Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo

Abstract

One of the challenges for modern neuroscience is to understand the rules of concerted neuronal function in vivo. This question can be addressed using noninvasive high-resolution imaging techniques like two-photon microscopy. This protocol describes a versatile approach for in vivo two-photon calcium imaging of neural networks, stained with membrane-permeant fluorescent-indicator dyes. It is based on a targeted pressure ejection of the dye into the tissue of interest and can be used for a large spectrum of indicator dyes, including Oregon Green 488 BAPTA-1 acetoxymethyl ester and Fura-2 acetoxymethyl ester. Through the use of dye mixtures and multicolor imaging, this technique allows the visualization of distinct neurons and glial cells up to 500 μm below the brain surface. It is suitable for staining the brain tissue of various different species (e.g., mouse, rat, cat and zebrafish) at all developmental stages. When combined with brain microendoscopy, it allows the monitoring of intracellular calcium signals in awake, behaving animals. The total time required to carry out the protocol, including dissection and cell staining, is 2 h. Thereafter, imaging experiments might be performed for at least 6 h.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Experimental arrangement for in vivo staining of neuronal populations with Ca2+-indicator dyes.
Figure 2: MCBL allows staining of cortical tissue at various developmental stages.
Figure 3: Assessment of the staining quality.
Figure 4: Spontaneous Ca2+ transients in the visual cortex of an adult mouse.
Figure 5: Ca2+ transients in the individual cortical neurons evoked by sensory stimulation.
Figure 6: Double staining and depth profile.
Figure 7: MCBL using a low concentration of the indicator dye.

References

  1. Tsien, R.Y. A non-disruptive technique for loading calcium buffers and indicators into cells. Nature 290, 527–528 (1981).

    Article  CAS  PubMed  Google Scholar 

  2. Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kerr, J.N., Greenberg, D. & Helmchen, F. Imaging input and output of neocortical networks in vivo. Proc. Natl. Acad. Sci. USA 102, 14063–14068 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ohki, K., Chung, S., Ch'ng, Y.H., Kara, P. & Reid, R.C. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005).

    Article  CAS  PubMed  Google Scholar 

  5. Nimmerjahn, A., Kirchhoff, F., Kerr, J.N.D. & Helmchen, F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat. Methods 1, 31–37 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Sullivan, M.R., Nimmerjahn, A., Sarkisov, D.V., Helmchen, F. & Wang, S.S. In vivo calcium imaging of circuit activity in cerebellar cortex. J. Neurophysiol. 94, 1636–1644 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Brustein, E., Marandi, N., Kovalchuk, Y., Drapeau, P. & Konnerth, A. 'In vivo' monitoring of neuronal network activity in zebrafish by two-photon Ca2+ imaging. Pflü gers Arch. 446, 766–773 (2003).

    Article  CAS  Google Scholar 

  8. Li, J. et al. Early development of functional spatial maps in the zebrafish olfactory bulb. J. Neurosci. 25, 5784–5795 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Niell, C.M. & Smith, S.J. Functional imaging reveals rapid development of visual response properties in the zebrafish tectum. Neuron 45, 941–951 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Yuste, R. in Imaging: A Laboratory Manual (eds. Yuste, R., Lanni, F. & Konnerth, A.) 34.1–34.9 (Cold Spring Harbor Press, Cold Spring Harbor, New York, 2000).

    Google Scholar 

  11. Wong, R.O. in Imaging: A Laboratory Manual (eds. Yuste, R., Lanni, F. & Konnerth, A.) 41.1–41.7 (Cold Spring Harbor Press, Cold Spring Harbor, New York, 2000).

    Google Scholar 

  12. Regehr, W.G. & Tank, D.W. Selective fura-2 loading of presynaptic terminals and nerve cell processes by local perfusion in mammalian brain slice. J. Neurosci. Methods 37, 111–119 (1991).

    Article  CAS  PubMed  Google Scholar 

  13. Christie, R.H. et al. Growth arrest of individual senile plaques in a model of Alzheimer's disease observed by in vivo multiphoton microscopy. J. Neurosci. 21, 858–864 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Neher, E. The use of fura-2 for estimating Ca2+ buffers and Ca2+ fluxes. Neuropharmacology 34, 1423–1442 (1995).

    Article  CAS  PubMed  Google Scholar 

  15. Adelsberger, H., Garaschuk, O. & Konnerth, A. Cortical calcium waves in resting newborn mice. Nat. Neurosci. 8, 988–990 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Flusberg, B.A. et al. Fiber-optic fluorescence imaging. Nat. Methods 2, 941–950 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Flecknell, P. Laboratory Animal Anaesthesia (Academic Press, San Diego, 2000).

    Google Scholar 

  18. Majewska, A., Yiu, G. & Yuste, R. A custom-made two-photon microscope and deconvolution system. Pflügers Arch. 441, 398–408 (2000).

    Article  CAS  PubMed  Google Scholar 

  19. Nikolenko, V. & Yuste, R. in Imaging in Neuroscience and Development: A Laboratory Manual (eds. Yuste, R. & Konnerth, A.) 75–78 (Cold Spring Harbor Press, Cold Spring Harbor, New York, 2005).

    Google Scholar 

  20. Paxinos, G. & Watson, C. The Rat Brain in Stereotaxic Coordinates (Academic Press, San Diego, CA, 2004).

    Google Scholar 

  21. Paxinos, G. & Franklin, K.B.J. The Mouse Brain in Stereotaxic Coordinates (Academic Press, San Diego, CA, 2001).

    Google Scholar 

  22. Vender, J.R., Hand, C.M., Sedor, D., Tabor, S.L. & Black, P. Oxygen saturation monitoring in experimental surgery: a comparison of pulse oximetry and arterial blood gas measurement. Lab. Anim. Sci. 45, 211–215 (1995).

    CAS  PubMed  Google Scholar 

  23. Eilers, J., Schneggenburger, R. & Konnerth, A. in Single-Channel Recording (eds. Sakmann, B. & Neher, E.) 213–229 (Plenum Press, New York, 1995).

    Book  Google Scholar 

  24. Xu, C. in Imaging: A Laboratory Manual (eds. Yuste, R., Lanni, F. & Konnerth, A.) 19.1–19.9 (Cold Spring Harbor Press, Cold Spring Harbor, New York, 2000).

    Google Scholar 

  25. Helmchen, F. & Denk, W. Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Ikegaya, Y. et al. Synfire chains and cortical songs: temporal modules of cortical activity. Science 304, 559–564 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Hirase, H., Qian, L., Bartho, P. & Buzsaki, G. Calcium dynamics of cortical astrocytic networks in vivo. PLoS Biol. 2, E96 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Soeller, C. & Cannell, M.B. Construction of a two-photon microscope and optimisation of illumination pulse duration. Pflügers Arch. 432, 555–561 (1996).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank M.A. Busche for help with experiments shown in Figure 6b. This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 391 and SFB 596) and the Bundesministerium für Bildung und Forschung (NGFN-2).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Arthur Konnerth.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.58

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing