显微镜快速自动对焦技术研究

发布时间：2018-10-20 08:40

【摘要】：作为显微成像系统的关键技术之一,自动对焦对于提升显微切片扫描技术的效率具有非常重要的意义。目前,在工业领域应用中,显微切片扫描技术主要采用离线对焦方法,该方法无法在任意横向位置快速获得离焦量,因此在扫描前需要对整个组织样品表面进行焦面建模,导致扫描效率低。一些在线自动对焦方法虽然解决了在任意横向位置获取离焦量,一定程度上提高了扫描效率,但仍存在对焦信号获取延时、结构复杂、成本高等问题。针对这些问题,本文提出一种基于倾斜摄像头和正置摄像头的自动对焦方法,该方法具有快速、无延时、结构简单等特点。本文主要对倾斜摄像头对焦的理论分析、对焦系统的模块化设计、对焦系统的实现等内容进行深入探索与研究,并测试分析了对焦系统的各项性能。主要研究内容如下:首先,从对焦系统的快速性出发,提出了倾斜摄像头如何获得特征值的方法,同时得出了实现倾斜对焦原理的理论关系,并分析了倾斜对焦系统的三个关键技术问题:摄像头倾斜角度的选取、倾斜摄像头和正置摄像头的位置关系以及特征值与离焦量的映射关系。深入研究了倾斜角度选取的理论和方法。讨论了倾斜对焦系统的影响因素,得出了景深、分辨率以及图像均匀度等通过改变系统参数可以减少对系统影响的结论,设计并研制了单透镜非球面聚光镜的照明系统。其次,在倾斜自动对焦理论的基础上,设计了倾斜摄像头的固定结构装置。分析并选取了评价特征图像的清晰度评价函数,对五种算法进行比较,确定了效率、单峰性、灵敏度等性能更优的Tenengrad算法。设计了合理的ROI区域,有效的提升了图像算法的计算效率。设计了以STM32微处理器为核心的运动控制系统,实现了载物台的运动控制,设计并分析了分段控制算法,减少了电机震荡、阈值选取等问题对成像稳定性的影响。再次,采用了Motic Calibration Slide样本对双摄像头位置标定,实现了特征图像的获取。通过实验标定了特征值与离焦量值的映射关系曲线,分析了理论曲线和实测曲线的误差问题,验证了倾斜对焦系统的可行性。研究并设计了人机交互界面,实现了一体化的对焦功能。最后,测试了倾斜对焦系统的各项性能。对焦效率方面,实现了1.08s的快速对焦,显微切片扫描效率提升一个数量级;灵敏度方面,实测的灵敏度为12pixel/μm,略低于理论值;工作范围方面,在10X、20X物镜下,实测的工作范围分别为105.2μm、48.8μm,均满足生物组织样品的测量。
[Abstract]:As one of the key techniques of microscopic imaging system, automatic focusing plays an important role in improving the efficiency of microslice scanning. At present, in the application of industrial field, the technology of microslice scanning mainly adopts off-line focusing method, which can not obtain the defocus quantity quickly at any transverse position, so it is necessary to model the focal plane of the whole tissue surface before scanning. The scanning efficiency is low. Some on-line auto-focusing methods solve the problems of obtaining defocus at any transverse position and improving scanning efficiency to a certain extent, but there are still some problems such as delay of acquisition of focusing signal, complex structure and high cost. Aiming at these problems, this paper presents an automatic focusing method based on tilted camera and forward camera, which has the advantages of fast speed, no delay, simple structure and so on. In this paper, the theoretical analysis of tilted camera focusing, the modular design of focusing system, the realization of focusing system and so on are explored and studied, and the performance of focusing system is tested and analyzed. The main research contents are as follows: firstly, from the point of view of the rapidity of the focusing system, the method of how to obtain the eigenvalue of the tilted camera is proposed, and the theoretical relationship of realizing the tilting focusing principle is obtained. Three key technical problems of tilted focusing system are analyzed, including the selection of tilt angle of camera, the position relationship between tilted camera and positive camera, and the mapping relationship between eigenvalues and defocus. The theory and method of tilt angle selection are studied in depth. This paper discusses the influence factors of tilting focusing system, and draws the conclusion that depth of field, resolution and image uniformity can reduce the influence on system by changing system parameters. The illumination system of single lens aspheric condenser is designed and developed. Secondly, on the basis of tilting auto-focus theory, the fixed structure of tilting camera is designed. This paper analyzes and selects the definition evaluation function of the evaluation feature image, compares the five algorithms, and determines the Tenengrad algorithm with better performance, such as efficiency, single peak, sensitivity and so on. A reasonable ROI region is designed to improve the computational efficiency of the image algorithm. A motion control system based on STM32 microprocessor is designed. The motion control of the platform is realized. The subsection control algorithm is designed and analyzed to reduce the influence of motor oscillation and threshold selection on the imaging stability. Thirdly, Motic Calibration Slide samples are used to calibrate the position of the two cameras, and the feature images are acquired. The mapping curves between eigenvalues and defocus values are calibrated experimentally. The errors between theoretical curves and measured curves are analyzed and the feasibility of tilted focusing system is verified. The man-machine interface is studied and designed, and the integrated focusing function is realized. Finally, the performance of the tilted focusing system is tested. In the aspect of focusing efficiency, the fast focusing of 1.08s is realized, and the scanning efficiency of microslice is increased by an order of magnitude. In sensitivity, the measured sensitivity is 12pixel/ 渭 m, which is slightly lower than the theoretical value. The measured range of work is 105.2 渭 m ~ (48. 8) 渭 m, which can meet the requirements of biological tissue samples.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TP391.41

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