超分辨高精度实时数字全息显微术及对活细胞动态成像研究

The project will focus on several key issues in digital holography, to reach the goal to enhance the imaging resolution, phase measurement accuracy and real-time reproducibility, under the background of the applications of digital holographic microscopy in living cell morphology and dynamic monitoring. Digital holographic-based high numerical aperture annular light cone lighting, real-time super-resolution imaging methods are proposed to realize the real real-time high-resolution imaging. By choosing 1/4 spectrum of the fixed area of the surface as a filter reconstruction area, to avoid the manual intervention of the reconstruction process, the real-time reconstruction will be achieved. The nonlinear reconstruction method based on homomorphic signal processing, along with the improvement and optimization of automatic phase distortion compensation method and the efficient phase unwrapping method, are used to achieve high-precision real-time reconstruction of holograms. To further improve the phase measurement accuracy, off-axis common path interference in the recording system based on the diffraction grating is used to reduce the phase noise introduced by the outside interference. Digital holographic recording system built from off-axis of the annular light cone lighting, real-time recording and reproduction by the image of the differential contrast techniques and animation, will clearly show the cellular details and living cells dynamic process. The implementation of this project will provide the accurate characterization of living cells on the magnitude of millisecond, ultrafast process and of several days process.

本项目以数字全息显微术在活细胞形态检测与动态监测中的应用为基本背景，以提高成像分辨率、位相测量精度及再现的实时性为目标，对数字全息显微中的几个关键问题进行研究。提出并研究基于高数值孔径环形光锥照明的数字全息实时超分辨成像方法，真正实现实时高分辨成像。通过选取1/4谱面的固定区域作为滤波重建区域，避免手工介入的重建过程，真正实现实时重建。采用并研究基于同态信号处理的非线性重建方法，同时对自动位相畸变补偿法及现有的高效位相解包裹方法进行改进和优化，实现全息图高精度实时重建。采用基于光栅衍射的离轴共路干涉记录系统，降低外界干扰引入的位相噪声，进一步提高位相测量精度。构建由环形光锥照明的离轴共路干涉像面数字全息记录系统，对生物活细胞动态变化过程进行实时记录与再现，通过图像微分对比技术及动画制作，清晰显示细胞细节及动态变化。本项目的实施可对细胞内ms量级的超快过程也可对长达数天的过程进行准确表征。

本项目主要基于高数值孔径环形空心光锥照明的显微成像系统超分辨成像的理论及实验验证展开研究。首先，研究了光瞳滤波技术在显微成像系统中对点扩散函数旁瓣抑制及主瓣锐化的方法，提出结构光照明方案。其次，研究了用非线性重建方法对平面参考光波记录全息图的精确重建方法。研究中采用了自动位相补偿方法、改进的最小二乘解包裹方法、质量图导向路径跟踪法，在消除噪声影响、提高重建质量和重建速度方面取得了进展。在信号处理方面研究了同态信号处理的相关理论和技术。所提出的用非线性重建方法对球面参考光波记录全息图的精确重建，提升了位相测量精度，并为相位测量的评价提供了依据。在实验研究中，完成了样品制作，并对活细胞进行全息记录与数值再现，获得动态位相变化信息。研究在特定药物作用下细胞的形态及活性变化情况，对所重建的细胞位相分布进行微分图像对比，完成细胞动态细节显示的动画制作，为活细胞的观测提供了技术支持。