激光共焦生物芯片扫描仪中的图像重建与分析
摘要
近10多年来，国内生物芯片技术的发展备受瞩目，在生命科学研究、疾病诊断、新药开发、生物武器、司法鉴定、食品卫生监督等等领域已越来越被广泛运用。然而在生物芯片技术中的重要组成部分之一——生物芯片的检测设备却几乎一直依赖国外的产品，其垄断性的昂贵价格，加上复杂的专业分析操作，对我们生物芯片的应用推广造成了许多障碍。因此，开发具有自主知识产权的检测扫描仪具有深远的意义。
本文提出了一种新颖的激光共焦生物芯片扫描仪。仪器基于激光共焦显微镜的理论，采用振镜和远心线性成像物镜实现X向扫描；精密导轨和步进电机实现Y向扫描的检测仪，经验证，扫描仪具有快速、操作简单、检测芯片能力良好等特征，有望弥补市场的空缺。
在本论文中，首先介绍了生物芯片技术的内涵，对检测系统的结构进行了比较分析，提出了检测仪的结构特点和存在的问题的解决方案。重点给出了检测系统研制中，信号的采集与传输的硬件和软件实现，实现了良好的无损数据采集和实时图像重建；然后通过采集成像实验，对扫描仪的性能参数即分辨率和灵敏度进行扫描分析，对系统结构加以调整和改进，最终达到了5um分辨率和接近1荧光分子／um~2的灵敏度指标。最后给出了深圳益生堂公司提供的丙肝病毒抗体蛋白芯片HCV芯片的检测实验结果。
In the present ten years, domestic biochip technique has been developed enormously, and applied to many fields, such as life science research diagnosis, new drug development, bio-weapon war, judicial evaluation, foods and environment supervise etc. However, the biochip analyzer, which is one important part of biochip technique, is almost all depended on foreign ones. And for the two factors of high price because of monopoly and complicated use method because of adapting to research field, it brings many obstruction to the application and spread of biochip technique in our country. So it is a great significance to research and develop a biochip analyzer that own ourselves intellectual copyright and/or patent.
A novel laser confocal biochip scanner was given in this paper. It was based on the principle of laser confocal microscope. And the two-dimensional scanning configuration was adopted by the optical scanner and the telecentric linear imaging objective lens of large numerical aperture to realize X-direction's scanning, and the conventional mechanical method using linear driver and linear guide track to Y-direction's. The experiment results indicate that the device can run smoothly and rapidly, be operated easily and detect fluorescence effectively.
The biochip technique and the detection methods were introduced detailedly firstly in the paper, then the characteristic of the scanner researched and the problem and resolution of it were put forward. Mainly the signal processing about the realization of signal data acquisition and real-time imaging, the hardware setting and software programming were presented. Next, on the basis of lots of experiments the performance parameters about the resolution and sensitivity of the scanner were discussed. And by adjusting and modifying the system configuration, its resolution reached 5 micron and sensitivity about one fluorescent molecule per square micron.. In the end the experiment results of the biochip which was given by Shenzhen Yishengtang Biological Products Co,.Ltd were interpreted.
本文提出了一种新颖的激光共焦生物芯片扫描仪。仪器基于激光共焦显微镜的理论，采用振镜和远心线性成像物镜实现X向扫描；精密导轨和步进电机实现Y向扫描的检测仪，经验证，扫描仪具有快速、操作简单、检测芯片能力良好等特征，有望弥补市场的空缺。
在本论文中，首先介绍了生物芯片技术的内涵，对检测系统的结构进行了比较分析，提出了检测仪的结构特点和存在的问题的解决方案。重点给出了检测系统研制中，信号的采集与传输的硬件和软件实现，实现了良好的无损数据采集和实时图像重建；然后通过采集成像实验，对扫描仪的性能参数即分辨率和灵敏度进行扫描分析，对系统结构加以调整和改进，最终达到了5um分辨率和接近1荧光分子／um~2的灵敏度指标。最后给出了深圳益生堂公司提供的丙肝病毒抗体蛋白芯片HCV芯片的检测实验结果。
In the present ten years, domestic biochip technique has been developed enormously, and applied to many fields, such as life science research diagnosis, new drug development, bio-weapon war, judicial evaluation, foods and environment supervise etc. However, the biochip analyzer, which is one important part of biochip technique, is almost all depended on foreign ones. And for the two factors of high price because of monopoly and complicated use method because of adapting to research field, it brings many obstruction to the application and spread of biochip technique in our country. So it is a great significance to research and develop a biochip analyzer that own ourselves intellectual copyright and/or patent.
