Nonlinear microscopy of common histological stains reveals third harmonic generation harmonophores
Nonlinear microscopy of common histological stains reveals third harmonic generation harmonophores
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DOI：
10.1039/c9an00267g
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发表时间：
2019-05-21
期刊：
影响因子：
4.2
通讯作者：
Wiseman, Paul W.
中科院分区：
文献类型：
--
作者：
Kazarine, Alexei;Gopal, Angelica A.;Wiseman, Paul W.
自一百多年前发明以来，使用有色染料染色的组织学分析仍然是组织病理学的黄金标准。虽然这些染色剂为各种诊断目的提供了关键信息，但它们提供的二维组织学信息有限。将经典的组织学分析扩展到三维需要新的成像方法，如多光子显微镜。多光子显微镜能够对组织学染色样本进行多模态三维成像。具体而言，三次谐波产生（THG），其中三个入射光子通过样品组合成一个的非线性光学过程，允许用吸收染料染色的组织的高对比度成像，吸收染料又充当谐波载体。虽然这种技术以前已被应用于苏木精和伊红（H & E）组织切片，我们将这种方法扩展到其他常用的组织学染色，以证明该技术的进一步潜在应用。我们展示了用H & E、Verhoeff-Van Gieson（VVG）和Picrosirius Red染色的人类皮肤和肝脏组织的THG成像。我们发现，这些染料提供了良好的对比度作为THG harmonophores，使组织学样本的高分辨率成像。Verhoeff染色的THG成像能够轻松检测弹性纤维，而苦天狼星红作为有效的谐波载体用于成像所有尺寸的胶原纤维。
Since its invention over a hundred years ago, histological analysis using coloured dye staining remains the gold standard for histopathology. While these stains provide critical information for a variety of diagnostic purposes, they offer limited two-dimensional histological information. Extending classical histological analysis to three dimensions requires novel imaging approaches such as multiphoton microscopy. Multiphoton microscopy enables multimodal, three-dimensional imaging of histologically stained samples. Specifically, third harmonic generation (THG), a nonlinear optical process in which three incident photons are combined into one by the sample, allows high contrast imaging of tissues stained with absorbing dyes, which in turn act as harmonophores. While this technique has previously been applied to hematoxylin and eosin (H&E) tissue sections, we extend this approach to other commonly used histological stains to demonstrate further potential applications of the technique. We demonstrate THG imaging of both human skin and liver tissue stained with H&E, Verhoeff-Van Gieson (VVG) and Picrosirius Red stains. We find that these stains provide excellent contrast as THG harmonophores, enabling high resolution imaging of histological samples. THG imaging of the Verhoeff stain enables easy detection of elastic fibers while Picrosirius Red acts as an effective harmonophore for imaging collagen fibers of all sizes.