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.

  • Letter
  • Published:

In vivo three-photon microscopy of subcortical structures within an intact mouse brain

Nature Photonics volume 7, pages 205–209 (2013)Cite this article

Subjects

Abstract

Two-photon fluorescence microscopy1 enables scientists in various fields including neuroscience2,3, embryology4 and oncology5 to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of two-photon fluorescence microscopy to the cortical layer within mouse brain, and imaging subcortical structures currently requires the removal of overlying brain tissue3 or the insertion of optical probes6,7. Here, we demonstrate non-invasive, high-resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein-labelled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher-order nonlinear excitation overcomes the limitations of two-photon fluorescence microscopy, enabling biological investigations to take place at a greater depth within tissue.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Wavelength-dependent attenuation length in brain tissue and measured laser characteristics.
Figure 2: In vivo 3PM images of Texas-Red–dextran labelled mouse brain vasculature.
Figure 3: In vivo 3PM images of RFP-labelled neurons in mouse brain.
Figure 4: Fluorescence signal attenuation curves of in vivo experiments.
Figure 5: Resolution characterization of 3PM.

Similar content being viewed by others

References

  1. Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    Article  ADS  Google Scholar 

  2. Kerr, J. N. D. & Denk, W. Imaging in vivo: watching the brain in action. Nature Rev. Neurosci. 9, 195–205 (2008).

    Article  Google Scholar 

  3. Dombeck, D. A., Harvey, C. D., Tian, L., Looger, L. L. & Tank, D. W. Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nature Neurosci. 13, 1433–1440 (2010).

    Article  Google Scholar 

  4. Olivier, N. et al. Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy. Science 329, 967–971 (2010).

    Article  ADS  Google Scholar 

  5. Williams, R. M. et al. Strategies for high-resolution imaging of epithelial ovarian cancer by laparoscopic nonlinear microscopy. Trans. Oncol. 3, 181–194 (2010).

    Article  Google Scholar 

  6. Jung, J. C., Mehta, A. D., Aksay, E., Stepnoski, R. & Schnitzer, M. J. In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy. J. Neurophysiol. 92, 3121–3133 (2004).

    Article  Google Scholar 

  7. Levene, M. J., Dombeck, D. A., Kasischke, K. A., Molloy, R. P. & Webb, W. W. In vivo multiphoton microscopy of deep brain tissue. J. Neurophysiol. 91, 1908–1912 (2004).

    Article  Google Scholar 

  8. Kleinfeld, D., Mitra, P. P., Helmchen, F. & Denk, W. Fluctuations and stimulus induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex. Proc. Natl Acad. Sci. USA 95, 15741–15746 (1998).

    Article  ADS  Google Scholar 

  9. Svoboda, K., Helmchen, F., Denk, W. & Tank, D. W. Spread of dendritic excitation in layer 2/3 pyramidal neurons in rat barrel cortex in vivo. Nature Neurosci. 2, 65–73 (1999).

    Article  Google Scholar 

  10. Theer, P., Hasan, M. T. & Denk, W. Two-photon imaging to a depth of 1000 µm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003).

    Article  ADS  Google Scholar 

  11. Kobat, D. et al. Deep tissue multiphoton microscopy using longer wavelength excitation. Opt. Express 17, 13354–13364 (2009).

    Article  ADS  Google Scholar 

  12. Kobat, D., Horton, N. G. & Xu, C. In vivo two-photon microscopy to 1.6-mm depth in mouse cortex. J. Biomed. Opt. 16, 106014 (2011).

    Article  ADS  Google Scholar 

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

    Article  Google Scholar 

  14. Balu, M. et al. Effect of excitation wavelength on penetration depth in nonlinear optical microscopy of turbid media. J. Biomed. Opt. 14, 010508 (2009).

    Article  ADS  Google Scholar 

  15. Sacks, Z. S., Kurtz, R., Juhasz, T., Spooner, G. & Mouroua, G. A. Subsurface photodisruption in human sclera: wavelength dependence. Ophthalmic Surg. Lasers Imag. 34, 104–113 (2003).

    Article  Google Scholar 

  16. Kou, L., Labrie, D. & Chylek, P. Refractive indices of water and ice in the 0.65- to 2.5-µm spectral range. Appl. Opt. 32, 3531–3540 (1993).

    Article  ADS  Google Scholar 

  17. Xu, C., Zipfel, W., Shear, J. B., Williams, R. M. & Webb, W. W. Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. Proc. Natl Acad. Sci. USA 93, 10763–10768 (1996).