A novel laser confocal biochip scanner was given in this paper. It was based on the principle of laser confocal microscope. And the two-dimensional scanning configuration was adopted by the optical scanner and the telecentric linear imaging objective lens of large numerical aperture to realize X-direction's scanning, and the conventional mechanical method using linear driver and linear guide track to Y-direction's. The experiment results indicate that the device can run smoothly and rapidly, be operated easily and detect fluorescence effectively.
The biochip technique and the detection methods were introduced detailedly firstly in the paper, then the characteristic of the scanner researched and the problem and resolution of it were put forward. Mainly the signal processing about the realization of signal data acquisition and real-time imaging, the hardware setting and software programming were presented. Next, on the basis of lots of experiments the performance parameters about the resolution and sensitivity of the scanner were discussed. And by adjusting and modifying the system configuration, its resolution reached 5 micron and sensitivity about one fluorescent molecule per square micron.. In the end the experiment results of the biochip which was given by Shenzhen Yishengtang Biological Products Co,.Ltd were interpreted.
引文
(1) Fodor S P, Massively parallel genomics. Science, 1997, 227:393-395
(2) Editoral. Nature Gentics, 1996, 14[4]:367-370
(3) Fodor S.P.A,Read J.L.,Thomas R.G.,et al, Light-directed spatially addressable paralled chemical synthesis, Science, 1991, 251:767-773
(4) Fodor S.P.A, Rava R.P, Huang X.C. et al, Multiplexed biochemical assays with biological chips. Nature, 1993.8, 364[6437]:555-556
(5) Pease A C, Solas D, Sullivan E J,et al. Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc Natl Acad Sci USA, 1994, 91:5022-5026
(6) 张天浩，张春平，张光寅．DNA芯片制作原理及其杂交信号检测方法。生物工程进展，2000，20[2]：61-68
(7) Hacia J G, Brody L C, et al. Nature genetics, 1996,14:441-447
(8) Christine Debouck, Peter N.Goodfellow. DNA microarrays in drug discovery and development. Nature genetics supplement, 1999,21:48-50
(9) 易霞，尚永丰。生物芯片技术发展及应用前景．科技前沿，2003．7，7[7]：51-55
(10) 卫新菊．生物芯片技术及其在农业上的应用．小麦研究，2002，2．3[4]：34-36
(11) Amersham Pharmacia Biotech Inc., Fluorescence Imaging principles and methods, Amersham pharmacia biotech technical manual, 2000
(12) 齐华，程光伟．基于荧光标记的生物芯片信号检测技术．现代电子技术，2003，5：41-44
(13) 王立强，陆祖康，林斌．生物芯片及其荧光信号检测．光学仪器，2002，24[4-5]：59-65
(14) 吴岚君．生物芯片[DNA微阵列]荧光扫描仪中的激光共聚焦扫描技术．基因快讯，2001[3]，http://www.ebiotrade.com/check2/f29.htm
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[17] 马立人，将中华．生物芯片，化学工业出版社，北京，2000．2：19-20，95-98
[18] CAMBRIDGE TECHNOLOGY, INC. Model 6450 Galvanometer optical Scanner Instruction Manual. Cambridge,Massachusetts, 1996
[19] 杨国光．现代光学测试技术．浙江大学出版社，杭州，1997：352-357
[20] Performance Advantage of a Geometric Beamsplitter in ScanArray Microarray Scanners. Parkard BioChip Technologies, http://www.packardbioscience.com
