Second harmonic generation (SHG) microscopy has emerged over the past two decades as a powerful tool for tissue characterization and diagnostics. Its main applications in medicine are related to mapping the collagen architecture of in-vivo, ex-vivo and fixed tissues based on endogenous contrast. In this work we present how H&E staining of excised and fixed tissues influences the extraction and use of image parameters specific to polarization-resolved SHG (PSHG) microscopy, which are known to provide quantitative information on the collagen structure and organization. We employ a theoretical collagen model for fitting the experimental PSHG datasets to obtain the second order susceptibility tensor elements ratios and the fitting efficiency. Furthermore, the second harmonic intensity acquired under circular polarization is investigated. The evolution of these parameters in both forward- and backward-collected SHG are computed for both H&E-stained and unstained tissue sections. Consistent modifications are observed between the two cases in terms of the fitting efficiency and the second harmonic intensity. This suggests that similar quantitative analysis workflows applied to PSHG images collected on stained and unstained tissues could yield different results, and hence affect the diagnostic accuracy.

中文翻译：

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苏木精伊红染色对固定组织切片二次谐波成像定量分析的影响

二次谐波发生 (SHG) 显微镜在过去的二十年中已经成为组织表征和诊断的强大工具。它在医学上的主要应用与基于内源性对比绘制体内、体外和固定组织的胶原结构有关。在这项工作中，我们展示了切除和固定组织的 H&E 染色如何影响特定于偏振分辨 SHG (PSHG) 显微镜的图像参数的提取和使用，已知这些参数可提供有关胶原结构和组织的定量信息。我们采用理论胶原模型来拟合实验 PSHG 数据集，以获得二阶磁化率张量元素比和拟合效率。此外，研究了在圆偏振下获得的二次谐波强度。这些参数在向前和向后收集的 SHG 中的演变是针对 H&E 染色和未染色的组织切片计算的。在拟合效率和二次谐波强度方面，在两种情况之间观察到一致的修改。这表明应用于在染色和未染色组织上收集的 PSHG 图像的类似定量分析工作流程可能会产生不同的结果，从而影响诊断准确性。