James Darian S, Campagnola Paul J
Department of Biomedical Engineering, University of Wisconsin-Madison, 1550 Engineering Dr, Madison, WI 53706, USA.
BME Front. 2021 Jan 25;2021:3973857. doi: 10.34133/2021/3973857. eCollection 2021.
Second harmonic generation (SHG) and third harmonic generation (THG) microscopies have emerged as powerful imaging modalities to examine structural properties of a wide range of biological tissues. Although SHG and THG arise from very different contrast mechanisms, the two are complimentary and can often be collected simultaneously using a modified multiphoton microscope. In this review, we discuss the needed instrumentation for these modalities as well as the underlying theoretical principles of SHG and THG in tissue and describe how these can be leveraged to extract unique structural information. We provide an overview of recent advances showing how SHG microscopy has been used to evaluate collagen alterations in the extracellular matrix and how this has been used to advance our knowledge of cancers, fibroses, and the cornea, as well as in tissue engineering applications. Specific examples using polarization-resolved approaches and machine learning algorithms are highlighted. Similarly, we review how THG has enabled developmental biology and skin cancer studies due to its sensitivity to changes in refractive index, which are ubiquitous in all cell and tissue assemblies. Lastly, we offer perspectives and outlooks on future directions of SHG and THG microscopies and present unresolved questions, especially in terms of overall miniaturization and the development of microendoscopy instrumentation.
二次谐波产生（SHG）和三次谐波产生（THG）显微镜已成为用于检查多种生物组织结构特性的强大成像方式。尽管SHG和THG源自截然不同的对比度机制，但二者相辅相成，通常可以使用经过改进的多光子显微镜同时采集。在本综述中，我们讨论了这些成像方式所需的仪器设备以及组织中SHG和THG的潜在理论原理，并描述了如何利用这些原理来提取独特的结构信息。我们概述了近期的进展，展示了SHG显微镜如何用于评估细胞外基质中的胶原蛋白变化，以及这如何被用于增进我们对癌症、纤维化和角膜的了解，以及在组织工程应用中的情况。文中突出了使用偏振分辨方法和机器学习算法的具体实例。同样，我们回顾了THG如何因其对折射率变化的敏感性而推动了发育生物学和皮肤癌研究，这种折射率变化在所有细胞和组织集合中都普遍存在。最后，我们对SHG和THG显微镜的未来发展方向提出了观点和展望，并提出了尚未解决的问题，特别是在整体小型化和微型内窥镜仪器开发方面。