[21] 郁道银，谈恒英．工程光学．机械工业出版社，北京，2001：256-264
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[23] Wilson T, Carlini A.R. Three-dimensional Imaging in Confocal Imaging Systems with Finite Sized Detectors. J.Microsc., 1998, 149[1]:51-66
[24] 高万荣，陶纯堪，杨晓春．共焦扫描显微成像的光学断层平面分辨率与信噪比之间的关系．光子学报，2002，31[6]：730-735
[25] 宋登元，孙同文．光电倍增管的特性、结构及选取考虑．中国仪器仪表，1995．5：33-35
[26] 雷肇棣．光电探测器原理及应用．物理，1994，23[4]：220-226
[27] HAMAMATSU. Photosensor Modules H7710/H7711/H7712 Series user's guide,2001, http://www.hamamatsu.com
[28] 王鑫，郭继燕，丁铁夫．基于PCI总线的高速数据采集卡的设计．微计算机信息，2003，19[5]：31-32
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[33] 王珏，葛冬梅．Windows下多幅大尺寸位图的显示与浏览．黑龙江工程学院学报，2002，16[1]：46-48
[34] 郑家亮，朱黎军，陈明．医疗设备图文档案格式分析．云南大学学报[自然科学报]，2000，22：49-51
[35] 贺京．TIFF分析及其应用。武测科技，1994，3：51-55
[36] 何斌，马天予，王运坚，朱红莲等．Visual C++数字图像处理．人民邮电出版社，北京，2001：4-20
[37] 马平，唐小萍，罗正全．生物芯片扫描仪图像处理过程及方法．微纳电子技术，2002，5：37-39
[38] Yee H Y, Michael J.Buckley, Sandrine Dudoit, et al. Comparison of methods for image analysis on eDNA microarray data. Technical Report, 2000.11
[39] Vincent Y.Jouenne ,Critical Issues in the Processing of eDNA Microarray Image. Computer and Apalication, 2001.6
[40] Jeremy Buhler, Trey Ideker, David Haynor. Dapple:Improved Techniques for Finding Spots on DNA Microarrays. UW CSE Technical Report UWTR, 2000.8
[41] Kenneth．R．Castleman， 朱志刚，林学阊，石定机等译．、数字图像处理；电子工业出版社，1998．9：387—418
[42] Axon Instruments,Inc. Genepix Pro 3.0 Microarray Acquisition and Analysis Software User's Guide. USA, 2000.9
[43] Pixel Resolution and Optical Resolution of Microarray Scanners. Packard Biochip Rechnologies, 2001, http://www.packardbioscience.com
[44] High Resolution Imaging for Accurate Microarray Quantitation. Packard Biochip Technologies. 2000.10, http://www.packardbiochip.com
[45] Full Moon Biosystems, Inc., Microarray Scanner Calibration Slide User's Guide. Part Number:FMB DS 01,2-9
(2) Editoral. Nature Gentics, 1996, 14[4]:367-370
(3) Fodor S.P.A,Read J.L.,Thomas R.G.,et al, Light-directed spatially addressable paralled chemical synthesis, Science, 1991, 251:767-773
(4) Fodor S.P.A, Rava R.P, Huang X.C. et al, Multiplexed biochemical assays with biological chips. Nature, 1993.8, 364[6437]:555-556
(5) Pease A C, Solas D, Sullivan E J,et al. Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc Natl Acad Sci USA, 1994, 91:5022-5026
(6) 张天浩，张春平，张光寅．DNA芯片制作原理及其杂交信号检测方法。生物工程进展，2000，20[2]：61-68
(7) Hacia J G, Brody L C, et al. Nature genetics, 1996,14:441-447
(8) Christine Debouck, Peter N.Goodfellow. DNA microarrays in drug discovery and development. Nature genetics supplement, 1999,21:48-50
(9) 易霞，尚永丰。生物芯片技术发展及应用前景．科技前沿，2003．7，7[7]：51-55
(10) 卫新菊．生物芯片技术及其在农业上的应用．小麦研究，2002，2．3[4]：34-36
(11) Amersham Pharmacia Biotech Inc., Fluorescence Imaging principles and methods, Amersham pharmacia biotech technical manual, 2000
(12) 齐华，程光伟．基于荧光标记的生物芯片信号检测技术．现代电子技术，2003，5：41-44
(13) 王立强，陆祖康，林斌．生物芯片及其荧光信号检测．光学仪器，2002，24[4-5]：59-65
