线结构光共焦扫描显微系统的研究
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摘要
共焦显微镜由于采用了“共焦”技术而获得了比普通光学显微镜更高的分辨率和信噪比,并且能够对样品进行三维成像。激光共焦扫描显微镜在生物医学、材料科学、微电子制造和精密测量等领域有着广泛的应用。
目前大多数共焦显微镜都采用点共焦扫描。本论文提出了一种新型线结构光共焦扫描显微系统,包括以下内容:
1、以傅立叶光学和光学传递函数理论为基础,讨论了点共焦显微术三维相干函数和线结构光共焦显微术的交叉传递函数,分析了影响成像分辨率的因素。
2、设计了线结构光共焦扫描显微成像光学系统,使用光学设计软件OSLO设计了专用的无限远像距半平场显微物镜和激光扩束准直镜。
3、设计了采用光栅反馈闭环控制的机械工作台移动平面扫描和步进电机控制、精密齿轮传动的物镜移动轴向扫描系统。
4、设计了线结构光共焦扫描显微系统整体结构,对线结构光共焦成像进行了实验研究,结果证明了线结构光共焦可以明显减小杂散光的影响,增强图像清晰度和对比度,且具有层析能力。
经过两年多的研究工作,掌握了共焦显微术的理论,并初步设计出线结构光共焦扫描显微实验装置,为接下来进一步提高成像分辨率和图像处理软件的开发打下良好的基础。
The confocal microscope can attain higher resolution and signal-to-noise ratio than ordinary optical microscope due to its“confocal”technique, and can make 3D images for the samples. Laser confocal scanning microscopes are widely applied in the areas of biomedicine, material science, microelectronic manufacture and precise measurement.
Most types of confocal microscopes are using point cofocal scanning, while this dissertation presents a new type of line constructive laser confocal scanning microscope system. The main topics including:
1、Point confocal microscopy 3D coherent transfer function(CTF) and line constructive laser confocal microscopy transmission cross-coefficient(TCC) are discussed based on Fourier optics and optical transfer function theory. The elements that affect the resolution of the image are analyzed.
2、The optical system of line constructive laser confocal scanning microscope is designed. The optical design software OSLO is used to design special infinite image distance half flat field objective as well as laser extender and collimating lens.
3、A machinery working platform movement flat scanning system that applies optical grating feedback closed loop control and an objective moving axial scanning system that applies stepping motor control and precise gear driving are developed.
4、The integer configuration of the ling construction beam confocal microscope system is designed. The effect of light constructive laser confocal image is researched in experiment. The result testified that the line constructive laser confocal imaging can diminish the effect of stray light obviously, and the image articulation and contrast are improved. The system also has the ability of optical sectioning.
During the two year’s research work, we mastered the theory of confocal microscopy, and designed an experimental line constructive laser confocal scanning microscope device. All these works formed a nicer basis for the next work of improving imaging resolution and the development of image processing software.
目前大多数共焦显微镜都采用点共焦扫描。本论文提出了一种新型线结构光共焦扫描显微系统,包括以下内容:
1、以傅立叶光学和光学传递函数理论为基础,讨论了点共焦显微术三维相干函数和线结构光共焦显微术的交叉传递函数,分析了影响成像分辨率的因素。
2、设计了线结构光共焦扫描显微成像光学系统,使用光学设计软件OSLO设计了专用的无限远像距半平场显微物镜和激光扩束准直镜。
3、设计了采用光栅反馈闭环控制的机械工作台移动平面扫描和步进电机控制、精密齿轮传动的物镜移动轴向扫描系统。
4、设计了线结构光共焦扫描显微系统整体结构,对线结构光共焦成像进行了实验研究,结果证明了线结构光共焦可以明显减小杂散光的影响,增强图像清晰度和对比度,且具有层析能力。
经过两年多的研究工作,掌握了共焦显微术的理论,并初步设计出线结构光共焦扫描显微实验装置,为接下来进一步提高成像分辨率和图像处理软件的开发打下良好的基础。
The confocal microscope can attain higher resolution and signal-to-noise ratio than ordinary optical microscope due to its“confocal”technique, and can make 3D images for the samples. Laser confocal scanning microscopes are widely applied in the areas of biomedicine, material science, microelectronic manufacture and precise measurement.
Most types of confocal microscopes are using point cofocal scanning, while this dissertation presents a new type of line constructive laser confocal scanning microscope system. The main topics including:
1、Point confocal microscopy 3D coherent transfer function(CTF) and line constructive laser confocal microscopy transmission cross-coefficient(TCC) are discussed based on Fourier optics and optical transfer function theory. The elements that affect the resolution of the image are analyzed.
2、The optical system of line constructive laser confocal scanning microscope is designed. The optical design software OSLO is used to design special infinite image distance half flat field objective as well as laser extender and collimating lens.
3、A machinery working platform movement flat scanning system that applies optical grating feedback closed loop control and an objective moving axial scanning system that applies stepping motor control and precise gear driving are developed.
4、The integer configuration of the ling construction beam confocal microscope system is designed. The effect of light constructive laser confocal image is researched in experiment. The result testified that the line constructive laser confocal imaging can diminish the effect of stray light obviously, and the image articulation and contrast are improved. The system also has the ability of optical sectioning.
During the two year’s research work, we mastered the theory of confocal microscopy, and designed an experimental line constructive laser confocal scanning microscope device. All these works formed a nicer basis for the next work of improving imaging resolution and the development of image processing software.
引文
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[31]唐志列,黄佐华等.共焦显微镜的纵向分辨率极限及其判据.量子电子学报, 2000, 17(3):199–204.
