Label-free imaging of Schwann cell myelination by third harmonic generation microscopy

Understanding the dynamic axon–glial cell interaction underlying myelination is hampered by the lack of suitable imaging techniques. Here we demonstrate third harmonic generation microscopy (THGM) for label-free imaging of myelinating Schwann cells in live culture and ex vivo and in vivo tissue. A 3D structure was acquired for a variety of compact and noncompact myelin domains, including juxtaparanodes, Schmidt–Lanterman incisures, and Cajal bands. Other subcellular features of Schwann cells that escape traditional optical microscopies were also visualized. We tested THGM for morphometry of compact myelin. Unlike current methods based on electron microscopy, g-ratio could be determined along an extended length of myelinated fiber in the physiological condition. The precision of THGM-based g-ratio estimation was corroborated in mouse models of hypomyelination. Finally, we demonstrated the feasibility of THGM to monitor morphological changes of myelin during postnatal development and degeneration. The outstanding capabilities of THGM may be useful for elucidation of the mechanism of myelin formation and pathogenesis.Myelin is a multiple-layered membrane sheath surrounding the axon. In myelinated nerves, the conduction of action potentials is much faster and the speed depends strongly on the structure of myelin. The structural integrity must be therefore tightly regulated for proper conduction of neuronal impulses, but the underlying axon–glial cell interaction is not well understood. Since the days of Ramón y Cajal, light microscopy has been widely used to visualize myelin morphology (1). A variety of fluorescent probes specifically binding to myelin components have allowed studies of the interaction between molecules (2–4). However, most such labeling methods are not suitable for unraveling in vivo dynamics of myelination because cell membranes are compromised during staining (especially immunohistochemistry) and/or the procedures are prohibitively time-consuming and invasive. It is thus of great interest to develop label-free methods. Recently, spectral confocal reflectance microscopy has been demonstrated for high-resolution in vivo imaging of myelin (5). There are also techniques of nonlinear optical microscopy, which are generally known to be more advantageous for imaging deep live tissue. Coherence anti-Stokes Raman scattering (CARS) microscopy, which requires two synchronized short pulse lasers for excitation, has been used for imaging in vivo myelin and detecting pathology (6, 7). Third harmonic generation (THG) microscopy is based on another nonlinear optical process of light emission, yielding distinguishable images from CARS. Though it has been demonstrated for imaging the white matter in the brain (8), so far few studies have applied THG microscopy (THGM) for elucidating the mechanism of myelin formation. Moreover, the omission of the peripheral nervous systems (PNS) in the previous studies is not trivial considering the significant departure from the CNS in terms of the myelin-forming glia and molecular subdomains: Schwann cells wrap individual internodes in the PNS, and oligodendrocytes form multiple myelin sheaths in the CNS. The basal lamina and nodal microvilli are unique to Schwann cells, and Schmidt–Lanterman incisures are more pronounced in the PNS (9). Here we demonstrate, to our knowledge for the first time, the utility of THGM as a method for morphological analysis of myelinating Schwann cells in live culture and ex vivo and in vivo tissue.