A new label-free method is presented for measuring myeloarchitecture of the murine cerebral cortex in vivo and ex vivo. Growing evidence suggests that cortical myelination plays significant roles in neuronal plasticity and pathologies, such as multiple sclerosis (MS), but illuminating the mechanism requires longitudinal imaging of the same brains. Here we demonstrate imaging unlabeled myelinated fibers in a live mouse brain by third-harmonic generation (THG). Contrary to other label-free microscopies based on reflectance, fibers of all orientations could be visualized, i.e., radial and tangential to the pia, which is suitable for revealing the three-dimensional connectivity. The depth of THG imaging in an intact brain was approximately 200 μm, so the network of myelinated fibers could be captured into layers 2/3 in vivo. THG provides a novel base for reconstruction of morphology. Semi-automatic tracing of THG-positive axons unraveled the depth-dependent distribution of the myelin lattice. Finally, a unique light property of THG was exploited for the estimation of the g-ratio. The demonstrated THG morphometry of the length density, orientation, and sheath thickness of cortical myelin could be useful for elucidating its function and how it is modulated during learning and disease.

中文翻译：

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一种通过本征三次谐波产生体内和离体测量小鼠脑皮质髓结构的方法。

提出了一种新的无标记方法，用于体内和离体测量鼠脑皮质的骨髓结构。越来越多的证据表明，皮质髓鞘形成在神经元可塑性和病理学（例如多发性硬化症（MS））中起着重要作用，但要阐明这种机制，需要对同一大脑进行纵向成像。在这里，我们展示了通过三次谐波产生（THG）在活的小鼠大脑中成像未标记的髓鞘纤维。与其他基于反射率的无标签显微技术相反，可以看到所有方向的纤维，即径向和与pia相切的纤维，适合显示三维连通性。完整大脑中THG成像的深度约为200μm，因此髓鞘纤维网络可以在体内捕获到2/3层。THG为形态重建提供了新的基础。THG阳性轴突的半自动跟踪揭示了髓鞘晶格的深度依赖分布。最后，THG的独特光特性被用于估计g比。皮质髓磷脂的长度密度，方向和鞘厚度的已证实的THG形态学可能有助于阐明其功能以及在学习和疾病过程中如何调节其功能。