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.myelin | Schwann cell | multiphoton microscopy | label-free imaging | morphometry M 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 s...

Label‐free non‐linear microscopy to measure myelin outcome in a rodent model of Charcot‐Marie‐Tooth diseases

“…Using CARS imaging, the myelin sheath is directly labelled thank to its rich content in lipids and it appears as a large band surrounding axons. These results are consistent with previous analysis by specialists in optics [18,19] and in peripheral nerve biology [7,14].…”

“…Using YFP axonal labelling, we found that this hatched signal came from a domain outlining the axon (red arrowheads, Figure 1I) and a domain outlining the myelin sheath (black arrowheads, Figure 1I). Taken together these data and previous reports in the literature [14] indicated that THG signal was produced at interfaces of the myelin with the axon (red arrowheads, Figure 1H,I) and of the myelin and the extracellular environment (black arrowheads, Figure 1H,I). These THG signals appeared as very large bands when the focus reached the surface of these interfaces (red and black lines, Figure 1H).…”

Label-free characterization of visual cortical areas in awake mice via three-photon microscopy reveals correlations between functional maps and structural substrates

“…Cortical areas with one sign (V1, AL, AM) may all represent similar thalamocortical (or intracortical) fiber orientations compared to areas with the opposite sign (RL, LM, PM). These fiber orientations would then be reflected in the orientations of myelin and even blood vessels 22,46,47 as measured with THG imaging that contribute to EALs.…”

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.…”