Implementation of artifact-free circular dichroism SHG imaging of collagen
Implementation of artifact-free circular dichroism SHG imaging of collagen
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DOI：
10.1364/oe.27.022686
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发表时间：
2019-08-05
期刊：
影响因子：
3.8
通讯作者：
Schanne-Klein, Marie-Claire
中科院分区：
文献类型：
--
作者：
Schmeltz, Margaux;Teulon, Claire;Schanne-Klein, Marie-Claire
二次谐波发生 (SHG) 能够对结缔组织中的纤维状胶原蛋白结构进行原位成像。最近，圆二色性 SHG (CD-SHG) 显微镜已被采用，以利用胶原蛋白手性来改善 3D 可视化。它测量左圆偏振与右圆偏振激发时获得的 SHG 信号的归一化差异。然而，CD-SHG 信号尚未得到很好的表征，并且文献中报道了截然不同的 CD-SHG 值。在这里，我们确定了 CD-SHG 实验中可能出现的两个主要伪影，并证明 CD-SHG 成像需要对实验装置进行彻底的优化和校准。值得注意的是，它需要仔细校准入射圆偏振和显微镜载物台的完美机械稳定性。最后，我们成功地在人角膜切片中记录 CD-SHG 图像，并证实该技术有效地揭示了焦平面外的胶原纤维。 (C) 2019 年美国光学学会根据 OSA 开放获取出版协议条款
Second harmonic generation (SHG) enables in situ imaging of fibrillar collagen architecture in connective tissues. Recently, Circular Dichroism SHG (CD-SHG) microscopy has been implemented to take advantage of collagen chirality to improve 3D visualization. It measures the normalized difference in the SHG signal obtained upon excitation by left versus right circular polarizations. However, CD-SHG signal is not well characterized yet, and quite different CD-SHG values are reported in the literature. Here, we identify two major artifacts that may occur in CD-SHG experiments and we demonstrate that thorough optimization and calibration of the experimental setup are required for CD-SHG imaging. Notably it requires a careful calibration of the incident circular polarizations and a perfect mechanical stabilization of the microscope stage. Finally, we successfully record CD-SHG images in human cornea sections and confirm that this technique efficiently reveals collagen fibrils oriented out of the focal plane. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement