Second-harmonic generation (SHG) microscopy is currently the preferred technique for visualizing collagen in intact tissues, but the usual implementations struggle to reveal collagen fibrils oriented out of the imaging plane. Recently, an advanced SHG modality, circular dichroism SHG (CD-SHG), has been proposed to specifically highlight out-of-plane fibrils. In this study, we present a theoretical analysis of CD-SHG signals that goes beyond the electric dipolar approximation to account for collagen chirality. We demonstrate that magnetic dipolar contributions are necessary to analyze CD-SHG images of human cornea sections and other collagen-rich samples. We show that the sign of CD-SHG signals does not reveal whether collagen fibrils point upwards or downwards as tentatively proposed previously. CD-SHG instead probes the polarity distribution of out-of-plane fibril assemblies at submicrometer scale, namely homogeneous polarity versus a mix of antiparallel fibrils. This makes CD-SHG a powerful tool for characterizing collagen organization in tissues, specifically the degree of disorder, which is affected during pathological remodeling. CD-SHG may thus serve to discriminate healthy and diseased collagen-rich tissues.

中文翻译：

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圆二色性二次谐波显微镜探测胶原原纤维的极性分布

目前，二次谐波生成（SHG）显微镜是使完整组织中的胶原蛋白可视化的首选技术，但是通常的实现方式很难揭示出成像平面之外的胶原蛋白原纤维。最近，已提出了一种先进的SHG形态，即圆二色性SHG（CD-SHG），以特别突出平面外原纤维。在这项研究中，我们提出了CD-SHG信号的理论分析，该信号超出了电偶极近似来说明胶原手性。我们证明，磁偶极子贡献对于分析人角膜切片和其他富含胶原蛋白的样品的CD-SHG图像是必要的。我们表明CD-SHG信号的迹象并不能揭示胶原纤维是向上还是向下指向，如先前尝试性地提出的那样。相反，CD-SHG探测亚微米级平面外原纤维组件的极性分布，即均匀极性与反平行原纤维的混合物。这使CD-SHG成为表征组织中胶原组织（特别是在病理重塑过程中受影响的疾病程度）的有力工具。因此，CD-SHG可以用来区分健康和患病的富含胶原蛋白的组织。