浅谈共聚焦显微技术

共聚焦显微镜以其高对比度、高分辨率及可重建三维图像的独特优势,在生物医学研究、微细加工、半导体和高分子材料的生产检测等领域获得广泛应用。常用的共聚焦技术方法有:传统的激光扫描共聚焦显微镜(LSCM),其特点是获得的图像对比度和分辨率高,但需要逐点扫描,帧成像时间长,系统复杂,体积大,价格昂贵;碟片共聚焦显微镜(SDCM)是采用多光束扫描的方法来获得共聚焦图像,速度可以大大提高,但牺牲了共聚焦图像的分辨率,系统更为复杂,且不能调整轴向分辨率;结构光显微镜(SIM)具有方法简单,可模块化设计,成本低,成像质量接近于激光扫描共聚焦显微镜,成像速度快,性价比较高。     

光学技术在细胞成像中的应用

本文对光学技术在细胞成像方面的应用进行了综述。着重介绍了激光扫描共聚焦显微镜、多光子荧光成像技术、全内反射荧光显微镜、近场扫描光学显微成像技术、光学相干层析成像技术的基本原理及其主要特点。体现了光学检测技术无侵入、无电离辐射,具有多种操作模式,可获得实时与定量等多种信息的特点,在生命科学研究中具有举足轻重的地位。     

激光共聚焦显微镜的应用技术

阐述LSM510激光扫描共聚焦显微镜的工作原理及主要功能,提出LSM510激光扫描共聚焦显微镜的使用方法及荧光探针的选择。     

提高激光扫描共焦显微镜反射镜扫描器定位精度的一种方法

介绍在激光扫描共焦显微镜三维扫描光路中，使用经微步细分的步进电机来完成其中的帧扫描，将MCS──51单片机引入微机控制，并采用电流补偿法来获得均匀的微细步，从而提高图象三维重建的质量。     

机械零件三维表面形貌测量与评定的研究

介绍了机械零件三维表面形貌的测量与评定,分析了激光干涉式位移传感器的光学原理和干涉条纹信号的细分方法.激光干涉式位移传感器的精度达到了5nm左右.另外,带计量系统的二维工作台也是整个测量系统的关键部分.因为采用了光栅尺作为二维工作台的计量系统,所以在表面形貌的测量过程中二维工作台在X和Y两个水平方向上都能获得精确的定位.     

扫描电化学显微镜压电工作台的建模与控制

为提高扫描电化学显微镜(SECM)微定位系统的运动定位精度,对其压电工作台的数学模型和控制器设计进行了研究.介绍了压电工作台的动态迟滞模型方程和采用Prandtl-Ishlinskii(PI)迟滞算子的动态迟滞模型,并在此基础上设计了压电工作台的复合控制方案.以CHI900B型扫描电化学显微镜的三维压电工作台为实验对象,对动态迟滞模型的具体建模过程进行了阐述,并验证了控制器的性能.在100 V/s和900 V/s两种不同输入电压速率下进行运动定位实验,动态迟滞模型平均误差分别为0.08μm和0.11μm,精度明中显优于压电工作台的线性动态模型和PI迟滞模型.复合控制方案下,系统跟踪±400μm/s任意三角波的平均误差为0.085μm,最大误差为0.105μm;跟踪复频波的平均误差为0.105μm,最大误差为0.115μm.控制效果较好.     

一种适用于皮肤荧光的成像检测系统探究

目前皮肤影像手段包括共聚焦激光扫描显微镜、光学相干断层扫描成像技术、高频超声、皮肤镜、荧光显微镜、反射式共聚焦显微镜、皮肤太赫兹成像等成像手段。每种成像手段只能突出皮肤肿瘤的部分特征,如超声能突出肿瘤的解剖结构成像,却不能准确定位肿瘤边缘;荧光成像能突出肿瘤边缘信息,但无法探查肿瘤深度等细节信息。国内目前的研究很少涉及不同皮肤影像手段的交叉研究。因此,本课题希望探究一种预留超声探头整合接口的荧光成像系统,为后续整合超声探头,进一步实现多模态皮肤肿瘤成像系统做贡献。课题研究包括皮肤肿瘤荧光原理探究,针对皮肤荧光的相机参数设计,整合超声探头的相机结构设计等部分,以及皮肤肿瘤荧光图像验证实验。     

显微观测技术的新进展及其应用

依据显微观测技术的发展过程 ,介绍了普通的光学显微镜和 2 0世纪流行的电子显微镜 ,详细阐述了以扫描隧道显微镜和激光扫描共焦显微镜为代表的新型显微镜系列的发展 ,以及各类显微镜的基本工作原理和应用情况     

