高空间分辨率光场三维显微镜系统研究

There is a wild application of microscopy in many areas such as medicine, biology, metal material and so on. With the increased awareness of three-dimensional micro-world for human, the traditional optical microscopy becomes hard to match the requirement of science research. Especially, with the development of research on the brain network, the urgent need for three-dimensional microscopy becomes serious. Until now, several types of microscopy have proposed to obtain the volume data of the specimens. The most popular one of them is the laser confocal scanning system. But the photobleaching for specimen limits its application only for non-live specimens. The resolution of light-sheet microscopy and optical projector tomography are mainly depended on the clearing technique. The light field microscopy can capture the multi-view image of specimen with no scanning mechanism. But due to the limitation size of microlens in the system, its resolution is low and hard to improve. Therefore, we propose a three-dimensional microscopy with one-snap-shot, non-invasive and high spatial resolution. Based on the principle of light field, we design a simply imaging path to capture multi-view images with high resolution. Then an effective reconstruction algorithm is developed to realize volumetric image of specimen. With the remarkable performance of dynamic images acquisition, the novel microscopy will be very helpful for the research on brain science area. The success of our project can provide a generation of distinctive novel three-dimensional microscopy, and promote the development of the three-dimensional microscopy for our country.

显微镜在医学、生物学、金属材料等领域的应用非常广泛。随着人们对三维微观世界认识的提高，传统显微镜越来越难以满足科研的需要，尤其是目前脑网络领域对三维显微镜的需求越来越迫切。目前发展有几种可对样本进行三维采集的显微镜，然而他们各有各的缺点。最常用的激光共聚焦扫面显微镜存在严重光漂白效应，难以对活体样本扫描；对光切片显微术和光学投影层析显微术，样本透明度又是其分辨率受影响的主要因素之一；光场显微镜可同时获得多个视角的图像，但受微透镜尺寸限制，其分辨率的提高成为技术瓶颈。因此本项目提出单次拍摄、非侵入式、高空间分辨率的光场三维显微镜。基于光场理论，设计出简单有效的光场采集光路，同时采集多个视角图像，经过合理的重构算法获得样本三维结构展示，可实现长时间动态三维图像的采集，为脑科学等相关前沿领域提供有效的研究工具。本项目推进我国三维显微镜技术发展，形成有特色、有影响力的新一代三维显微镜光场采集系统。

传统的二维显微系统由于缺乏三维深度信息，越来越难以满足医学、生物学等领域的需要了。然而当前的最常用的可进行三维信息采集的激光共聚焦显微镜，往往存在严重的光漂白效应，难以对活体样本进行扫描。基于光场理论的三维显微镜可有效实现非侵入式的信息采集。但是Levoy提出的光场显微镜分辨率受到微透镜尺寸限制而难以提高。为此，本项目提出一款新型的高分辨率光场显微系统，并展开如下研究工作：1）设计完成基于透镜阵列的光场显微系统；2）开发出微观三维重构算法、基于光流法的超分辨率图像处理算法等；3）理论推导并实际测量系统的各项性能，并对静态及动态样本成功地进行图像采集以及三维重构。本项目的研究成果对新型三维显微技术发展及其应用具有重要的研究意义。