自动化显微超声切割方法研究
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摘要
随着现代生物及相关技术的飞速发展，显微操作技术已成为研究的热点，其中有关显微切割的操作是常用的重要操作之一。目前，在生物医学领域获得特定组织区域或单个细胞，大多是通过手工或者机械辅助的方法来完成的，这样就避免不了操作人员在显微镜下因长时间工作，工作强度大，易疲劳等因素所造成的人为误差且精度低，可控性差。所以研究显微切割技术不仅具有理论必要还具有实践意义。
本文基于超声振动显微切割的需求，将超声振动引入显微切割领域。在介绍超声主要特性及其应用的基础上，分析了超声振动显微切割机理。为进一步研制超声显微切割系统打下理论基础。
鉴于不同厚度的样品对能量的需求不同，选择合适的超声发生装器及压电陶瓷，便于调整切割工具的振动频率与幅值。对选用的压电陶瓷进行震动测试及一阶模态分析。针对显微切割环境和工具的特殊性设计切割工具的整体结构及三种切割针的连接方式供选择。同时选择具有高品质图像处理能力的basler公司的A601f摄像头和高精度的电动操作手及相应的控制器并对控制器进行必要的调试工作。
以VC6.0编程软件为开发平台，利用微软提供的类库MFC和具有丰富视觉处理算法的计算机视觉库OpenCV。研究了在线图像匹配算法及控制方案，并开发具备图像动态采集存储可实现微操作工具的自动搜索，切割针针尖位置检测，具有实时跟踪针尖功能的程序文档，可实现将切割针定位在组织切片上方任意位置。开发工作台、操作手等控制程序以实现切割针的精确定位,准确完成路径规划及切割操作。
最后，利用研制的自动化显微超声切割系统，对厚4um的大肠肿瘤组织及乳腺癌组织切片进行显微切割实验。通过对理论和实验结果的分析，得出了4um厚的大肠肿瘤组织切片切割的最佳参数。在该参数条件下可以完成任意形状的切割，切割效果理想，还可实现拾取目标组织便于后续处理。实验证明深度可控自动压电超声显微切割系统增加了显微切割的自动化程度，明显提高了显微切割工作的准确性与效率。
Along with the rapid development of modern biology and the related techniques, micromanipulation technique has become a research focus, including micro-dissection which is frequently referred to as one of the important operations. At present, in biomedical field the specific organizations or single cell are mostly obtained by manual or machine assisted method, with unavoidable human errors due to long working at the microscope, heavy working intensity, easily fatiguing and so on and low accuracy, poor controllability. Therefore, micro-dissection technology is provided with not only theoretical necessity but practical significance.
Based on the requirements of ultrasonic vibration micro-dissection, ultrasonic vibration is introduced into the microscopic cutting field in this paper. Ultrasonic vibration's main characteristics and some actual applications are presented based on the analysis of the ultrasonic vibration micro-dissection theory. And theoretical basis is provided for further development of ultrasonic micro-dissection system.
In view of different thickness of dissecting samples requirements different energy, select a suitable signal generator and piezoelectric ceramic that is suitable for adjustment of dissect tool's vibration frequency and amplitude. Vibration test and the first-order modal analysis of selected piezoelectric ceramic are performed. Contraposing the particularity of micro-dissection environment and dissection tool, we not only designed the overall structure of the dissection tool but designed three connection ways of dissection needle to choose. Meanwhile we select the A601f camera of German's basler company with high quality and image processing, high accuracy manipulator, the corresponding controller, precise air pump, etc.
With VC6.0(programming software) for the development platform, using Microsoft's MFC and computer vision library (OpenCV) with rich visual processing algorithm, the high speed image matching algorithm and control scheme are researched, procedure document with dynamic image collection and storage is developed, which can realize automatic searching micro tools, detecting the position of the cutting's needle tip and tracking the needle visually in real time, thus successfully setting the needle in any position over the tissue. The control procedures of work stage and manipulator are developed to achieve precise positioning of dissection needle, accurately completing path planning and dissection operation.
Finally, we use the depth-controlled and automated piezoelectric ultrasonic vibration micro-dissection system to dissect the4micron colon cancer tissues and Breast cancer tissues. Through analysis the theory and the experimental results, we got the best dissection parameters of the4um colon cancer tissues slice. In this circumstance, dissection for any shape can be accomplished ideally, also the post-dissection tissue can be taken away for further process. Experiments prove that depth-controlled automated piezoelectric ultrasonic vibration microscopic cutting system increases the automation degree of microscopic cutting, and obviously improves the accuracy and efficiency of dissection work.
