激光共聚焦扫描显微镜技术的研究

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摘要

激光共聚焦扫描显微镜与传统的光学显微镜相比，系统的横向分辨率和轴向

分辨率有了显著的提高，并能够建立三维立体图像，实现对样品的实时观测。激

光共聚焦扫描显微镜具有体积小，分辨率高，操作简单，非接触测量等特点，因

此，共聚焦显微镜越来越被研究人员重视，被广泛的应用到生物，微电子，材料

和医学等领域中。

本文首先对共聚焦显微镜的工作原理进行了理论和实验上的分析，推导出了

共聚焦显微镜横向分辨率和纵向分辨率的影响因素，并设计和确定了共聚焦显微

系统的总体方案；分析了选择系统中各个元件的理论依据。通过研究，得出了共

聚焦显微镜的分辨率和光束直径，物镜的数值孔径，探测针孔等有很大的关系，

并证明了共聚焦显微镜系统测量的可行性。

在实验室搭建了一套激光共聚焦扫描显微镜系统，并对各模块进行调节，尽

量减少系统中存在的误差和干扰。通过搭建的实验系统对硬币进行二维扫描，得

到了硬币表面扫描区域的图像信息，通过串口输出数据，最后在计算机上合成灰

度图像。最后，通过中值滤波，拉普拉斯滤波，直方图均衡化等图像处理方法对

采集到的图像进行处理，得到了一幅清晰的硬币表面的图像。实验结果证明了共

聚焦显微镜工作原理理论上的正确性和实验室搭建共聚焦显微系统的可行性，最

后提出了本系统的改进方案和未来的发展趋势。

关键字：共聚焦扫描成像分辨率图像处理

ABSTRACT

Compared with traditional optical microscope, laser confocal scanning microscope

significantly improved the system’s lateral resolution and axial resolution and can

rebuild three-dimensional image, achieve real-time observation of samples. Laser

confocal scanning microscope has characteristic of small size, high resolution, simple

operation, non-contact measurement and so on. Confocal microscope attract more and

more attention by researcher and has been applied to biology, microelectronics,

materials and medicine field.

First, the paper analysis the principle of confocal microscope in theory and

experiment, inferential reasoning the influencing factors of system’s lateral resolution

and axial resolution, design and determine the overall program of confocal microscope

system. Through the research, we know that the resolution of confocal microscope have

the great relation of the beam diameter, objective lens’ numerical aperture, probe

pinhole aperture, and prove the feasibility of confocal microscope system measurement.

We build a set of laser confocal scanning microscope system in the laboratory,

and adjust each module, try my best to reduce the error and interference in the system.

Through two-dimensional scans of the coins by this system, we get the image

information of coin surface scanning area, and output data by serial, synthetic gray

image in the computer; process the collected image by median filtering, laplacian

filtering, histogram equalization and get a clear image of the surface of the coin.

Experimental results show that the principle of confocal microscope is correct in theory

and setting up confocal microscope in the laboratory is feasibility. Finally, propose this

system improvement program and future development treads.

Keyword: confocal, scanning image, resolution, image processing

中文摘要

ABSTRACT

第一章绪论 ·······················································································1

§1.1 课题的来源和研究意义 ······························································ 1

§1.2 国内外的研究状况 ·····································································2

§1.3 激光共聚焦扫描显微镜的应用 ····················································· 4

§1.4 论文的主要研究内容 ··································································4

第二章共聚焦显微镜的成像理论 ··························································· 6

§2.1 共聚焦显微镜的基本结构 ··························································· 6

§2.2 共聚焦显微镜的成像理论 ··························································· 7

§2.2.1 薄透镜的透过率 ····································································7

§2.2.2 菲涅耳近似理论 ····································································8