(14) 吴岚君．生物芯片[DNA微阵列]荧光扫描仪中的激光共聚焦扫描技术．基因快讯，2001[3]，http://www.ebiotrade.com/check2/f29.htm
(15) J.Montagu, P.Honkanen. Fluorescence ArrayScanner. SPIE, 1999.7, 3779:284-292
(16) Mehta D.S., Lee C.Y., Chiou A., Multipoint parallel excitation and CCD-based imaging system for high-throughput fluorescence detection of biochip micro-
arrays, Optics Communications, 2001,190:59-68
[17] 马立人，将中华．生物芯片，化学工业出版社，北京，2000．2：19-20，95-98
[18] CAMBRIDGE TECHNOLOGY, INC. Model 6450 Galvanometer optical Scanner Instruction Manual. Cambridge,Massachusetts, 1996
[19] 杨国光．现代光学测试技术．浙江大学出版社，杭州，1997：352-357
[20] Performance Advantage of a Geometric Beamsplitter in ScanArray Microarray Scanners. Parkard BioChip Technologies, http://www.packardbioscience.com
[21] 郁道银，谈恒英．工程光学．机械工业出版社，北京，2001：256-264
[22] Gu M., Sheppard C.J.R., Effects of a finite-sized detector on the OTF of Confocal Fluorescent Microscopy, Optik, 1991, 89[2]: 65-69
[23] Wilson T, Carlini A.R. Three-dimensional Imaging in Confocal Imaging Systems with Finite Sized Detectors. J.Microsc., 1998, 149[1]:51-66
[24] 高万荣，陶纯堪，杨晓春．共焦扫描显微成像的光学断层平面分辨率与信噪比之间的关系．光子学报，2002，31[6]：730-735
[25] 宋登元，孙同文．光电倍增管的特性、结构及选取考虑．中国仪器仪表，1995．5：33-35
[26] 雷肇棣．光电探测器原理及应用．物理，1994，23[4]：220-226
[27] HAMAMATSU. Photosensor Modules H7710/H7711/H7712 Series user's guide,2001, http://www.hamamatsu.com
[28] 王鑫，郭继燕，丁铁夫．基于PCI总线的高速数据采集卡的设计．微计算机信息，2003，19[5]：31-32
[29] ADLINK Technology INC. NuDAQ PCI-9111DG/HR Multi-functions Data Acquistion Card User's Guide, 2000.11
[30] ADLINK Technology INC. NuDAQ ACLD Series Termination Boards User's Guide, 2000.6
[31] ADLINK Technology INC. ADLINK PCI/CompactPCI DAQ Cards Software Installation Guide, 2002.3
[32] Charles Petzold，北京博彦科技发展有限公司译．Windows程序设计[第五版]，北京大学出版社，北京，1999．11
[33] 王珏，葛冬梅．Windows下多幅大尺寸位图的显示与浏览．黑龙江工程学院学报，2002，16[1]：46-48
[34] 郑家亮，朱黎军，陈明．医疗设备图文档案格式分析．云南大学学报[自然科学报]，2000，22：49-51
[35] 贺京．TIFF分析及其应用。武测科技，1994，3：51-55
[36] 何斌，马天予，王运坚，朱红莲等．Visual C++数字图像处理．人民邮电出版社，北京，2001：4-20
[37] 马平，唐小萍，罗正全．生物芯片扫描仪图像处理过程及方法．微纳电子技术，2002，5：37-39
[38] Yee H Y, Michael J.Buckley, Sandrine Dudoit, et al. Comparison of methods for image analysis on eDNA microarray data. Technical Report, 2000.11
[39] Vincent Y.Jouenne ,Critical Issues in the Processing of eDNA Microarray Image. Computer and Apalication, 2001.6
[40] Jeremy Buhler, Trey Ideker, David Haynor. Dapple:Improved Techniques for Finding Spots on DNA Microarrays. UW CSE Technical Report UWTR, 2000.8
[41] Kenneth．R．Castleman， 朱志刚，林学阊，石定机等译．、数字图像处理；电子工业出版社，1998．9：387—418
[42] Axon Instruments,Inc. Genepix Pro 3.0 Microarray Acquisition and Analysis Software User's Guide. USA, 2000.9
[43] Pixel Resolution and Optical Resolution of Microarray Scanners. Packard Biochip Rechnologies, 2001, http://www.packardbioscience.com
[44] High Resolution Imaging for Accurate Microarray Quantitation. Packard Biochip Technologies. 2000.10, http://www.packardbiochip.com
[45] Full Moon Biosystems, Inc., Microarray Scanner Calibration Slide User's Guide. Part Number:FMB DS 01,2-9