基于多层光片体全息的三维非扫描层析成像新方法研究

It is very difficult to detect the living cells for a long time by using the fluorescence microscopy because of the photobleaching and the optical damage. To solve this problem, this project proposes a new optical microscopic method, the multi-light-sheet volume holographic imaging, which can obtain a 3D image in one exposure time. The main research contents include: 1. Build the mathematical model of the multi-light-sheet volume holographic optical method based on the Kogelink coupled wave theory and Fourier Optics, analyze the mechanism of the optical property change, which is influenced by the multi-light-sheet illumination. 2. Build a multi-light-sheet volume holographic imaging system to verify the theoretical analysis conclusions, realize the fast 3D imaging function. 3. Develop the 3D volume holographic reconstruction algorithms based on the deconvolution method. There is a certain novelty value in our proposed method to develop the multi-light-sheet illumination and combine it with the volume holography to raise the optical sectioning of the imaging system. There is no 3D scanning process in our method, so the image speed will be raised largely, and the photobleaching and the optical damage which are induced by the overlap region in the 3D scanning process can also be avoided. Last but not least, because all of the parts used in the system are common optical elements, the cost is very cheap, the environmental requirements are greatly reduced, and the system is easy to use. Because of these advantages, our method has extensive application in many deferent research areas such as the study on neurology and the embryonic development.

针对使用荧光成像技术观测活体细胞时，由于光漂白和光损伤造成的无法长时间跟踪观测的问题，本申请提出研究一种单次曝光即可三维成像的多层光片体全息成像新方法。主要研究内容包括：采用Kogelink耦合光波理论和傅立叶光学，建立多层光片体全息成像方法的数学模型，分析多层光片照明对体全息成像光学性能的影响机理；建立一套多层光片体全息成像系统，验证理论模型、实现快速三维成像功能；研究发展基于反卷积技术的三维体全息影像重构算法。本申请提出将单层光片拓展为多层光片照明，并与体全息成像相结合，可有效提高成像系统层析能力，具有较好的新意。该成像方法可免去扫描获得样品三维信息的过程，在大幅度提高图像采集速度的同时，可减少活细胞样品受到光漂白与光毒性的损伤。此外系统制造成本低、对环境要求宽松、操作方便。本申请的实施可为诸如神经学、胚胎发育生物学等领域提供新的观察方法，具有广泛的应用前景。

研究基于多层光片体全息技术的三维非扫描层析成像的基础理论与方法，其主要内容包括：研究设计了基于空间光调制技术的单次曝光多层结构光片照明系统；研究了照明系统参数对多层光片厚度、层间距、光强分布等的影响；将照明系统的光片位置与体全息成像系统焦距进行了匹配，搭建了一套多层结构光片体全息成像系统，并进行相应的性能测试。结合Hilo重构算法，对各类样品进行了成像实验。与传统反射式体全息成像系统相比，项目所设计的多层结构光片体全息成像系统的层析有很大提高，其轴向分辨率达到了8.7um。但与此同时，也发现了所搭建系统对散射样品无法构建精细的结构片光，限制了其应用范围。我们经研究分析后已开始探索此问题的解决途径。