基于荧光偏振的生物分子三维取向分布显微成像技术研究

The organizational structure between biological molecules plays a crucial role in a large number of biological processes. As an important parameter, the orientation distribution of biological molecules can directly reflect their organizational structure. Hence, quantitatively retrieving the orientation distribution of biological molecules is very significant for revealing the biological process and understanding the interaction mechanism. Based on the polarization property of fluorescence, some techniques have been explored to quantitatively obtain the orientation distribution of bio-molecules. However, present techniques are limited to obtain the planar projection in sample plane of the orientation distribution and suffer limitations in biological applications due to the lack of obtaining the real and complete orientation distribution. In this proposal, a method to probe the real 3D orientation distribution of biological molecules was proposed for the first time by using a new fluorescence exciting mechanism with the 3D polarization control. Here, the key technique of this method will be investigated, as well as the related theoretical analysis and experimental verification. This method will be used for the first time to achieve the 3D orientation distribution of biological molecules, which will promote the technique of fluorescence polarization microscopy to a higher level, so as to provide a powerful tool for numerous biomedical researches involving molecular organizational structure.

在大量生物过程中，生物分子间的组织结构发挥着至关重要的作用，生物分子的取向分布作为重要参数能够直接反映生物分子间的组织结构，因此定量获取此信息对揭示相关生物过程和作用机制有着极其重要的意义。研究人员基于荧光偏振特性提出了定量获取生物分子取向分布的方法，并取得了阶段性进展。但现有方法仅限于提供分子取向在样本平面投影的二维分布，不能真实完整地反映生物分子组织结构信息，极大地限制了其在生物科学研究中的应用。为此本课题首次提出基于荧光三维偏振激发定量获取分子三维取向分布的方法，并围绕该方法的关键技术、理论模型以及实验验证进行研究。本项目将首次实现定量获取生物分子的三维取向分布，推动荧光偏振显微技术进入更成熟阶段，为众多涉及分子组织结构的生物医学研究提供有力工具。

本研究提出基于三维荧光偏振调制获取生物分子三维取向分布的方法，对相关技术进行了系统研究。首先基于线偏振光和径向偏振光的相干叠加实现显微物镜聚焦光场三维偏振方向控制，利用矢量传播理论建立计算模型对该方法进行了系统研究，研究结果表明该方法具有可行性，基于该方法成功构建对应的三维偏振控制光路；其次基于矢量传播理论通过数学建模给出了三维荧光偏振的计算模型，利用该计算模型验证了基于三维荧光偏振调制获取分子三维取向分布方法的可行性，同时给出了一种基于傅里叶分析的荧光调制响应快速处理方法，利用该数据处理方法能够快速获取分子三维取向分布参数；最后搭建了荧光偏振显微系统，在系统构建过程中开发了光学元件偏振特性检测工具和矢量偏振光偏振分布检测工具，同时基于搭建的荧光偏振显微系统进行了巨型囊泡验证实验和健康软骨组织的测试实验，实验表明系统的正确性和其在生物分子结构领域的应用价值。