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.

  • Technical Report
  • Published:

Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy

Nature Medicine volume 17, pages 223–228 (2011)Cite this article

Subjects

Abstract

The combination of intravital microscopy and animal models of disease has propelled studies of disease mechanisms and treatments. However, many disorders afflict tissues inaccessible to light microscopy in live subjects. Here we introduce cellular-level time-lapse imaging deep within the live mammalian brain by one- and two-photon fluorescence microendoscopy over multiple weeks. Bilateral imaging sites allowed longitudinal comparisons within individual subjects, including of normal and diseased tissues. Using this approach, we tracked CA1 hippocampal pyramidal neuron dendrites in adult mice, revealing these dendrites' extreme stability and rare examples of their structural alterations. To illustrate disease studies, we tracked deep lying gliomas by observing tumor growth, visualizing three-dimensional vasculature structure and determining microcirculatory speeds. Average erythrocyte speeds in gliomas declined markedly as the disease advanced, notwithstanding significant increases in capillary diameters. Time-lapse microendoscopy will be applicable to studies of numerous disorders, including neurovascular, neurological, cancerous and trauma-induced conditions.

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: Chronic mouse preparation for repeated imaging of deep brain tissues using microendoscopy.
Figure 2: Time-lapse two-photon microendoscopy of CA1 hippocampal neurons.
Figure 3: Time-lapse microendoscopy of CA1 microvasculature shows normal blood vessel morphologies are stable over time.
Figure 4: Time-lapse imaging of glioma angiogenesis in mouse CA1 reveals progressive distortions to vascular geometry and reduced microcirculatory speeds.
Figure 5: Quantitative tracking of glioma angiogenesis in CA1 hippocampus shows tumor vessels broaden in diameter but undergo marked declines in flow speed.

Similar content being viewed by others

References

  1. Misgeld, T. & Kerschensteiner, M. In vivo imaging of the diseased nervous system. Nat. Rev. Neurosci. 7, 449–463 (2006).

    Article  CAS  Google Scholar 

  2. Melder, R.J., Salehi, H.A. & Jain, R.K. Interaction of activated natural killer cells with normal and tumor vessels in cranial windows in mice. Microvasc. Res. 50, 35–44 (1995).

    Article  CAS  Google Scholar 

  3. Lichtman, J.W., Magrassi, L. & Purves, D. Visualization of neuromuscular junctions over periods of several months in living mice. J. Neurosci. 7, 1215–1222 (1987).

    Article  CAS  Google Scholar 

  4. Grutzendler, J., Kasthuri, N. & Gan, W.B. Long-term dendritic spine stability in the adult cortex. Nature 420, 812–816 (2002).

    Article  CAS  Google Scholar 

  5. Trachtenberg, J.T. et al. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature 420, 788–794 (2002).

    Article  CAS  Google Scholar 

  6. Kerschensteiner, M., Schwab, M.E., Lichtman, J.W. & Misgeld, T. In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat. Med. 11, 572–577 (2005).

    Article  CAS  Google Scholar 

  7. Bareyre, F.M., Kerschensteiner, M., Misgeld, T. & Sanes, J.R. Transgenic labeling of the corticospinal tract for monitoring axonal responses to spinal cord injury. Nat. Med. 11, 1355–1360 (2005).

    Article  CAS  Google Scholar 

  8. Tsai, J., Grutzendler, J., Duff, K. & Gan, W.B. Fibrillar amyloid deposition leads to local synaptic abnormalities and breakage of neuronal branches. Nat. Neurosci. 7, 1181–1183 (2004).

    Article  CAS  Google Scholar 

  9. McLellan, M.E., Kajdasz, S.T., Hyman, B.T. & Bacskai, B.J. In vivo imaging of reactive oxygen species specifically associated with thioflavine S–positive amyloid plaques by multiphoton microscopy. J. Neurosci. 23, 2212–2217 (2003).

    Article  CAS  Google Scholar 

  10. Meyer-Luehmann, M. et al. Rapid appearance and local toxicity of amyloid-β plaques in a mouse model of Alzheimer's disease. Nature 451, 720–724 (2008).

    Article  CAS  Google Scholar 

  11. Yuan, F. et al. Vascular permeability and microcirculation of gliomas and mammary carcinomas transplanted in rat and mouse cranial windows. Cancer Res. 54, 4564–4568 (1994).

