基于三维光强传输的无标记、非干涉、高分辨率相位层析显微成像研究

Based on the basic principle and methods in the field of the noninterferometic transport-of-intensity phase retrieval and phase tomography, the main focus of the proposed research is the study of label-free, non-interferometric, high-resolution phase micro-tomography based on three-dimensional (3D) intensity transport. There are three main themes within the proposed research. The first is to extend the conventional 2D transport-of-equation theory to 3D, explore the relations between the 3D intensity transport with the 3D phase distribution of the measured specimen, and establish the mathematical model of the phase 3D point spread function (PSF) and 3D optical transfer function (OTF) for bright field microscopic systems. The second theme is to develop accurate and efficient phase deconvolution algorithm for 3D intensity transport based on the new developments in the areas of image and signal processing, such as the image restoration and the compressive sensing. The third theme is to explore the new experimental configurations for realizing high-resolution phase micro-tomography by drawing on the experience from the fields of the fluorescence deconvolution microscopy and wide-field super-resolution microscopy. The research in this direction leads towards new approaches to reconstruct high-precision high-resolution 3D phase or refractive index distribution of biological samples, such as cells and tissues, in a label-free, non-interferometric manner. In this way the fruits of the research will be full of significance for basic biology research, but also will provide a powerful tool and a new imaging methodology for clinical diagnostic imaging, drug development, cell dynamics, and pathology research.

本项目以光强传输方程非干涉相位恢复与相位断层扫描的基本原理和方法为基础，拟探索一种基于三维光强传输的无标记、非干涉、高分辨率相位层析显微成像方法。借鉴现有的二维光强传输方程相关理论，研究部分相干照明下明场显微系统的三维光强传输与三维相位分布之间的关联，建立相应的相位三维点扩散函数与三维光学传递函数模型；借鉴图像反卷积复原、压缩感知等信号处理领域的新技术，发展出针对三维光强传输的高效、准确的三维相位反卷积重构方法；借鉴荧光反卷积显微、宽场超分辨率显微的新成果，发展出高分辨、非干涉三维层析显微成像的新结构。通过创新研究，突破各项关键问题，最终实现生物细胞相位与三维折射率分布的无标记、非干涉、高精度、高分辨率定量成像与表征。相关研究成果不仅对基础生物学有重要意义，而且将为临床医学诊断、药物开发、细胞动力学以及病理研究等提供强有力的手段和新的影像学方法，对推动其相关研究具有重要的价值和作用。

本项目瞄准了国际上相位恢复与定量显微成像发展的前沿方向，以光强传输方程非干涉相位恢复与相位断层扫描的基本原理和方法为基础，开展基于三维光强传输的无标记、非干涉、高分辨率相位层析显微成像研究。通过3年研究攻关，主要取得的成果包括以下三个方面：.① 提出了基于最优化环形照明的光强传输方程定量相位成像与三维折射率层析成像方法，通过照明光场调制实现了相位传递函数响应与支持域的最优化，实现了横向分辨率达208nm的无标记非干涉定量动态定量相位成像以及，横向分辨率与轴向分辨率达到200nm与645nm的三维折射率层析。.② 提出了基于高数值孔径可编程照明的大视场超分辨显微成像技术，并搭建了满足空域频域最优采样率的高数值孔径可编程照明显微成像系统。在0.4数值孔径10倍物镜2.34mm2大视场的前提下，有效合成数值孔径达到了1.6，在波长435nm照明下的重构分辨率达到了154nm（半宽分辨率），最终的空间带宽积为0.985亿像素。相比于使用相同物镜的传统显微成像系统，空间带宽积提高了近50倍。.③ 提出了一种自适应松弛系数的无透镜超分辨显微成像技术，并搭建了基于轴向扫描的无透镜显微成像系统，实现了在29.85mm2大视场下770nm的横向半宽分辨率，达到了相机像素尺寸限制的采样分辨率的2.17倍。.上述研究成果实现高速、高分辨率的非干涉三维相位层析显微成像，为细胞生物学，病理学，临床医学研究与诊断提供一种崭新的无标记影像学手段。.在本项目的资助下，发表期刊论文39篇（SCI收录论文32篇），发表论文WOS引用次数435次。会议论文24篇，项目在研期间授权发明专利16件，申请发明专利24件，超额完成了项目的预期目标。