    Article  ADS  Google Scholar 

  18. Hell, S. W. et al. Three-photon excitation in fluorescence microscopy. J. Biomed. Opt. 1, 71–74 (1996).

    Article  ADS  Google Scholar 

  19. Wokosin, D. L., Centonze, V. E., Crittenden, S. & White, J. Three-photon excitation fluorescence imaging of biological specimens using an all-solid-state laser. Bioimaging 4, 208–214 (1996).

    Article  Google Scholar 

  20. Zysset, B., Beaud, P. & Hodel, W. Generation of optical solitons in the wavelength region 1.37–1.49 µm. Appl. Phys. Lett. 50, 1027–1029 (1987).

    Article  ADS  Google Scholar 

  21. Wang, K. & Xu, C. Tunable high-energy soliton pulse generation from a large-mode-area fiber and its application to third harmonic generation microscopy. Appl. Phys. Lett. 99, 071112 (2011).

    Article  ADS  Google Scholar 

  22. Limpert, J. et al. High-power rod-type photonic crystal fiber laser. Opt. Express 13, 1055–1058 (2005).

    Article  ADS  Google Scholar 

  23. Barad, Y., Eisenberg, H., Horowitz, M. & Silberberg, Y. Nonlinear scanning laser microscopy by third harmonic generation. Appl. Phys. Lett. 70, 922–924 (1997).

    Article  ADS  Google Scholar 

  24. Müller, M., Squier, J., Wilson, K. R. & Brakenhoff, G. J. 3D microscopy of transparent objects using third-harmonic generation. J. Microsc. 191, 266–274 (1998).

    Article  Google Scholar 

  25. Farrar, M. J., Wise, F. W., Fetcho, J. R. & Schaffer, C. B. In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy. Biophys. J. 100, 1362–1371 (2011).

    Article  ADS  Google Scholar 

  26. Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

    Article  ADS  Google Scholar 

  27. Franklin, K. B. J. & Paxinos, G. Mouse Brain in Stereotaxic Coordinates (Academic Press, 2008).

  28. Murphy, P. A. et al. Notch4 normalization reduces blood vessel size in arteriovenous malformations. Sci. Transl. Med. 4, 117ra8 (2012).

    Article  Google Scholar 

  29. Pologruto, T. A., Sabatini, B. L. & Svoboda, K. ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003).

    Article  Google Scholar 

  30. Binding, J. et al. Brain refractive index measured in vivo with high-NA defocus corrected full-field OCT and consequences for two-photon microscopy. Opt. Express 19, 4833–4847 (2011).

    Article  ADS  Google Scholar 

  31. Bacallao, R., Sohrab, S. & Phillips, C. in Handbook of Biological Confocal Microscopy (ed. Pawley, J. B.) 368–380 (Springer, 2006).

  32. Rosidi, N. L. et al. Cortical microhemorrhages cause local inflammation but do not trigger widespread dendrite degeneration. PLoS ONE 6, e26612 (2011).

    Article  ADS  Google Scholar 

Download references

Acknowledgements

This work is partially funded by grants from the National Institutes of Health (NIH; R01CA133148, R01EB014873 and R21RR032392). N.G.H. is supported by the National Science Foundation Graduate Research Fellowship Program (DGE-0707428). The authors acknowledge discussions with D. Dombeck, as well as N. Nishimura and J. Rubin, regarding preparation of the ex vivo brain slices.

Author information

Author notes

  1. Nicholas G. Horton, Ke Wang and Demirhan Kobat: These authors contributed equally to this work

Authors and Affiliations

  1. School of Applied and Engineering Physics, Cornell University, Ithaca, 14853, New York, USA

    Nicholas G. Horton, Ke Wang, Demirhan Kobat, Frank W. Wise & Chris Xu

  2. Department of Biomedical Engineering, Cornell University, Ithaca, 14853, New York, USA

    Catharine G. Clark & Chris B. Schaffer

Authors
  1. Nicholas G. Horton
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ke Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Demirhan Kobat
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Catharine G. Clark
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Frank W. Wise
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Chris B. Schaffer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chris Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.X. initiated and supervised the study. N.G.H., K.W., D.K. and C.G.C. performed the experiments and data analysis. N.G.H., K.W., D.K. and C.X. contributed to the writing and editing of the manuscript. C.B.S. and C.X. contributed to the design of the experiments. F.W. and C.X. contributed to the laser source design.

Corresponding author

Correspondence to Chris Xu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary information

Supplementary information (PDF 982 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Horton, N., Wang, K., Kobat, D. et al. In vivo three-photon microscopy of subcortical structures within an intact mouse brain. Nature Photon 7, 205–209 (2013). https://doi.org/10.1038/nphoton.2012.336

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nphoton.2012.336

This article is cited by

Search

Advanced 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