Williams Rebecca M, Zipfel Warren R, Webb Watt W
Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA.
Biophys J. 2005 Feb;88(2):1377-86. doi: 10.1529/biophysj.104.047308. Epub 2004 Nov 8.
Fibrillar collagen, being highly noncentrosymmetric, possesses a tremendous nonlinear susceptibility. As a result, second-harmonic generation (SHG) microscopy of collagen produces extremely bright and robust signals, providing an invaluable tool for imaging tissue structure with submicron resolution. Here we discuss fundamental principles governing SHG phase matching with the tightly focusing optics used in microscopy. Their application to collagen imaging yields several biophysical features characteristic of native collagen structure: SHG radiates from the shell of a collagen fibril, rather than from its bulk. This SHG shell may correspond to the supporting element of the fibril. Physiologically relevant changes in solution ionic strength alter the ratio of forward-to-backward propagating SHG, implying a resulting change in the SHG shell thickness. Fibrillogenesis can be resolved in immature tissue by directly imaging backward-propagating SHG. Such findings are crucial to the design and development of forthcoming diagnostic and research tools.
纤维状胶原蛋白具有高度的非中心对称性，具有极大的非线性极化率。因此，胶原蛋白的二次谐波产生（SHG）显微镜能够产生极其明亮且稳定的信号，为亚微米分辨率的组织结构成像提供了极为宝贵的工具。在此，我们讨论与显微镜中使用的紧聚焦光学器件相关的SHG相位匹配基本原理。它们在胶原蛋白成像中的应用产生了几个天然胶原蛋白结构特有的生物物理特征：SHG从胶原纤维的外壳辐射，而非从其主体辐射。这种SHG外壳可能对应于纤维的支撑元件。溶液离子强度的生理相关变化会改变向前传播与向后传播的SHG的比例，这意味着SHG外壳厚度会相应改变。通过直接对向后传播的SHG成像，可以在未成熟组织中分辨出纤维形成过程。这些发现对于未来诊断和研究工具的设计与开发至关重要。