§2.2.3 焦平面上的衍射模式 ······························································9

§2.2.4 非焦平面上的衍射模式 ························································· 10

§2.2.5 薄透镜的相干成像和点扩散函数 ············································· 11

§2.3 扫描显微镜的成像公式 ····························································· 13

§2.4 激光共聚焦扫描显微镜的分辨率 ·················································14

§2.4.1 共聚焦显微镜的横向分辨率 ··················································· 14

§2.4.2 共聚焦显微镜的纵向分辨率 ··················································· 15

§2.5 本章小结 ··············································································· 18

第三章激光共聚焦扫描显微镜的系统设计 ··············································19

§3.1 共聚焦显微镜系统的基本类型 ····················································19

§3.2 共聚焦显微镜系统的整体架构 ····················································21

§3.3 系统的主要模块 ······································································ 22

§3.3.1 光源系统模块 ····································································· 22

§3.3.2 扫描系统模块 ····································································· 23

§3.3.3 显微系统模块 ····································································· 28

§3.3.4 探测系统模块 ····································································· 29

§3.4 本章小结 ··············································································· 31

第四章软件控制和图像处理算法 ·························································· 32

§4.1 共聚焦显微镜的软件设计 ·························································· 32

§4.2 扫描模块的控制系统设计 ·························································· 32

§4.3 数据传输模块的控制系统设计 ····················································33

§4.3.1 RS232 串口数据传输 ···························································· 33

§4.3.2 通信协议的设定 ·································································· 33

§4.3.3 数据的采集 ········································································ 34

§4.4 图像处理算法 ········································································· 35

§4.4.1 中值滤波 ··········································································· 35

§4.4.2 拉普拉斯滤波 ····································································· 36

§4.4.3 直方图均衡化 ····································································· 37

§4.4 本章小结 ··············································································· 38

第五章实验测量结果和分析 ································································ 39

§5.1 焦平面附近微小位移对探测信号影响的实验结果 ····························39

§5.2 对实际样品进行行扫描 ····························································· 41

§5.3 对实际样品进行二维扫描 ·························································· 43

§5.4 图像预处理的实验结果 ····························································· 44

§5.5 本章小结 ··············································································· 46

第六章总结与展望 ············································································ 47

参考文献 ··························································································· 49

在读期间公开发表的论文和承担科研项目及取得成果 ··································52

致谢 ································································································· 53