本文基于超声振动显微切割的需求，将超声振动引入显微切割领域。在介绍超声主要特性及其应用的基础上，分析了超声振动显微切割机理。为进一步研制超声显微切割系统打下理论基础。
鉴于不同厚度的样品对能量的需求不同，选择合适的超声发生装器及压电陶瓷，便于调整切割工具的振动频率与幅值。对选用的压电陶瓷进行震动测试及一阶模态分析。针对显微切割环境和工具的特殊性设计切割工具的整体结构及三种切割针的连接方式供选择。同时选择具有高品质图像处理能力的basler公司的A601f摄像头和高精度的电动操作手及相应的控制器并对控制器进行必要的调试工作。
以VC6.0编程软件为开发平台，利用微软提供的类库MFC和具有丰富视觉处理算法的计算机视觉库OpenCV。研究了在线图像匹配算法及控制方案，并开发具备图像动态采集存储可实现微操作工具的自动搜索，切割针针尖位置检测，具有实时跟踪针尖功能的程序文档，可实现将切割针定位在组织切片上方任意位置。开发工作台、操作手等控制程序以实现切割针的精确定位,准确完成路径规划及切割操作。
最后，利用研制的自动化显微超声切割系统，对厚4um的大肠肿瘤组织及乳腺癌组织切片进行显微切割实验。通过对理论和实验结果的分析，得出了4um厚的大肠肿瘤组织切片切割的最佳参数。在该参数条件下可以完成任意形状的切割，切割效果理想，还可实现拾取目标组织便于后续处理。实验证明深度可控自动压电超声显微切割系统增加了显微切割的自动化程度，明显提高了显微切割工作的准确性与效率。
Along with the rapid development of modern biology and the related techniques, micromanipulation technique has become a research focus, including micro-dissection which is frequently referred to as one of the important operations. At present, in biomedical field the specific organizations or single cell are mostly obtained by manual or machine assisted method, with unavoidable human errors due to long working at the microscope, heavy working intensity, easily fatiguing and so on and low accuracy, poor controllability. Therefore, micro-dissection technology is provided with not only theoretical necessity but practical significance.
Based on the requirements of ultrasonic vibration micro-dissection, ultrasonic vibration is introduced into the microscopic cutting field in this paper. Ultrasonic vibration's main characteristics and some actual applications are presented based on the analysis of the ultrasonic vibration micro-dissection theory. And theoretical basis is provided for further development of ultrasonic micro-dissection system.
In view of different thickness of dissecting samples requirements different energy, select a suitable signal generator and piezoelectric ceramic that is suitable for adjustment of dissect tool's vibration frequency and amplitude. Vibration test and the first-order modal analysis of selected piezoelectric ceramic are performed. Contraposing the particularity of micro-dissection environment and dissection tool, we not only designed the overall structure of the dissection tool but designed three connection ways of dissection needle to choose. Meanwhile we select the A601f camera of German's basler company with high quality and image processing, high accuracy manipulator, the corresponding controller, precise air pump, etc.
With VC6.0(programming software) for the development platform, using Microsoft's MFC and computer vision library (OpenCV) with rich visual processing algorithm, the high speed image matching algorithm and control scheme are researched, procedure document with dynamic image collection and storage is developed, which can realize automatic searching micro tools, detecting the position of the cutting's needle tip and tracking the needle visually in real time, thus successfully setting the needle in any position over the tissue. The control procedures of work stage and manipulator are developed to achieve precise positioning of dissection needle, accurately completing path planning and dissection operation.
Finally, we use the depth-controlled and automated piezoelectric ultrasonic vibration micro-dissection system to dissect the4micron colon cancer tissues and Breast cancer tissues. Through analysis the theory and the experimental results, we got the best dissection parameters of the4um colon cancer tissues slice. In this circumstance, dissection for any shape can be accomplished ideally, also the post-dissection tissue can be taken away for further process. Experiments prove that depth-controlled automated piezoelectric ultrasonic vibration microscopic cutting system increases the automation degree of microscopic cutting, and obviously improves the accuracy and efficiency of dissection work.
引文
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[2]王振宁,张学,徐惠绵等.激光捕获显微切割技术的原理及应用[J].国外医学遗传学分册,2001,24(2)：61-64.
[3]Nicole L.Simone, Robert F.Bonner, John W.Gillespie.Laser-capture microdissection: opening the microscopic frontier to molecular analysis[J]. Trends in genetics:TIG,1998,14(7):272-276.
[4]Nath Chandra, Rahman Mustafizur, Ken Soon Neo. Modeling of the Effect of Machining Parameters on Maximum Thickness of Cut in Ultrasonic Elliptical Vibration Cutting[J].Journal of manufacturing science and engineering,2011,133:1087-1357.
[5]Rosa I. Gallagher, Steven R. Blakely, Lance A. Liotta, etal. Laser Capture Microdissection:ArcturusXT Infrared Capture and UV Cutting Methods[J].Methods in Molecular Biology,2012,823:157-178.
[6]CHEN L G, LIU Y X, SUN L N. Piezo-powered Micro-dissection system with ultrasonic vibration[C]. Proceedings of the30th Chinese Control Conference, yantai, P.R. China:ICEMS,2011:6067-6071.
[7]罗泊涛,苏维勇,唐泽立.手工组织显微切割后的快速DNA提取技术[J].华北煤炭医学院学报,2010,12(4)：471-472.
[8]王修杰,袁淑兰,李昌平等.显微切割术分离切片DNA用于分子病理学研究[J].中华病理学杂志,2001,30(3)：229-231.
[9]温遇华.微操作机器人自动作业的几项关键技术研究[D].天津：南开大学,2004.
[10]毕树生,宗光华,赵玮,张建勋.生物工程中的微操作机器人系统[J].高技术通讯,1998.1：53-57.
[11]郭福政,邱淑兰,肖雪媛.LCM激光捕获显微切割技术及其应用[J].现代仪器,2003,1：40-42.
[12]邓小玲,胡盛平.组织显微切割技术及其应用[J].汕头大学医学院学报, 2005,(15)3：167-169.
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