激光共聚焦显微镜与光学显微镜之比较

激光扫描共聚焦显微镜在活细胞的动态检测、光学切片和三维结构重建等方面较光学显微镜有质的飞跃。本文对激光扫描共聚焦显微镜和光学显微镜进行了比较和讨论,并简单介绍多光子激光扫描显微镜。     

激光扫描共聚焦显微镜在植物学中的应用

激光扫描共聚焦显微镜(LSCM)是普通光学显微镜与激光和计算机及其相应的软件技术组合的产物,实现了连续光学切片,能在亚细胞水平观察细胞骨架的动态变化、细胞内特异蛋白、钙等离子的变化,并结合电生理等技术观察细胞生理活动与细胞形态及运动变化的相互关系。并广泛应用于生物三维结构重组及动态分析。本文综述了应用激光扫描共聚焦显微镜技术在植物细胞学、植物发育、组织化学以及基因表达、检测等领域取得的进展。     

Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope

By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as “easy-STED”, achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating.     

真菌的自发荧光现象研究

本试验用激光共聚焦扫描显微镜对三株不同真菌的菌丝、分子孢子以及子实体的自发荧光现象进行了观察,结果发现：处于相同培养条件的三株真菌的荧光分布特点不一,并且同一真菌在不同培养基条件下所表现的荧光现象亦有差异,说明真菌的自发荧光具有种属特异性并受到培养基质等因素的影响,而培养时间对荧光强度及类别的影响不显著。化学试剂的处理试验表明,弱碱性物质对真菌的荧光具有减弱效应,而强碱性物质对真菌的荧光则表现为增强,提示不同真菌细胞内的自发荧光物质结构上和组分上的差异,其变化可导致荧光性质的改变。     

激光扫描共聚焦显微镜在花粉研究中的应用

激光扫描共聚焦显微镜（Laser scanning confocal microscope,LSCM）是普通光学显微镜、激光、计算机及其相应的软件技术结合的产物,能实现连续光学切片和生物三维结构重组及动态分析.作为一种先进的技术手段,LSCM技术已经为花粉生物学的研究提供了有价值的新资料,大大地推动了植物生殖生物学的发展.本文综述了LSCM技术在花粉粒形态（形状、大小及三维重建）、花粉的内部结构（如细胞骨架）、花粉的遗传学、花粉的发育、花粉的萌发、花粉自发荧光特性、孢粉研究等方面中的应用,并指出了LSCM的不足之处,最后提出了LSCM技术在花粉研究中的应用展望.未来LSCM技术应该与多光子技术、活细胞工作站技术相结合使用,才能更准确地进行花粉动态研究的实时监测.     

人膀胱癌细胞纤维肌动蛋白共聚焦激光扫描显微镜观察

探讨膀胱癌细胞纤维肌动蛋白（F－actin)的空间结构。采用共聚焦激光扫描显微镜光学切片技术结合异硫酸氢荧光素－鬼笔环肽（FITC－phalloidin)标记纤维肌动蛋白和碘化丙啶（PI）和标记核酸的荧光探针双重标记技术对膀胱癌细胞纤维肌动蛋白进行形态学观察,结果可见膀胱癌细胞内纤维肌动蛋白微丝形态完整,成细束或细丝状,平行排列地整个细胞或细胞突起,在胞质边缘处较密集。     

Automated Confocal Laser Scanning Microscopy and Semiautomated Image Processing for Analysis of Biofilms

The purpose of this study was to develop and apply a quantitative optical method suitable for routine measurements of biofilm structures under in situ conditions. A computer program was designed to perform automated investigations of biofilms by using image acquisition and image analysis techniques. To obtain a representative profile of a growing biofilm, a nondestructive procedure was created to study and quantify undisturbed microbial populations within the physical environment of a glass flow cell. Key components of the computer-controlled processing described in this paper are the on-line collection of confocal two-dimensional (2D) cross-sectional images from a preset 3D domain of interest followed by the off-line analysis of these 2D images. With the quantitative extraction of information contained in each image, a three-dimensional reconstruction of the principal biological events can be achieved. The program is convenient to handle and was generated to determine biovolumes and thus facilitate the examination of dynamic processes within biofilms. In the present study, Pseudomonas fluorescens or a green fluorescent protein-expressing Escherichia coli strain, EC12, was inoculated into glass flow cells and the respective monoculture biofilms were analyzed in three dimensions. In this paper we describe a method for the routine measurements of biofilms by using automated image acquisition and semiautomated image analysis.     

油杉小孢子发生的激光扫描共聚焦显微镜观察

应用激光扫描共聚焦显微镜研究了油杉小孢子的发生过程。油杉样品经曙红-Hoechst 33342双染和冬青油透明后,分别以UV、488 nm激光进行单扫描或系列扫描,并对系列扫描图像进行三维重建。观察结果表明,LSCM图像更能清晰地展示油杉花粉母细胞减数分裂的过程。粗线期染色体密集并缠绕成一团,占据核的一部分,核仁靠在这一团染色体的一边;减数分裂存在扩散双线期;在减数分裂过程中胞质分裂为同时型。观察到中期I、Ⅱ形成的纺锤体和四分体时期胼胝质壁的沉积等。研究结果表明,曙红-Hoechst 33342双染法可用于花粉发育阶段的鉴定,LSCM是研究花粉生物学的理想工具。研究为探究其濒危机制提供有意义资料,为其保护生物学和系统分类学提供胚胎学依据。     

人鼻咽癌细胞纤维肌动蛋白的共聚焦激光扫描显微镜观察

为探讨高分化和低分化鼻咽癌细胞纤维肌动蛋白（F-actin)的空间结构,采用共聚焦激光扫描显微镜光学切片技术结合异硫酸氢荧光素－鬼笔环肽（FITC－phalloidin)标记F-actin、碘化吡啶（PI）标记核酸的荧光探针双重标记技术,对鼻咽癌细胞F－actin进行光学切片、三维重组及形态学观察。实验结果可见高分化鼻咽癌细胞F－actin呈芒刺状分布于细胞表面,而在细胞突起末端细胞间连接处呈束状,放射状密集分布;代分化鼻咽癌细胞F－actin明显少于高分化鼻咽癌细胞,仅沿细胞膜表面呈弯曲细小绒毛状分布。结果表明F－actin在细胞内的空间分布与鼻咽癌的分化类型有关,肿瘤或恶性转化细胞的F－actin在形态和结构方面有异常改变;共聚焦激光扫描显微镜光学切片结合荧光双重标记技术是研究细菌骨架结构的理想方法。     

用激光共聚焦显微镜鉴别可疑笔迹与印章

本文采用激光共聚焦显微镜鉴别可疑笔迹和印章及其它的文件。法医在鉴别可疑文件时通常都是凭经验和用普通光学显微镜,找出其中的一些物理特征。但还是有许多文件的笔迹顺序无法确定,尤其是墨水写的笔迹,为解决法医的这一问题,我们用激光共聚焦显微镜鉴别了72例不同铅笔和圆珠笔写的肉眼难于鉴别的交叉笔顺和其他的一些文字文件。在激光共激光共聚焦显微镜下大多数笔迹和印章都能发出荧光,因此很容易鉴别其笔顺和印章的特征,必要时还可以进行笔顺的三维图象构建,以帮助鉴别。结论：激光共聚焦显微镜可以更准确地鉴别可疑笔迹和印章。印章和笔迹的交叉也很容易分辨出来。     

抗砷细胞荧光染色后的激光共聚焦显微镜检测

目的:应用激光共聚焦显微镜检测活细胞内荧光物质含量.方法:传代培养长期低剂量砷诱导的抗砷细胞,用荧光染料Rhodamine-123对细胞染色30min,实验组与维拉帕米(Verapamil)共同孵育,对照组为单加Rhodamine-123的抗砷细胞.应用激光共聚焦显微镜采集Rhodamine-123的荧光图像动态序列,并且记录不同时间段的细胞内荧光强度.结果:实验组细胞染色12h,24h,36h,48h,60h后,荧光强度依次为(51.567±0.7572)、(46.533±0.7095)、(39.557±0.601)、(38.6±0.6245)和(38.505±0.718),明显高于同时间段对照组的荧光强度,差异均有显著性(P＜0.01).结论:应用激光共聚焦显微成像技术能进行活细胞水平荧光物质实时定量检测.     

Histological Observations of Dental Tissues Using the Confocal Laser Scanning Microscope

To investigate the time course of mineralization in undecalcified dental tissues, calcein-and tetracycline-labeled rat maxillary molar sections were stained with Villanueva bone stain en bloc, embedded in methyl-methacrylate (MMA), ground to 50 μm thickness, and observed by confocal laser scanning microscopy (CLSM). This method allowed observation of dental structures including odontoblasts, pulp cells and periodontal ligament, and dentinal tubules and enamel rods at high resolution; labeled enamel, dentine, and cementum could be observed simultaneously regardless of section thickness. CLSM permitted simultaneous observation of both the components of calcified tissue and the cellular components of dental tissues, and assessment of the mineralization time course of hard tissues labeled by tetracycline or calcein. The technique is useful for both assessing the elements composing dental structure and observing the histological dynamics by which dental structure develops.     

Histological Observations of Dental Tissues Using the Confocal Laser Scanning Microscope

To investigate the time course of mineralization in undecalcified dental tissues, calcein-and tetracycline-labeled rat maxillary molar sections were stained with Villanueva bone stain en bloc, embedded in methyl-methacrylate (MMA), ground to 50 μm thickness, and observed by confocal laser scanning microscopy (CLSM). This method allowed observation of dental structures including odontoblasts, pulp cells and periodontal ligament, and dentinal tubules and enamel rods at high resolution; labeled enamel, dentine, and cementum could be observed simultaneously regardless of section thickness. CLSM permitted simultaneous observation of both the components of calcified tissue and the cellular components of dental tissues, and assessment of the mineralization time course of hard tissues labeled by tetracycline or calcein. The technique is useful for both assessing the elements composing dental structure and observing the histological dynamics by which dental structure develops.     

激光扫描共聚焦显微术在生物医学中的研究

激光扫描共聚焦显微技术的发展,为形态学、分子细胞生物学、神经科学、肿瘤医学、药理学、遗传学等领域的研究提供了新的先进研究方法和手段。本文对激光扫描共聚焦显微术在粘附细胞分选,动态荧光分析测定,显微细胞外科和光陷阱技术,荧光光漂白恢复技术以及三维图像重建功能等方面的研究进展进行了综述。     

激光扫描共聚焦显微镜（LSCM）及其生物学应用

激光扫描共聚焦显微镜（ＬＳＣＭ）有效地排除了非焦平面信息,提高了分辨率及对比度,使图像更为精确清晰;与计算机及相应的软件技术组合,ＬＳＣＭ 实现了连续光学切片,广泛应用于生物三维结构重组及动态分析。目前,激光共聚焦显微技术已成功应用于生物芯片技术、激光显微操作系统、细胞骨架研究、生理生化及胚胎学研究、基因定位等领域。多光子技术的发展,进一步改善了ＬＳＣＭ 成像清晰度,拓宽了ＬＳＣＭ 在生物学领域中的应用。本文叙述了ＬＳＣＭ 的基本原理及其在生物学研究中的应用。     

用激光扫描共聚焦显微镜观察雪松花粉和花粉管

为更直观地观察和显示花粉和花粉管中细胞结构及其细胞核的状态与行为。雪松花粉和花粉管经卡诺液固定,分别以埃氏苏木精、曙红、Hoechst 33243单染和曙红-Hoechst 33342双染后,用冬青油整体透明,在激光扫描共聚焦显微镜下观察。4种染色法观察效果不同;以曙红-Hoechst 33342双染的样品观察效果最佳,在紫外光激发下清晰地显示出细胞核,在488 nm激光激发下不仅能清晰看到花粉和花粉管壁结构,且能分辨管细胞、柄细胞及体细胞的结构特点和空间位置关系。建立了一种快速简便的适于在激光扫描共聚焦显微镜下观察花粉和花粉管中成员细胞结构及其细胞核的状态、行为的制片技术;激光扫描共聚焦显微镜具有独特的共轭成像装置、连续光学扫描、图像三维重组和多通道检测等功能,极好地展示了雪松花粉和花粉管的结构特点,相比于传统的光学显微镜和荧光显微镜,其观察到的图像更清晰、更直观、更具立体感。     

电离辐射对血管内皮细胞骨架蛋白F-actin影响的激光共聚焦显微镜观察

目的： 观察电离辐射对血管内皮细胞骨架蛋白F-actin影响,探讨血管内皮细胞电离辐射损伤的机理.方法： 用Co60 γ射线对体外培养的血管内皮细胞进行0、2、4、6、8、10、12 Gy照射,于照射后6小时后用激光共聚焦显微镜对其细胞骨架蛋白F-actin进行观察.结果： 细胞骨架蛋白F-actin随着辐射剂量的增加而解聚增加,这种变化呈剂量依赖性.结论： 电离辐射对血管内皮细胞骨架蛋白F-actin的破坏可能是血管内皮细胞辐射损伤机体机理之一.     

利用激光扫描共聚焦显微镜的肿瘤血管在体成像研究

传统的观察血管的方法需将组织制成切片,然后通过光学显微镜进行观察。显示的只是血管的某一片段而无法观察到血管的全貌。应用激光扫描共聚焦显微镜,可对活体动物血管进行断层成像,从而再现血管的结构。本方法为对肿瘤等病变组织血管进行研究提供了一种新的检测手段。     

利用激光扫描共聚焦显微镜研究植物细胞发育形态学变化

通过激光扫描共聚焦显微镜,利用不同种类(波长)的激光研究植物细胞发育形态学变化。结果表明,利用紫外激光(351 nm)扫描可以清楚地观察到拟南芥叶片表皮细胞的形态及其变化,在已分化的叶片表皮上可观察到包括“铺垫”表皮细胞(epidermal pavement cells)、气孔保卫细胞(guard cell)、气孔伴胞(subsidiarycells)、表皮毛细胞(trichomes)和表皮毛的足细胞(socket cells)等多种形态不同的细胞种类;利用蓝光激光(488nm)辅助曙红浅染,可清晰地显示出拟南芥根生长区内部的各种原始细胞,包括静止区(quiescent center)细胞、皮层/内皮层原始细胞(cortex/endodermal initial cell)、表皮/根冠原始细胞(epidermal/root cap initial cell)和中柱/根冠原始细胞(columella/root cap initial cell)等。利用双光子激光(800 nm)连续扫描30 s可以诱发叶绿体产生自发荧光,并可观察到叶绿体在叶肉细胞中的运动轨迹。结果说明激光扫描共聚焦显微镜在植物细胞形态及发育研究上具有独特的功能。     

利用激光扫描共聚焦显微镜研究拟南芥气孔发生与发育

通过激光扫描共聚焦显微镜,利用不同种类(波长)的激光研究拟南芥叶片气孔发生与发育。结果表明,利用紫外激光(351nm)扫描可以清楚观察到拟南芥表皮各种细胞及其发生发育的形态变化,包括表皮毛细胞、副卫细胞、保卫细胞、铺垫表皮细胞等。气孔发生过程中,首先原表皮细胞不对称分裂产生拟分生组织和副卫细胞,接着分化出保卫细胞母细胞,进一步发育形成保卫细胞,最终形成气孔器。气孔分化完成后,保卫细胞在紫外激光下不产生荧光,但利用蓝光激发(488nm)辅助荧光素染色,可清晰地看到保卫细胞。结果表明,激光扫描共聚焦显微镜在拟南芥叶表皮细胞形态研究上有独特的功能。