成像流式是对流动中的细胞进行高速、高通量显微成像的技术，在药物筛选、免疫应答、癌症研究等领域具有广泛的应用前景。成像流式的挑战在于需要在短时间内对大量以数米每秒运动的细胞进行成像。近年来，成像流式主要遵循基于阵列探测器和单像素探测器的两种技术路线。其中阵列探测器式成像方案使用CCD或CMOS器件作为探测器，受限于器件读出速率，难以同时获得较高的通量和灵敏度；而单像素探测器成像方案使用单个光电探测器作为探测器件，具有灵敏度高，响应速度快的特点，正成为成像流式技术发展的重要方向。对于单像素探测成像方式而言，核心在于图像的编码和解码方式。目前现有的单像素探测器方式存在光路系统复杂、解算过程复杂、可扩展性差等问题。本文提出了基于线阵光斑扫描成像的方法，通过光斑的空间排布及细胞运动可以将图像编码到时域，通过信号分割重组即可实现解码。这种方式可同时适应于明场、暗场和荧光成像，具有实现简单、可扩展性好、通量高(>5000eps)的优势。本文研究了线阵光斑的产生方式和排列准则。线阵光斑通过衍射光学器件产生，且受到衍射场尺寸、成像分辨率、物镜视场大小的约束。本文以此为理论基础，设计加工了衍射光学器件，并搭建了流式成像光路系统，验证了成像方案的可行性及优化了成像分辨率和光路结构。本文进一步实现了双激光五通道成像流式系统，用于满足流式检测多激光和多通道检测的需求。为此，首先研究和校准了衍射光学器件边缘减弱的问题，同时提出了明场成像的方法。最终搭建了488nm和638nm激光同时激发的，具备明场、FITC、PE、PI和APC五个成像通道的流式成像系统，并使用荧光微球测试了其成像性能。本文进行了基础的生物学实验。实验中对Hela细胞内的荧光蛋白分布、细胞器分布等进行了成像。针对流式中常见的细胞周期分析，借助于本系统实现了全细胞周期的检测。同时通过图像特征提取并结合高维流式的t-SNE数据分析方法，可以实现细胞自动、准确地分类。总体而言，本文提出的线阵光斑扫描成像方法，为分析型成像流式、光谱成像流式和基于图像的高通量细胞筛选奠定了基础。
Imaging flow cytometry (IFCM) is a technique for high-speed, high-throughput imaging of cells in flow, with promising applications in areas such as drug screening, immune response, and cancer research. The challenge of imaging flow is the need to image a large number of cells moving at several meters per second in a short period of time. In recent years, IFCM has followed two main technical routes based on array detectors and single-pixel detectors. The array detector imaging scheme uses CCD or CMOS devices as detectors, which are limited by the serial readout rate of the devices, making it difficult to obtain high throughput and sensitivity at the same time; while the single-pixel detector imaging scheme uses a single photodetector as the detector device, and is becoming an important direction in the development of IFCM technology.For single-pixel methods, the core lies in the image encoding and decoding process. The existing single-pixel detector method mainly suffer from complex optical path system, complicated decoding process and poor scalability. In this work, we propose a method based on line array spot scanning, which can encode the image into the time domain through the spatial arrangement of the spots and cell movement, and decode the image through signal segmentation and recombination. This approach can be extended to bright-field, dark-field and fluorescence imaging at the same time, and has the advantages of simplicity, good scalability and high throughput (>5000 eps).The linear array spot is generated by diffractive optics and is constrained by the diffractive field size, imaging resolution, and objective field of view size. In this work, we designed and processed diffractive optics and built an IFCM system to verify the feasibility of the imaging scheme and optimize the resolution and optical path structure.In this work, a dual-laser five-channel IFCM system is further implemented to meet the requirements of multi-laser and multi-channel detection. For this purpose, the edge weakening of laser spots is investigated and calibrated, and the bright-field imaging method is proposed. Finally, an IFCM system with five imaging channels of bright field, FITC, PE, PI and APC, excited by 488 nm and 638 nm lasers simultaneously, was built and its imaging performance was tested using fluorescent microbeads.Some basic biological experiments were performed in this paper. The fluorescent protein distribution and organelle distribution in Hela cells were imaged in the experiments. For cell cycle analysis, which is commonly used in flow cytometry, the detection of the whole cell cycle was achieved with the help of our system. Also, by image feature extraction and combining with the t-SNE data analysis method, the cells can be automatically and accurately classified.Overall, the linear array spot scanning imaging method proposed in this paper lays the foundation for analytical IFCM, spectral IFCM and image-based high-throughput cell sorting.