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[2]梁作舟,白也武.扫描隧道显微镜的发展史.广西物理,1995,16(5):93–96.
[3]姚骏恩.创造探索微观世界的有力工具(今年诺贝尔物理学奖获得者的贡献).中国科技报,1986-12-08 (3).
[4]J.Z.Young, F.Roberts. A flying-spot microscopy. Nature, 1951,Vo1.167:231–232.
[5]Minsky M., Microscopy apparatus. US Patant:3013467, 1961.
[6]M.D.Egger M.Petran, Science, 1967,157 (8)305.
[7]Petran M, Hadravsky M., Egger M.D., Galambos R., Tandemscanning reflected-light microscopy. J.Opt. Soc. 1968, 58:661–664.
[8]Svishchev, G.M., 1969. Microscope for the study of transparent lightscattering objects in incident light. Opt. Spectrosc. 26, 171–172.
[9]P.Davidovits and M.D.Egger. Scanning lasering nucroscopy for biological investi- gation. Appl Opt, 1971,10: 1615–1619.
[10]P.Davidovits and Egger M.D, US Patent: 3643015, 1972.
[11]Barry R.Masters. David Maurice’s contributions to optical ophthalmic instrument- ation: roots of the scanning slit clinical confocal microscope. Experimental Eye Resea- rch 2004,78:315–326.
[12][澳]M.顾.共焦显微术的三维成像原理[M].北京:新时代出版社,2000.
[13]Koerter C.J., Scanning mirror microscope with optical sectioning characteristic, Appl Opt, 1980,19:1749–1757.
[14]Koester, C.J. Scanning microscope apparatus. United States Patent, 4,241,257. 1980.
[15]Guy Cox. Biology confocal microscopy. Materialstoday, 2002, 34–41.
[16]美国 Bio-Rad 公司主页 http://www.bio-rad.com.
[17]德国 Zeiss 公司主页 http://www.zeiss.com.
[18]德国 Leica 公司主页 http://www.leica.com
[19]日本 Olympus 公司主页 http://www.olympus-global.com
[20]杨永田.首台国产激光共焦扫描显微镜问世.科学时报, 2001.12.25.
[21]田明丽,包正康等.激光共焦扫描显微镜及其应用.光学仪器, 2001, 23(1):16–19.
[22]丁志华,包正康等.共焦扫描显微术中影响轴向分辨率的因素分析.中国激光, 2000, 27 (6):531–536.
[23]孔兵,王昭,谭玉山.利用狭缝的共焦测量系统.仪器仪表学报,2001,22(增刊):132–133.
[24]李明周,王昭,赵宏.点扫描激光共焦测量研究,2003,37(6):39–41.
[25]孔兵,王昭等.基于针孔阵列的多光束共焦三维测量系统.中国激光,2002,29(3): 263–266.
[26]陈华,金伟其等.数字共焦显微技术及其处理算法的进展.光电子技术与信息, 2005,18 (2) :1–5.
[27]高万荣,陶纯堪等.共焦扫描光学显微镜的高分辨率.光学技术,1999,3:4–7.
[28]胡茂海,陶纯堪等.激光共焦扫描显微镜数据场的三维重建.中国激光,2002, 29 (6): 573–575.
[29]戚永军,迟泽英.共焦扫描显微成像系统的点扩散函数再商讨.光子学报,2002, 31 (18):1029–1031.
[30]高万荣,杨晓春等.共焦扫描荧光显微镜的信噪比与信息量,光学学报.2002,22 (10):1229–1235.
[31]唐志列,黄佐华等.共焦显微镜的纵向分辨率极限及其判据.量子电子学报, 2000, 17(3):199–204.
[32]唐志列,梁瑞生等.偏振共焦扫描激光显微镜的成像特性研究.光学学报,1999, 19 (8):1118–1122.
[33]张建寰,谭久彬.应用共焦显微镜原理测量倾斜工程表面.光学学报,2003,23(4): 446–450.
[34]周拥军,陈德强等.共聚焦激光扫描荧光显微镜扫描系统研制.光学 精密工程, 2002,10(6):582–587.
[35]王永红,余晓芬等.多光束并行共焦探测系统特性的研究.计量学报,2004,25(2): 100–103.
[36]杨莉松,王桂英等.共焦扫描成像研究.中国激光,1999,26(10):931–934.
[37]刘力,杨莉松.利用光纤共焦扫描显微镜的轴向响应曲线测定压电陶瓷的逆压电伸缩效应.光学学报,2001,Vol.21(3):380~382.
[38]邓小强,刘力等.超分辨在共焦三维形貌检测术中的应用.光学学报,2001,21(7): 853–856.
[39]Ilko K.Ilev, Ronald W. Waynant. A high-resolution fiber-optic confocal microsco- pe. Proceedings of SPIE, Vol.4261 (2001):7–13.
[40]C.J.R. Sheppard, Hao Zhou. Confocal interference microscopy. SPIE Vol. 2984 (1997):85–89.
[41]Mitsuhiro Ishihara, Hiromi Sasakj. High-speed 3D shape measurement using a non-scanning multiple-beam confocal imaging system. SPIE Vol.3478(1998):68–75.
[42]M.Minsky. Scanning.1988, 10:128.
[43]C.J.R.Sheppard and A.Choudhury, Optica Acta, 24(1977)1051.
[44]Zeno Foldes-Papp, Ulrike Demel, Gernot P.Tilz. Laser scanning confocal fluores-cence microscopy: an overview. International Immunopharmacology 3(2003):1715–1729.
[45]宋登元.共焦激光扫描显微镜及其应用.激光与红外,1998,28(1):19–22.
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