面向微纳材料的激光扫描二维成像系统

在对微纳材料光学特性表征中,需要获得分辨率更高的波长和强度的荧光图像。普通的显微镜无法满足测试的要求,因此将普通的成像显微镜、光谱仪以及纳米移动台组成激光扫描显微镜成像系统,并利用LabVIEW开发了一套完整的集二维扫描采集与信号图像处理一体的系统上位机软件。扫描采集过程使用了低通滤波等数字信号处理方法消除光谱仪信号噪声的影响。利用本系统测量硒化镉纳米带、单层二硫化钼得到了荧光强度图像以及荧光峰值波长图像,能分辨出最小波长为0.03 nm的荧光。将采集长度与实际长度进行比较并分析荧光强度差异,取得了较好的效果。     

一种新型三维表面形貌测量仪的研制及应用

介绍了一种新型的基于直线相位光栅干涉三维表面形貌测量仪,该测量仪具有高分辨率、大量程、低成本的特点.该三维表面形貌测量仪由基于直线相位衍射光栅干涉原理的微位移传感器、X-Y二维工作台、立柱、光电探测器以及信号处理电路、计算机及数据处理软件组成.该轮廓仪工作台的工作范围为50 mm×50 mm,最小步距为0.2μm,工作台计量系统的分辨率为0.05μm,微位移传感器理论垂直分辨率可达到0.12 nm,实际测量量程为2 mm,通过更换测杆可以达到6 mm的测量量程.     

光机二维扫描技术在激光共聚焦生物芯片扫描仪中的应用

介绍了应用于激光共聚焦生物芯片扫描仪中的光机二维扫描技术 ,即用振镜和f- θ扫描物镜构成其中一维的光扫描系统 ,用步进电机驱动扫描工作台移动构成另一维机械扫描系统 ,并在此基础上分析研究了光机二维扫描控制系统的设计。为快速、高精度激光共聚焦生物芯片扫描仪的研制作了新的有益尝试 ,并取得了初步的实验结果     

激光扫描共聚焦光谱成像系统

在激光扫描共聚焦显微成像技术基础上引入了光谱成像技术以便区分生物组织中的不同荧光成分。采用分光棱镜对荧光进行光谱展开,在光谱谱面处设置两个可移动缝片形成出射狭缝,两个步进电机带动安装其上的两个缝片设置系统在整个工作波长（400~700 nm）内的光谱带宽,其最小光谱带宽优于5 nm。用488 nm激光和低压汞灯实际测量了几条谱线对应的狭缝位置并和理论值做了比较,结果显示实际狭缝位置和理论值的差值均小于0.1 mm。在全光谱和50 μm出射狭缝（对应2.5 nm光谱带宽）对老鼠肾脏组织进行了共聚焦光谱成像实验,获得了老鼠肾脏组织中DAPI标定的细胞核图像和Alexa Fluor^®488标定的肾脏小球曲管图像,实现了对老鼠肾脏组织不同成分的区分。实验结果表明：提出的系统能够进行共聚焦光谱成像,扩大了共聚焦显微镜的适用范围。     

Intensity correction of fluorescent confocal laser scanning microscope images by mean-weight filtering

This paper addresses the problem of intensity correction of fluorescent confocal laser scanning microscope images. Confocal laser scanning microscope images are frequently used in medicine for obtaining 3D information about specimen structures by imaging a set of 2D cross sections and performing 3D volume reconstruction afterwards. However, 2D images acquired from fluorescent confocal laser scanning microscope images demonstrate significant intensity heterogeneity, for example, due to photo‐bleaching and fluorescent attenuation in depth. We developed an intensity heterogeneity correction technique that (a) adjusts the intensity heterogeneity of 2D images, (b) preserves fine structural details and (c) enhances image contrast, by performing spatially adaptive mean‐weight filtering. Our solution is obtained by formulating an optimization problem, followed by filter design and automated selection of filtering parameters. The proposed filtering method is experimentally compared with several existing techniques by using four quality metrics: contrast, intensity heterogeneity (entropy) in a low frequency domain, intensity distortion in a high frequency domain and saturation. Based on our experiments and the four quality metrics, the developed mean‐weight filtering outperforms other intensity correction methods by at least a factor of 1.5 when applied to fluorescent confocal laser scanning microscope images.     

Inexpensive, high-quality optical relay for use in confocal scanning beam imaging

An inexpensive, high optical-quality relay lens made up of two eyepieces arranged in an afocal assembly for use in confocal scanning laser imaging is described. In the past we have used relays, within our confocal microscopes, made up of achromats with long focal lengths (> or = 10 cm), which take up large optical tracks and suffer from significant amounts of astigmatism and curvature of field. We quantify aberrations associated with achromat and eyepiece relays using CODE V optical design and analysis software. The eyepiece relay is found to be more compact, better corrected, and not significantly more expensive than its achromat counterpart. In addition to being used to interconnect two scanning mirrors optically as well as scanning mirrors with microscope objectives, it can form part of the optics in a confocal scanning laser MACROscope-Microscope system (Biomedical Photometrics, Inc., Waterloo, Ontario, Canada). Due to design constraints, the MACROscope-Microscope system cannot incorporate a conventional wide-field microscope into its structure such as is done in most commercial confocal microscopes. The eyepiece relay is used as a stand-alone, compact optical link between the scanning mirrors and the microscope objective. This consequently makes the MACROscope-Microscope system more compact and easier to commercialize.     

A 2D MEMS mirror with sidewall electrodes applied for confocal MACROscope imaging

This paper presents microelectromechanical system micromirrors with sidewall electrodes applied for use as a Confocal MACROscope for biomedical imaging. The MACROscope is a fluorescence and brightfield confocal laser scanning microscope with a very large field of view. In this paper, a microelectromechanical system mirror with sidewall electrodes replaces the galvo-scanner and XYZ-stage to improve the confocal MACROscope design and obtain an image. Two micromirror-based optical configurations are developed and tested to optimize the optical design through scanning angle, field of view and numerical aperture improvement. Meanwhile, the scanning frequency and control waveform of the micromirror are tested. Analysing the scan frequency and waveform becomes a key factor to optimize the micromirror-based confocal MACROscope. When the micromirror is integrated into the MACROscope and works at 40 Hz, the micromirror with open-loop control possesses good repeatability, so that the synchronization among the scanner, XYZ-stage and image acquisition can be realized. A laser scanning microscope system based on the micromirror with 2 μm width torsion bars was built and a 2D image was obtained as well. This work forms the experimental basis for building a practical confocal MACROscope.     

Analysis of digitizing errors of a laser scanning system

The digitizing errors of a high-speed 3D laser scanning system are analyzed and characterized in this paper. As the laser scanner is an electro-optical device and based on the principle of optical triangulation, the measurement accuracy is affected by the measured part geometry and its position within the scanning window. Commercial laser scanners are often calibrated in the scanning plane to account for variation of the incident angle of the laser beam. The effects of the scan depth and the projected angle, characterizing the surface normal of the measured part external to the scanning plane, on the measurement accuracy are not considered in the standard calibration process and have been identified by experiments in the present work. Experimental results indicate that the random error of the scanned data is close to the nominal value provided by the manufacturer. The systematic error shows a bilinear relationship with the scan depth and the projected angle and has a maximum value of about 160 μm. The developed empirical model correctly predicts the systematic error with a maximum deviation of only 25 μm.     

激光共焦显微光束的偏转扫描

为了提高激光共聚焦系统的扫描速度,本文提出一种逐场扫描的场同步扫描方法。构建了激光共焦显微系统,将美国THORLABS公司的GVS002型二维检流计振镜应用于该系统,根据光学系统参数以及扫描范围要求计算振镜的整场扫描波形。借助NI公司的PCIe6353多功能数据采集卡,输出行同步的扫描波形,同时,对共焦显微系统共焦位置上针孔处的光强信号进行采集,先后扫描一幅256×256和512×512的图像,记录扫描图像和成像时间;然后,在相同的硬件结构下,以场同步的方式输出扫描波形,记录扫描图像和成像时间。实验结果表明:场同步方式扫描256×256图像的速度可提高10倍,扫描512×512图像的速度可提高5倍,且满足共焦显微成像的清晰、抗干扰能力强等要求。与行同步扫描方法相比,场同步扫描方法可以消除行与行之间转换的停留时间,在不改变硬件的情况下大幅提高扫描速度。     

Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1

Two-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6–17, which enhances the contrast by a factor of 6–21; (2) a lower rate of photobleaching by a factor of 2–4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations, probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0.18-mW laser power with a 2.φ pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-φ pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.     

Three‐dimensional visualization of dermal skin structure using confocal laser scanning microscopy

The properties and performance of collagen‐based materials may be affected by the collagen fibre bundle pattern, orientation and weave. The aim of this study was to develop and apply methods to visualize the dermis using confocal laser scanning microscopy from thin tissue sections stained with haematoxylin and eosin. The data was processed to allow three‐dimensional (3‐D) visualization on a PC and using a 3‐D immersive technology system. The 3‐D visualization of the confocal microscope image stacks allowed the evaluation of the collagen macromolecular structure including the collagen fibre bundles. The methods developed provide a novel way of viewing complex organic structures with further potential applications in the medical field.     

Three-dimensional visualization of insect morphology using confocal laser scanning microscopy

Cuticular structures of insects are often microscopic and intricately complex; among the most complex structures are male genitalia. Genitalic structures are essential in taxonomic and phylogenetic studies of insects. Using well‐described species from two disparate dipteran genera, we demonstrate the utility of confocal laser scanning microscopy for studying the morphological characters of fly genitalia by taking advantage of the autofluorescent properties of cuticle material. Reconstructions of confocal data sets obtained from genitalic structures embedded in two commonly used entomological mounting media (euparal and glycerin jelly) are presented. Aberration artefacts often observed in confocal data obtained from thick specimens were analysed and strategies for their minimization are discussed. Our results indicate that confocal laser scanning microscopy and 3D reconstruction are excellent techniques for visualizing small, complex, autofluorescent structures in flies. These techniques could have a profound impact on the quality of information provided by 3D representations of insect structures over more traditional methods of visualization.