We demonstrate the use of wide-field high-throughput second-harmonic (SH) microscopy for investigating cytoskeletal morphological changes on the single-cell level. The method allows for real-time, in vitro, label-free measurements of cytoskeletal changes that can, under certain conditions, be quantified in terms of orientational distribution or in terms of changes in the number of microtubules. As SH generation is intrinsically sensitive to noncentrosymmetrically structured microtubules, but not to isotropic or centrosymmetric materials, we use it to probe the microtubule structure in the cytoskeleton when it undergoes dynamic changes induced by the application of nocodazole, a well-known microtubule-destabilizing drug that reversibly depolymerizes microtubules. In addition, the orientational directionality of microtubules in neurites and cell bodies is determined label-free using SH polarimetry measurements. Finally, we use spatiotemporal SH imaging to show label-free, real-time nocodazole-induced morphological changes in neurons of different age and in a single axon.

中文翻译：

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无标记的广域二次谐波成像在神经元细胞骨架中实时形态变化的映射：诺考达唑的案例研究。

我们展示了广域高通量二次谐波（SH）显微镜的使用，用于研究单细胞水平的细胞骨架形态变化。该方法允许对细胞骨架变化进行实时，体外，无标记的测量，在某些条件下，可以根据方向分布或根据微管数量的变化进行定量。由于SH生成本质上对非中心对称结构的微管敏感，但对各向同性或中心对称材料不敏感，因此当它受到因使用已知的微管稳定药物nocodazole引起的动态变化时，我们用它来探测细胞骨架中的微管结构。可逆地解聚微管。此外，使用SH旋光法测量无标记的神经突和细胞体内微管的定向方向性。最后，我们使用时空SH成像显示在不同年龄和单个轴突中无标记的，实时的诺考达唑诱导的形态变化。