    CAS  Google Scholar 

  12. 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 

  13. 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 

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

    Article  CAS  Google Scholar 

  15. Jung, J.C. & Schnitzer, M.J. Multiphoton endoscopy. Opt. Lett. 28, 902–904 (2003).

    Article  Google Scholar 

  16. Hsiung, P.L. et al. Detection of colonic dysplasia in vivo using a targeted heptapeptide and confocal microendoscopy. Nat. Med. 14, 454–458 (2008).

    Article  CAS  Google Scholar 

  17. Llewellyn, M.E., Barretto, R.P., Delp, S.L. & Schnitzer, M.J. Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans. Nature 454, 784–788 (2008).

    Article  CAS  Google Scholar 

  18. Feng, G. et al. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28, 41–51 (2000).

    Article  CAS  Google Scholar 

  19. Lee, W.-C.A. Dynamic remodeling of dendritic arbors in GABAergic interneurons of adult visual cortex. PLoS Biol. 4, 271–280 (2006).

    Article  CAS  Google Scholar 

  20. Chow, D.K. et al. Laminar and compartmental regulation of dendritic growth in mature cortex. Nat. Neurosci. 12, 116–118 (2009).

    Article  CAS  Google Scholar 

  21. Li, Y., Mu, Y. & Gage, F.H. Development of neural circuits in the adult hippocampus. Curr. Top. Dev. Biol. 87, 149–174 (2009).

    Article  CAS  Google Scholar 

  22. Lim, D.A. et al. Relationship of glioblastoma multiforme to neural stem cell regions predicts invasive and multifocal tumor phenotype. Neuro-oncol. 9, 424–429 (2007).

    Article  Google Scholar 

  23. Larjavaara, S. et al. Incidence of gliomas by anatomic location. Neuro-oncol. 9, 319–325 (2007).

    Article  Google Scholar 

  24. Ramnarayan, R., Dodd, S., Das, K., Heidecke, V. & Rainov, N.G. Overall survival in patients with malignant glioma may be significantly longer with tumors located in deep grey matter. J. Neurol. Sci. 260, 49–56 (2007).

    Article  Google Scholar 

  25. Jang, T. et al. A distinct phenotypic change in gliomas at the time of magnetic resonance imaging detection. J. Neurosurg. 108, 782–790 (2008).

    Article  Google Scholar 

  26. Jain, R.K. et al. Angiogenesis in brain tumours. Nat. Rev. Neurosci. 8, 610–622 (2007).

    Article  CAS  Google Scholar 

  27. Kashiwagi, S. et al. Perivascular nitric oxide gradients normalize tumor vasculature. Nat. Med. 14, 255–257 (2008).

    Article  CAS  Google Scholar 

  28. Garkavtsev, I. et al. The candidate tumour suppressor protein ING4 regulates brain tumour growth and angiogenesis. Nature 428, 328–332 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  30. Xu, H.T., Pan, F., Yang, G. & Gan, W.B. Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nat. Neurosci. 10, 549–551 (2007).

    Article  CAS  Google Scholar 

  31. Mizrahi, A., Crowley, J.C., Shtoyerman, E. & Katz, L.C. High-resolution in vivo imaging of hippocampal dendrites and spines. J. Neurosci. 24, 3147–3151 (2004).

    Article  CAS  Google Scholar 

  32. Dierssen, M. & Ramakers, G.J. Dendritic pathology in mental retardation: from molecular genetics to neurobiology. Genes Brain Behav. 5, Suppl 2, 48–60 (2006).

    Article  CAS  Google Scholar 

  33. Li, P. & Murphy, T.H. Two-photon imaging during prolonged middle cerebral artery occlusion in mice reveals recovery of dendritic structure after reperfusion. J. Neurosci. 28, 11970–11979 (2008).

    Article  CAS  Google Scholar 

  34. Shapiro, L.A., Ribak, C.E. & Jessberger, S. Structural changes for adult-born dentate granule cells after status epilepticus. Epilepsia 49, Suppl 5, 13–18 (2008).

    Article  Google Scholar 

  35. Murayama, M., Perez-Garci, E., Luscher, H.R. & Larkum, M.E. Fiberoptic system for recording dendritic calcium signals in layer 5 neocortical pyramidal cells in freely moving rats. J. Neurophysiol. 98, 1791–1805 (2007).

    Article  Google Scholar 

  36. Pyapali, G.K., Sik, A., Penttonen, M., Buzsaki, G. & Turner, D.A. Dendritic properties of hippocampal CA1 pyramidal neurons in the rat: intracellular staining in vivo and in vitro. J. Comp. Neurol. 391, 335–352 (1998).

