In vivo imaging of myelin in the vertebrate central nervous system using third harmonic generation microscopy |
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| Authors: | Farrar Matthew J Wise Frank W Fetcho Joseph R Schaffer Chris B |
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| Institution: | †Department of Biomedical Engineering, Cornell University, Ithaca, New York;‡Department of Physics, Cornell University, Ithaca, New York;§Department of Applied and Engineering Physics, Cornell University, Ithaca, New York;¶Department of Neurobiology and Behavior, Cornell University, Ithaca, New York |
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| Abstract: | Loss of myelin in the central nervous system (CNS) leads to debilitating neurological deficits. High-resolution optical imaging of myelin in the CNS of animal models is limited by a lack of in vivo myelin labeling strategies. We demonstrated that third harmonic generation (THG) microscopy—a coherent, nonlinear, dye-free imaging modality—provides micrometer resolution imaging of myelin in the mouse CNS. In fixed tissue, we found that THG signals arose from white matter tracts and were colocalized with two-photon excited fluorescence (2PEF) from a myelin-specific dye. In vivo, we used simultaneous THG and 2PEF imaging of the mouse spinal cord to resolve myelin sheaths surrounding individual fluorescently-labeled axons, and followed myelin disruption after spinal cord injury. Finally, we suggest optical mechanisms that underlie the myelin specificity of THG. These results establish THG microscopy as an ideal tool for the study of myelin loss and recovery. |
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