第一章绪论

随着激光技术，光纤，计算机，集成电路，材料工程，生命科学，精密机械

和光电子技术的快速发展，显微技术也进入了一个快速发展的阶段。世界各个显

微镜的强国都推出了各种型号和类型的显微镜。激光共聚焦扫描显微镜（Laser

Confocal Scanning Microscope——LCSM）就是其中一种新型的显微镜。激光共聚

焦扫描显微镜是一种光电技术结合的新型显微镜，和传统的光学显微镜相比，在

分辨率，放大率，灵敏度和信号的信噪比上都有了很大的提高。目前，它已成为

微电子，材料工程，生命科学等领域测量的主要工具，受到了国内外科研人员的

广泛关注和研究。

§1.1 课题的来源和研究意义

2009年6月4日出版的《自然》杂志刊发社论—《显微镜奇迹》（Microscopic

marvels）。社论强调，显微镜使得生物学研究面目焕然一新，研究人员要有所作为，

就必须有创新和协作，包括研究工具的创新和使用。本期的《自然》杂志展示了5

项显微镜领域的创新工作：从超高分辨率的光学显微镜（其分辨率可与电子显微

镜相匹敌）到较厚样品观察的电子显微镜（其样品厚度要求可与光学显微镜想匹

敌）[1]。而激光共聚焦扫描显微镜（LCSM）是介于传统光学显微镜以及现代电子

显微镜之间的，是在20世纪80年代发展起来的一项具有划时代意义的高科技新产

品，从图1-1来看，激光共聚焦扫描显微镜从分辨率和放大倍数两方面填补了传统

和现代电子显微镜之间的空白，是当今世界最先进的细胞生物学分析仪器[2]。

图1-1 激光共聚焦扫描显微镜和其他显微镜的比较

激光共聚焦扫描显微镜和传统的光学显微镜相比，它具有超过传统光学显微

激光共聚焦扫描显微镜技术的研究

镜的横向分辨率，又克服了景深小的缺点，取得了较高的纵向分辨率，从而实现

了光学的层析[3,4]，这是传统的光学显微镜达不到的。共聚焦的含义就是将焦平面

以外的成像信号过滤，使其不影响最终的成像结果，首先将光束照射到样品的一

点，然后对所要观察的样品平面进行扫描，焦平面以外的信号通过探测针孔滤除。

共聚焦扫描显微镜独特的成像特性来自于探测小孔的使用，探测小孔可以抑制焦

平面以外的杂散光进入探测器，因此，显微镜焦平面的图像不会受到焦平面以外

的杂散光线的干扰，从而可以得到比传统显微镜更加清晰的图像；另外，共聚焦

显微镜通过纵向的微调，可以将一系列二维焦平面的图像通过计算机合成三维的

立体图像，这种通过非接触式测量就得到了高分辨率的图像，避免了以往科研人

员需要切片，漂洗，染色等花费大量的时间来准备样本，并且样品在切片过程中

会破坏原有样品的物理结构，另外，还有很多样品本身是不可切割的[5]。使用共聚

焦显微镜可以有效的避免上述问题，可以实时的记录样品的图像，使科研人员可

以从容的研究和观察样品的情况。所以它问世以来在生命科学，材料工程和微电

子器件领域得到了广泛的应用[6]。

自1985年激光共聚焦扫描显微镜问世以来，该项技术一直在飞速发展和完善

中。共聚焦显微镜价格昂贵，但在生物科学，材料工程检测方面起着非常大的作

用，并且在近些年来形成了巨大的市场价值。每年我国相关单位都需要花费巨额

外汇来购买国外的产品，动辄几十万美元。共聚焦显微技术的研究以及产品的研

制主要在国外，尤其是德国的卡尔蔡司（CarlZeiss）,日本的奥林巴斯（Olympus）,

尼康（Nikon）等大型公司。

激光共聚焦扫描技术是光学，机械和电子结合的前沿交叉课题，我们希望可

以研制出显微镜的样机，进而进行成品生产，打破欧美等国家在高端显微镜领域

的垄断，填补国家在这方面的空白。

本课题来自上海市优秀青年教师基金项目：激光共聚焦显微镜的研究

（shg-07006）。

§1.2 国内外的研究状况

在19 世纪，德国物理学家 Ernst Abee 得出光学显微镜的分辨率受到衍射现象

的限制，其最小分辨率不小于半个光波长，这就是著名的阿贝准则[7]。为了突破光

学显微镜的衍射极限，科学家不断的寻找新的方法来提高显微镜的分辨率。1931

年，Ruska 和Knoll 研制成功了第一台电子显微镜（Electron Microscopy），突破了

光学显微镜分辨率的极限。随后科学家有发明了分辨率更高的原子力显微镜和扫

描隧道显微镜[2]。但是它们也有很多不足之处[3]，如价格昂贵，准备样品操作复杂，

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摘要：

摘要激光共聚焦扫描显微镜与传统的光学显微镜相比，系统的横向分辨率和轴向分辨率有了显著的提高，并能够建立三维立体图像，实现对样品的实时观测。激光共聚焦扫描显微镜具有体积小，分辨率高，操作简单，非接触测量等特点，因此，共聚焦显微镜越来越被研究人员重视，被广泛的应用到生物，微电子，材料和医学等领域中。本文首先对共聚焦显微镜的工作原理进行了理论和实验上的分析，推导出了共聚焦显微镜横向分辨率和纵向分辨率的影响因素，并设计和确定了共聚焦显微系统的总体方案；分析了选择系统中各个元件的理论依据。通过研究，得出了共聚焦显微镜的分辨率和光束直径，物镜的数值孔径，探测针孔等有很大的关系，并证明了共聚焦显微镜系统测量...

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