Chronic in vivo imaging in the mouse spinal cord using an implanted chamber

Journal name:
Nature Methods
Year published:
Published online


Understanding and treatment of spinal cord pathology is limited in part by a lack of time-lapse in vivo imaging strategies at the cellular level. We developed a chronically implanted spinal chamber and surgical procedure suitable for time-lapse in vivo multiphoton microscopy of mouse spinal cord without the need for repeat surgical procedures. We routinely imaged mice repeatedly for more than 5 weeks postoperatively with up to ten separate imaging sessions and observed neither motor-function deficit nor neuropathology in the spinal cord as a result of chamber implantation. Using this chamber we quantified microglia and afferent axon dynamics after a laser-induced spinal cord lesion and observed massive microglia infiltration within 1 d along with a heterogeneous dieback of axon stumps. By enabling chronic imaging studies over timescales ranging from minutes to months, our method offers an ideal platform for understanding cellular dynamics in response to injury and therapeutic interventions.

At a glance


  1. An imaging chamber for longitudinal optical access to mouse spinal cord without the need for repeated surgeries.
    Figure 1: An imaging chamber for longitudinal optical access to mouse spinal cord without the need for repeated surgeries.

    (a) Photograph of the imaging chamber. (b) Schema showing the implantation of the imaging chamber in mice at the T11–T12 vertebra, just below the dorsal fat pad (taupe). (c) Photograph showing the spinal cord imaged through the implanted chamber 144 d after the surgery. (d) Photograph of a mouse with an implanted chamber (same mouse as in c).

  2. Longitudinal 2PEF imaging of axons and blood vessels over many weeks after surgery.
    Figure 2: Longitudinal 2PEF imaging of axons and blood vessels over many weeks after surgery.

    (a) Projections of 2PEF image stacks of afferent axons expressing EYFP (teal) and blood vessels labeled with intravenously injected Texas Red dextran (red) taken over 9 weeks after chamber implantation. Asterisks indicate the location of red autofluorescence from invading, likely inflammatory, cells located above the spinal cord at later time points. Arrows denote landmark features of the axons that were visible at all time points. (b) High-resolution 2PEF imaging of EYFP-expressing axons from the same region as in a. (c) Profile and fit (Online Methods, equation (3)) across maximum intensity projections of selected axon segments shown in the boxed region in b and in the inset; scale bar, 30 μm). A.u., arbitrary units. (d,e) Image contrast (d) and lateral spatial resolution (e) as functions of time after surgery from the fits for all axon segments for two mice (separate curves for each mouse, ~10 axons measured at each time point for each mouse). Error bars, s.d.

  3. Histological analysis of reactive microglia and astrocytes, and tissue morphology after chamber implantation.
    Figure 3: Histological analysis of reactive microglia and astrocytes, and tissue morphology after chamber implantation.

    (a,b) Wide-field fluorescence images of 30-μm-thick coronal tissue sections from the laminectomy site 1 d and 1 week after implantation and in non-surgical controls for mice expressing EGFP in microglia (a) or astrocytes (b). (c) Hematoxylin and eosin–stained tissue section taken 7 d after implantation. Magnifications of the left and right boxed regions show the fibrous connective tissue that covered the dorsal aspect of the spinal cord under the implant and the neural tissue, respectively. (d,e) Microglia (d) and astrocyte (e) densites in spinal cord sections 1 and 7 d after implantation for sections one vertebra rostral to the surgical site (rost.), at the surgical site (surg.) and one vertebra caudal to the surgical site (caud.) and in controls (*P = 0.012; **P = 0.0010; ***P = 0.0098; #P < 0.0001; n ≥ 15 measurements per segment per time point; 3 mice per time point). Error bars, s.e.m.

  4. Imaging and quantification of microglial scar formation at the site of a laser-induced SCI.
    Figure 4: Imaging and quantification of microglial scar formation at the site of a laser-induced SCI.

