In Vivo Imaging of Myelin in the Vertebrate Central Nervous System Using Third Harmonic Generation Microscopy

• Published in: Biophysical journal Vol. 100; no. 5; pp. 1362 - 1371
• Main Authors: Farrar, Matthew J., Wise, Frank W., Fetcho, Joseph R., Schaffer, Chris B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02-03-2011; Biophysical Society; The Biophysical Society
• Subjects: animal models; Animals; axons; Axons - metabolism; Biophysics; Body fat; Fluorescence; image analysis; Larva - cytology; Mice; Microscopy; Microscopy - methods; Molecular Imaging - methods; myelin sheath; Myelin Sheath - metabolism; Myelin Sheath - physiology; Nervous system; Optical Phenomena; Optics; Rodents; Spectroscopy, Imaging, and Other Techniques; spinal cord; Spinal Cord - cytology; Spinal Cord - physiology; Spinal cord injuries; Spinal Cord Injuries - pathology; Spinal Cord Injuries - physiopathology; Vertebrates; Zebrafish
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2011.01.031

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.