Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 50(6):823–839. doi:10.1016/j.neuron.2006.05.019

    Article  CAS  Google Scholar 

  7. Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2(12):932–940. doi:10.1038/nmeth818

    Article  CAS  Google Scholar 

  8. Garaschuk O, Milos RI, Konnerth A (2006) Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo. Nat Protoc 1(1):380–386. doi:10.1038/nprot.2006.58

    Article  CAS  Google Scholar 

  9. Smith GB, Sederberg A, Elyada YM, Van Hooser SD, Kaschube M, Fitzpatrick D (2015) The development of cortical circuits for motion discrimination. Nat Neurosci 18(2):252–261. doi:10.1038/nn.3921

    Article  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgments

We would like to thank Dominique Ouimet for helpful discussions on surgical procedures, Yishai Elyada for the headplate design and discussions on cranial window implants, and Daniel Wilson for images of dendritic spines and for comments on this manuscript.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to David Fitzpatrick .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Smith, G.B., Fitzpatrick, D. (2016). Viral Injection and Cranial Window Implantation for In Vivo Two-Photon Imaging. In: Schwartzbach, S., Skalli, O., Schikorski, T. (eds) High-Resolution Imaging of Cellular Proteins. Methods in Molecular Biology, vol 1474. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6352-2_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6352-2_10

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6350-8

  • Online ISBN: 978-1-4939-6352-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics