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Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy

Abstract

We report a newly developed technique for high-resolution in vivo imaging of myelinated axons in the brain, spinal cord and peripheral nerve that requires no fluorescent labeling. This method, based on spectral confocal reflectance microscopy (SCoRe), uses a conventional laser-scanning confocal system to generate images by merging the simultaneously reflected signals from multiple lasers of different wavelengths. Striking color patterns unique to individual myelinated fibers are generated that facilitate their tracing in dense axonal areas. These patterns highlight nodes of Ranvier and Schmidt-Lanterman incisures and can be used to detect various myelin pathologies. Using SCoRe we carried out chronic brain imaging up to 400 μm deep, capturing de novo myelination of mouse cortical axons in vivo. We also established the feasibility of imaging myelinated axons in the human cerebral cortex. SCoRe adds a powerful component to the evolving toolbox for imaging myelination in living animals and potentially in humans.

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Figure 1: In vivo imaging of mouse cortex using spectral confocal reflectance microscopy (SCoRe).
Figure 2: SCoRe signal is dependent on myelination.
Figure 3: Transcranial time-lapse imaging of the mouse cortex reveals progressive age-dependent myelination.
Figure 4: Multicolor reflection spectrum reveals distinct myelin structures in the spinal cord and sciatic nerve in vivo.
Figure 5: Myelin pathology and human myelinated axons imaged with SCoRe.

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Acknowledgements

This study was supported by the following grants: R01AG027855 and R01HL106815 (J.G.). We would like to thank J. Bewersdorf and D. Toomre for helpful discussions and P. Yuan for critical reading of the manuscript. We thank A. Nishiyama (University of Connecticut) and F. Kirchhoff (University of Saarland) for providing PLPDsRed mice. Postmortem human specimen was obtained from the brain bank in the Cognitive Neurology and Alzheimer's disease Center (CNADC) at Northwestern University (grant AG13854).

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Authors

Contributions

A.J.S. and J.G. conceived and designed the initial project. A.J.S. and R.A.H. carried out the experiments. All authors contributed to experimental design, data analysis and manuscript. J.G. supervised the project.

Corresponding author

Correspondence to Jaime Grutzendler.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 15505 kb)

Three dimensional rendering of a SCoRe image captured in vivo in a Thy1-YFP transgenic mouse.

A single YFP-labeled (green) reflective (magenta) axon (arrow) is shown among many non myelinated YFP-labeled axons and dendrites. (AVI 8154 kb)

Representative 450 μm z stack of the mouse cortex acquired in vivo with SCoRe.

Video demonstrates the depth capabilities of SCoRe which maintains a high signal to noise ratio even at ~400 μm. The decrease in the number of reflected fibers with depth indicates the drop-off in the number of myelinated fibers just below cortical layer 1 projection axons in addition to the change in the orientation of some of the myelinated axons as SCoRe is not able to efficiently detect myelinated axons running orthogonally. Step size 1 μm, depth indicated in upper left corner and video displayed at 10 frames per second. (AVI 23477 kb)

Three dimensional rendering of a SCoRe image from the sciatic nerve.

Video shows the unique reflected spectrum from individual axons after removing the layers of the highly reflective and disordered signal from the sciatic epineurium. (AVI 6094 kb)

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Schain, A., Hill, R. & Grutzendler, J. Label-free in vivo imaging of myelinated axons in health and disease with spectral confocal reflectance microscopy. Nat Med 20, 443–449 (2014). https://doi.org/10.1038/nm.3495

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