University of Florence, Department of Evolutionary Biology Leo Pardi, Florence, Italy.
J Biomed Opt. 2012 Jun;17(6):060901. doi: 10.1117/1.JBO.17.6.060901.
Second-harmonic-generation (SHG) microscopy has emerged as a powerful tool to image unstained living tissues and probe their molecular and supramolecular organization. In this article, we review the physical basis of SHG, highlighting how coherent summation of second-harmonic response leads to the sensitivity of polarized SHG to the three-dimensional distribution of emitters within the focal volume. Based on the physical description of the process, we examine experimental applications for probing the molecular organization within a tissue and its alterations in response to different biomedically relevant conditions. We also describe the approach for obtaining information on molecular conformation based on SHG polarization anisotropy measurements and its application to the study of myosin conformation in different physiological states of muscle. The capability of coupling the advantages of nonlinear microscopy (micrometer-scale resolution in deep tissue) with tools for probing molecular structure in vivo renders SHG microscopy an extremely powerful tool for the advancement of biomedical optics, with particular regard to novel technologies for molecular diagnostic in vivo.
二次谐波产生（SHG）显微镜已成为一种强大的工具，可用于对未经染色的活组织进行成像，并探测其分子和超分子结构。本文综述了 SHG 的物理基础，强调了二次谐波响应的相干叠加如何导致偏振 SHG 对焦点体积内发射器的三维分布的灵敏度。基于对该过程的物理描述，我们研究了探测组织内分子组织及其对不同生物医学相关条件的反应的实验应用。我们还描述了基于 SHG 偏振各向异性测量获取分子构象信息的方法，以及将其应用于研究不同肌肉生理状态下肌球蛋白构象的方法。将非线性显微镜（在深部组织中具有微米级分辨率）的优势与体内探测分子结构的工具相结合的能力，使得 SHG 显微镜成为生物医学光学领域的一个极其强大的工具，特别是在体内分子诊断的新技术方面。
