Second harmonic generation (SHG) microscopy is a commonly used technique to study the organization of collagen within tissues. However, individual collagen fibrils, which have diameters much smaller than the resolution of most optical systems, have not been extensively investigated. Here we probe the structure of individual collagen fibrils using polarization-resolved SHG (PSHG) microscopy and atomic force microscopy. We find that longitudinally polarized light occurring at the edge of a focal volume of a high numerical aperture microscope objective illuminated with linearly polarized light creates a measurable variation in PSHG signal along the axis orthogonal to an individual collagen fibril. By comparing numerical simulations to experimental data, we are able to estimate parameters related to the structure and chirality of the collagen fibril without tilting the sample out of the image plane, or cutting tissue at different angles, enabling chirality measurements on individual nanostructures to be performed in standard PSHG microscopes. The results presented here are expected to lead to a better understanding of PSHG results from both collagen fibrils and collagenous tissues. Further, the technique presented can be applied to other chiral nanoscale structures such as microtubules, nanowires, and nanoribbons.

中文翻译：

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高数值孔径成像允许使用偏振二次谐波发生显微镜测量单个胶原原纤维的手性


二次谐波发生 (SHG) 显微镜是研究组织内胶原组织的常用技术。然而，单个胶原原纤维的直径远小于大多数光学系统的分辨率，尚未得到广泛研究。在这里，我们使用偏振分辨二次谐波 (PSHG) 显微镜和原子力显微镜探测单个胶原原纤维的结构。我们发现，在用线性偏振光照射的高数值孔径显微镜物镜的焦点体积边缘发生的纵向偏振光沿与单个胶原纤维正交的轴产生可测量的 PSHG 信号变化。通过将数值模拟与实验数据进行比较，我们能够估计与胶原原纤维的结构和手性相关的参数，而无需将样品倾斜出图像平面或以不同角度切割组织，从而能够对单个纳米结构进行手性测量在标准 PSHG 显微镜中。这里提出的结果预计将有助于更好地了解胶原原纤维和胶原组织的 PSHG 结果。此外，所提出的技术可以应用于其他手性纳米级结构，例如微管、纳米线和纳米带。