    (a,b) Projections of 2PEF image stacks of EYFP-labeled axons (teal) and EGFP-labeled microglia (mauve) before (a), 1 d (b) and 1 week (c) after producing a ~200-μm-long laser-induced microlesion in the spinal cord. (d,e) Boxplots of the number of microglia (d) and the microglial scar size (e) in the 300-μm field of view over time (four lesions in two mice). Horizontal red lines denote the median, blue boxes bound the 25th and 75th percentiles of the data, and the whiskers denote non-outlier extrema (defined as outside the box by less than 1.5 times the interquartile range).

  5. 2PEF imaging and quantification of axon dieback after a laser-induced SCI.
    Figure 5: 2PEF imaging and quantification of axon dieback after a laser-induced SCI.

    (a) Projection of a 2PEF image stack from mice expressing EYFP (teal) in a subset of DRG neurons with the vasculature labeled with Texas Red dextran (red). (b) Projections of 2PEF image stacks of EYFP-expressing axons shown in the boxed region in a before and at indicated times after a lesion produced by translating high-energy, tightly focused femtosecond laser pulses through the cord. Mauve circles indicate easily recognizable patterns in spared axons that were identified at all time points and provide a point of origin. Yellow arrows, axon that exhibited rapid degeneration; blue arrows, axon that died back more slowly; red arrows, axon that persisted near the lesion site and made an ultimately aborted growth response (the morphology of this axon's tip is shown in the insets; scale bar in inset, 10 μm); and *, location of early sprouting responses that did not persist over time. (c) Position of axon endings over time after the lesion, with positive and negative values corresponding to positions rostral and caudal to the lesion site, respectively (see schematic in inset) (107 individual axon trajectories over nine lesions in five mice). Axon trajectories in color correspond to the locations marked by respectively colored arrows in b. (d) Speed of axon-tip dieback for axons remaining in the field of view over time after the lesion. Black circles denote measurements of dieback speed from individual axon tips, horizontal red lines represent the median, the blue boxes bound the 25th to 75th percentage of the data, and the whiskers extend 1.5 times the interquartile range beyond the boxes. Points outside the whiskers were considered outliers and have a red cross through them. Because axons died back beyond the imaging field over time, the dieback speed at early times includes data from ~100 axons, and the last time point includes data for only 16 axons.


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Author information


  1. Department of Physics, Cornell University, Ithaca, New York, USA.

    • Matthew J Farrar
  2. Department of Biomedical Engineering, Cornell University, Ithaca, New York, USA.

    • Matthew J Farrar,
    • Ida M Bernstein &
    • Chris B Schaffer
  3. Department of Biomedical Sciences, Cornell University, Ithaca, New York, USA.

    • Donald H Schlafer
  4. Department of Psychology, Cornell University, Ithaca, New York, USA.

    • Thomas A Cleland
  5. Department of Neurobiology and Behavior, Cornell University, Ithaca, New York, USA.

    • Joseph R Fetcho


M.J.F., T.A.C., J.R.F. and C.B.S. conceived and designed the experiments. M.J.F. performed surgeries and imaging experiments, I.M.B. performed behavioral assays, and D.H.S. performed histopathology. M.J.F., I.M.B., J.R.F. and C.B.S. analyzed data. J.R.F., T.A.C. and C.B.S. contributed reagents and materials. M.J.F., J.R.F. and C.B.S. wrote the paper.

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The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (2M)

    Supplementary Figures 1–5, Supplementary Notes 1–7, Supplementary Protocol


  1. Supplementary Video 1 (8M)

    Rendering of MicroCT image data from a mouse taken 6 d after implantation of the imaging chamber, showing normal spine alignment and no vertebral damage.

  2. Supplementary Video 2 (8M)

    Video of a mouse taken 2 weeks after implanting the imaging chamber, showing locomotion, grooming and exploratory behavior.

  3. Supplementary Video 3 (30M)

    A series of 2PEF image stacks taken at different times after a laser-induced spinal cord injury, showing GFP-expressing axons (green) and Texas Red-dextran labeled vasculature (red).

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