Technical Report | Published:

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

Nature Medicine volume 17, pages 223228 (2011) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

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

  2. 2.

    , & Interaction of activated natural killer cells with normal and tumor vessels in cranial windows in mice. Microvasc. Res. 50, 35–44 (1995).

  3. 3.

    , & Visualization of neuromuscular junctions over periods of several months in living mice. J. Neurosci. 7, 1215–1222 (1987).

  4. 4.

    , & Long-term dendritic spine stability in the adult cortex. Nature 420, 812–816 (2002).

  5. 5.

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

  6. 6.

    , , & In vivo imaging of axonal degeneration and regeneration in the injured spinal cord. Nat. Med. 11, 572–577 (2005).

  7. 7.

    , , & Transgenic labeling of the corticospinal tract for monitoring axonal responses to spinal cord injury. Nat. Med. 11, 1355–1360 (2005).

  8. 8.

    , , & Fibrillar amyloid deposition leads to local synaptic abnormalities and breakage of neuronal branches. Nat. Neurosci. 7, 1181–1183 (2004).

  9. 9.

    , , & In vivo imaging of reactive oxygen species specifically associated with thioflavine S–positive amyloid plaques by multiphoton microscopy. J. Neurosci. 23, 2212–2217 (2003).

  10. 10.

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

  11. 11.

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

  12. 12.

    , , , & In vivo multiphoton microscopy of deep brain tissue. J. Neurophysiol. 91, 1908–1912 (2004).

  13. 13.

    , , , & In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy. J. Neurophysiol. 92, 3121–3133 (2004).

  14. 14.

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

  15. 15.

    & Multiphoton endoscopy. Opt. Lett. 28, 902–904 (2003).

  16. 16.

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

  17. 17.

    , , & Minimally invasive high-speed imaging of sarcomere contractile dynamics in mice and humans. Nature 454, 784–788 (2008).

  18. 18.

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

  19. 19.

    Dynamic remodeling of dendritic arbors in GABAergic interneurons of adult visual cortex. PLoS Biol. 4, 271–280 (2006).

  20. 20.

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

  21. 21.

    , & Development of neural circuits in the adult hippocampus. Curr. Top. Dev. Biol. 87, 149–174 (2009).

  22. 22.

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

  23. 23.

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

  24. 24.

    , , , & 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).

  25. 25.

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

  26. 26.

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

  27. 27.

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

  28. 28.

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

  29. 29.

    & Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

  30. 30.

    , , & Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex. Nat. Neurosci. 10, 549–551 (2007).

  31. 31.

    , , & High-resolution in vivo imaging of hippocampal dendrites and spines. J. Neurosci. 24, 3147–3151 (2004).

  32. 32.

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

  33. 33.

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

  34. 34.

    , & Structural changes for adult-born dentate granule cells after status epilepticus. Epilepsia 49, Suppl 5, 13–18 (2008).

  35. 35.

    , , & Fiberoptic system for recording dendritic calcium signals in layer 5 neocortical pyramidal cells in freely moving rats. J. Neurophysiol. 98, 1791–1805 (2007).

  36. 36.

    , , , & Dendritic properties of hippocampal CA1 pyramidal neurons in the rat: intracellular staining in vivo and in vitro. J. Comp. Neurol. 391, 335–352 (1998).

  37. 37.

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

  38. 38.

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

  39. 39.

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

  40. 40.

    , & In vivo fluorescence imaging with high-resolution microlenses. Nat. Methods 6, 511–512 (2009).

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

    • Robert P J Barretto
    • , Tony H Ko
    •  & Juergen C Jung

    These authors contributed equally to this work.

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. Search for Robert P J Barretto in:

  2. Search for Tony H Ko in:

  3. Search for Juergen C Jung in:

  4. Search for Tammy J Wang in:

  5. Search for George Capps in:

  6. Search for Allison C Waters in:

  7. Search for Yaniv Ziv in:

  8. Search for Alessio Attardo in:

  9. Search for Lawrence Recht in:

  10. Search for Mark J Schnitzer in:

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mark J Schnitzer.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–4 and Supplementary Methods

Videos

  1. 1.

    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.

  2. 2.

    Supplementary Video 2

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

  3. 3.

    Supplementary Video 3

    High-speed imaging of microcirculation in a hippocampal glioma using one-photon microendoscopy.

About this article

Publication history

Received

Accepted

Published

DOI

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

Further reading