    Article  CAS  Google Scholar 

  37. Louis, D.N. Molecular pathology of malignant gliomas. Annu. Rev. Pathol. 1, 97–117 (2006).

    Article  CAS  Google Scholar 

  38. Szatmari, T. et al. Detailed characterization of the mouse glioma 261 tumor model for experimental glioblastoma therapy. Cancer Sci. 97, 546–553 (2006).

    Article  CAS  Google Scholar 

  39. Flusberg, B.A. et al. High-speed, miniaturized fluorescence microscopy in freely moving mice. Nat. Methods 5, 935–938 (2008).

    Article  CAS  Google Scholar 

  40. Barretto, R.P., Messerschmidt, B. & Schnitzer, M.J. In vivo fluorescence imaging with high-resolution microlenses. Nat. Methods 6, 511–512 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was initiated under National Institute on Drug Abuse CEBRA DA017895 and further supported by National Institute of Neurological Disorders and Stroke R01NS050533, National Cancer Institute P50CA114747 and a research contract with Mauna Kea Technologies. We gratefully acknowledge support from the Stanford–US National Institutes of Health Biophysics Program (R.P.J.B.) and a Machiah postdoctoral fellowship (Y.Z.). We thank M. Lim and J. Weimann for help with GL261 cells, S. Kim, T. Jang, A. Lui, J. Li and M. Ramkumar for technical assistance with histology and image processing, E. Mukamel for helpful conversations and B. Colyear and B. Wilt for help with graphic illustration.

Author information

Author notes

  1. Robert P J Barretto, Tony H Ko and Juergen C Jung: These authors contributed equally to this work.

Authors and Affiliations

  1. James H. Clark Center for Biomedical Engineering & Sciences, Stanford University, Stanford, California, USA

    Robert P J Barretto, Tony H Ko, Juergen C Jung, Tammy J Wang, George Capps, Allison C Waters, Yaniv Ziv, Alessio Attardo & Mark J Schnitzer

  2. Department of Neurology and Neurological Sciences, Stanford University, Stanford, California, USA

    Lawrence Recht

  3. Department of Neurosurgery, Stanford University, Stanford, California, USA

    Lawrence Recht

  4. Howard Hughes Medical Institute, Stanford University, Stanford, California, USA

    Mark J Schnitzer

  5. CNC Program, Stanford University, Stanford, California, USA

    Mark J Schnitzer

Authors
  1. Robert P J Barretto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tony H Ko
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Juergen C Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tammy J Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. George Capps
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Allison C Waters
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yaniv Ziv
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Alessio Attardo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Lawrence Recht
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Mark J Schnitzer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.P.J.B. designed experiments, developed tracking of neuronal dendrites, performed the study on CA1 neuron stability, analyzed the neuronal histology data, validated the algorithm for computing erythrocyte speeds and computed relationships between vessel diameters and speeds. T.H.K. designed experiments, performed the glioma experiments and computed flow speeds and vessel sizes. J.C.J. designed experiments, developed the chronic preparation and tested it for imaging neurons and gliomas. T.J.W. and G.C. performed neuronal imaging and contributed to the glioma experiments. A.C.W. developed bilateral imaging, performed neuronal imaging and contributed to the glioma experiments. Y.Z. developed and performed striatal imaging. A.A. performed histological analyses and analyzed vessel branching ratios. L.R. designed experiments and supervised the glioma study. M.J.S. designed experiments, performed statistical testing, initiated and supervised the project and wrote the paper. All authors edited the paper.

Corresponding author

Correspondence to Mark J Schnitzer.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Methods (PDF 1262 kb)

Supplementary Video 1

Three-dimensional image stack of hippocampal blood vessels acquired in a live mouse by two-photon microendoscopy and intravascular injection of fluorescein-dextran. (MOV 2999 kb)

Supplementary Video 2

Hippocampal microcirculation in normal tissue imaged by high-speed one-photon microendoscopy and intravascular injection of fluorescein-dextran. (MOV 7787 kb)

Supplementary Video 3

High-speed imaging of microcirculation in a hippocampal glioma using one-photon microendoscopy. (MOV 6488 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Barretto, R., Ko, T., Jung, J. et al. Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy. Nat Med 17, 223–228 (2011). https://doi.org/10.1038/nm.2292

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.2292

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer