Label-free Imaging of Schwann Cell Myelination by Third Harmonic Generation Microscopy

Abstract

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 THGMbased 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.yelin 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 timeconsuming 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 ...

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“…Oligodendrocytes and Schwann cells are two types of cells existing in the CNS and peripheral nervous system, respectively. Both of them are capable of promoting myelination (Lim et al, 2014;Wegener et al, 2015). Then, the following BBB scale scores showed that partial recovery of spinal-cord-injured rats 4 weeks' posttransplantation and the gaps between the proximal and distal stumps were 100% closed in transected saphenous nerve rats posttransplantation.…”

Third harmonic generation microscopy of cells and tissue organization

“…In ex vivo and in vivo preparations of sciatic nerves of mice and rats, THG can be used to further resolve subdomains of myelinated peripheral nerves, including nodes of Ranvier, SchmidtLanterman incisures and Cajal bands. By using THG, myelination characteristics are amenable to rapid acquisition and bias-free automated quantification over large imaging fields (Lim et al, 2014). Therefore, THG might be useful to trace myelin loss and recovery in live tissue; for example, the degeneration of myelin sheets in multiple sclerosis, which causes deficits in sensory and motor functions of the demyelinated nerves.…”

Uncovering the biology of myelin with optical imaging of the live brain

“…Similar to OCM's dependence on differential tissue scattering, THG microscopy is a nonlinear imaging approach that generates a signal at lipid–water interfaces due to nonlinear coherent scattering (Weigelin, Bakker, & Friedl, ). Again based on the unique biophysical properties of myelin, THG can be used for label‐free imaging of myelin in vivo (Farrar et al, ; Lim et al, ). THG imaging requires high intensity long wavelength lasers and high laser power in order to generate bright signals thus photodamage or thermal injury is a possibility.…”