Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues
Second harmonic and sum frequency generation imaging of fibrous astroglial filaments in ex vivo spinal tissues
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DOI：
10.1529/biophysj.106.089011
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发表时间：
2007-05-01
影响因子：
3.4
通讯作者：
Cheng, Ji-Xin
中科院分区：
文献类型：
--
作者：
Fu, Yan;Wang, Haifeng;Cheng, Ji-Xin
本文观察了豚鼠脊髓白质螺旋纤维的和频产生(SFG)和二次谐波产生(SHG)。通过结合SFG和相干反斯托克斯拉曼散射显微镜，可以显示有髓轴突，这些纤维被发现分布在脊髓表面附近，相邻轴突之间，并沿着血管分布。利用20微米厚的组织切片，发现来自大束的倍频信号与来自小的单束的倍频信号的比值要大得多，这表明相干显微镜中存在位相匹配效应。根据纤维间的强度分布和同一纤维的前向和后向信号的大小依赖关系，我们得出结论：主要的倍频信号直接来自纤维，而不是表面倍频效应。进一步对倍频信号的偏振分析表明，纤丝的对称性可以用圆柱模型很好地描述。SHG信号与胶质纤维酸性蛋白免疫染色的双光子激发荧光(TPEF)共同定位，表明SHG信号来源于星形胶质细胞。哀叹。SHG对比信号与来自星形胶质细胞突起的TPEF信号的共同定位进一步支持了这一指定，该信号由钙离子指示剂和磺胺荷丹明101标记。这项工作表明，三种非线性光学成像技术的组合-相干反斯托克斯拉曼散射、TPEF和倍频(SFG)显微镜-允许同时显示复杂生物系统中的不同结构。
Sum frequency generation (SFG) and second harmonic generation (SHG) were observed from helical fibrils in spinal cord white matter isolated from guinea pigs. By combining SFG with coherent anti-Stokes Raman scattering microscopy, which allows visualization of myelinated axons, these fibers were found to be distributed near the surface of the spinal cord, between adjacent axons, and along the blood vessels. Using 20-mu m-thick tissue slices, the ratio of forward to backward SHG signal from large bundles was found to be much larger than that from small single fibrils, indicating a phase-matching effect in coherent microscopy. Based on the intensity profiles across fibrils and the size dependence of forward and backward signal from the same fibril,we concluded that the main SHG signal directly originates from the fibrils, but not from surface SHG effects. Further polarization analysis of the SHG signal showed that the symmetry property of the fibril could be well described with a cylindrical model. Colocalization of the SHG signal with two-photon excitation fluorescence (TPEF) from the immunostaining of glial fibrillary acidic protein demonstrated that SHG arises from astroglial. laments. This assignment was further supported by colocalization of the SHG contrast with TPEF signals from astrocyte processes labeled by a Ca2+ indicator and sulforhodamine 101. This work shows that a combination of three nonlinear optical imaging techniques-coherent anti-Stokes Raman scattering, TPEF, and SHG (SFG) microscopy-allows simultaneous visualization of different structures in a complex biological system.