Membrane Potential Changes Visualized in Complete Growth Media through Confocal Laser Scanning Microscopy of bis-Oxonol-Loaded Cells

Confocal laser scanning microscopy (CLSM) was employed to visualize and measure membrane potential changes in several types of cultured adherent cells, such as human fibroblasts, mouse mammary tumor C127 cells, and human saphenous vein endothelial cells, preloaded with the anionic dye bis-1,3,-diethylthiobarbituratetrimethineoxonol (bis-oxonol). The fluorescence of cell-associated bis-oxonol was detected in a single confocal plane. An original flow-chamber apparatus was employed to replace the extracellular medium, avoiding alterations of the plane selected for observation. In all the cell types and the experimental situations tested the intracellular distribution of the dye was typical; perinuclear zones accumulated the dye which, conversely, was excluded by the nucleus. Fluorescence was calibrated versus the membrane potential by varying the extracellular concentration of sodium in the presence of gramicidin. With this approach membrane potential was measured (i) in cultured human fibroblasts incubated under anisotonic conditions, (ii) in heterogeneous cell populations which respond unevenly to potential perturbing conditions, and (iii) in human macrovascular endothelial cells maintained in high-serum, complete growth medium. The results obtained indicate that CLSM can be successfully employed to measure changes of membrane potential in single, bis-oxonol-loaded adherent cells under experimental conditions which severely hinder conventional spectrofluorimetric approaches.     

双光子及共聚焦激光扫描显微术研究天花粉蛋白对人绒癌细胞的作用机制

天花粉蛋白(trichosanthin, TCS)是从中草药栝楼根中提取的一种核糖体失活蛋白,具有抗肿瘤和抗HIV功能.应用双光子及共聚焦激光扫描显微术结合特异性荧光探针Hoechst 33342、2′,7′-二氯荧光黄双乙酸酯 (DCFH-DA)、Indo-1和Fluo 3-AM,首次同时观察了TCS诱导人绒癌细胞（JAR细胞）凋亡过程中活性氧自由基（ROS）和细胞内钙离子浓度（［Ca²⁺］_i）的变化,实验结果表明TCS引起的［Ca²⁺］_i升高和ROS形成参与了TCS诱导的JAR细胞凋亡,并且ROS形成和［Ca²⁺］_i升高有关.共聚焦激光扫描显微术的研究结果表明,［Ca²⁺］_i升高不是导致ROS形成的主要原因,TCS诱导产生的ROS可能是通过TCS与JAR细胞膜表面受体作用介导的.     

活细胞钙动态的共聚焦扫描显微镜检测技术

共聚焦激光扫描显微镜（Confocal Laser Scarming Microscope,CLSM）广泛应用于活细胞内钙敏感探针标记的钙水平的动态测量。较之传统的显微镜CLSM在钙成像分析上有着不可比拟的优越性,但也存在一些缺陷,近些年陆续出现了一些针对这些缺陷的改善措施,如比率法、葡聚糖探针及其他一些新技术与共聚焦显微镜的联合应用等,并且出现了诸如双光子显微镜等新型激光共聚焦显微镜。随着共聚焦钙成像技术的不断发展进步,其今后的应用前景将会越越广阔。     

激光扫描共聚焦显微镜对人类染色体三维结构的观察

为了获得染色体内部结构的多种信息,以及对染色体形态构建提供有益的尝试,本试验利用荧光染料的特异性标记及激光扫描共聚焦显微镜的连续断层扫描和三维重建的特点,对人类染色体的形态结构进行观测,结果显示：本方法不仅能显示染色体的荧光带纹的分布状况,而且能作染色体内部的一系列光学切片和染色体三维结构的观测。     

激光扫描共聚焦显微镜术中活细胞标本的制备

激光扫描共聚焦显微镜是以单个的、活性的、贴壁的细胞标本为主要的研究对象。为了获得适合共聚焦显微镜分析的组织细胞标本,本文讨论了标本制备存在的一些问题并提出了改进的方法。结果显示：组织细胞外环境中盐溶液、ｐＨ值、温度、氧气等均为影响细胞活性的重要因素;而且细胞的贴壁效果也是观测分析的关键条件之一。本文对激光扫描共聚焦显微镜术中的组织细胞学方法进行了探讨,并为此提供一些有实效的实验方法。     

基于激光共聚焦扫描显微技术分析激光对扁藻的影响

本文以亚心形扁藻为样品,波长1 341 nm的Nd∶YAP激光为光源,通过激光共聚焦扫描显微技术,研究Nd∶YAP激光辐照亚心形扁藻对亚心形扁藻叶绿体自体荧光强度和叶绿体面积大小的影响。Nd∶YAP激光辐照后的亚心形扁藻通过488 nm Ar^+激光激发获得亚心形扁藻自体荧光图像及其荧光光谱。结果表明,试验中除(10 W,60 s)辐照剂量组外,其余辐照剂量组均提高了亚心形扁藻的自体荧光强度,且所有的辐照剂量组均增大了亚心形扁藻的叶绿体面积。Nd∶YAP激光可刺激亚心形扁藻的叶绿体发育,促进藻细胞的生长,改善叶绿体光合作用的活性。     

Scanning Electron Microscopy of Microbial Cells on Membrane Filters

Scanning electron micrographs of a Pseudomonas species, Staphylococcus aureus, and Bacillus subtilis on two membrane filtration systems are compared.     

激光扫描共聚焦显微镜荧光探针的选择和应用

激光扫描共聚焦显微镜是检测生物荧光信号的最新技术手段。不仅广泛用于荧光定性、定量测量,还可用于活细胞动态荧光监测、组织细胞断层扫描、三维图象重建、共聚焦图象分析、荧光光漂白恢复、激光显微切割手术等。本文拟就激光扫描共聚焦显微镜常用的检测内容及其相关荧光探针的选择和应用做一简单的介绍。     

A morphological analysis of growth cones of DRG neurons combining Atomic Force and Confocal Microscopy

We have analyzed the morphology of growth cones of differentiating neurons from rat dorsal root ganglia (DRG) with conventional Laser Scanning Confocal Microscopy (LSCM) and Atomic Force Microscopy (AFM). Images of immunofluorescent DRG growth cones colabeled for actin and tubulin were superimposed to images obtained with AFM at different scanning forces. In order to reduce changes of the image surface caused by the pressure of the AFM tip, we have developed a procedure to obtain 0 pN AFM images. Further analysis of these images revealed topographical structures with nanoscale dimensions, referred to as “invaginations” or “holes”. These holes had an area varying from 0.01 to 3.5 μm² with a depth varying from 2 to 178 nm. Comparative analysis with LSCM images showed that these holes correspond to regions where staining of both actin and tubulin was negligible. Filopodia height varied from 40 to 270 nm and their diameter from 113 to 887 nm. These results show that the combination of LSCM and AFM reveal structural details with a nanoscale dimension of DRG growth cones, difficult to resolve with conventional microscopy.     

激光对DNA作用机理的AFM研究

激光作用质粒DNA和小牛胸腺DNA产生损伤效应,导致DNA结构变化,利用一种改进的试样制备过程和纳米显微镜－－原子力显微镜（AFM）能够获得可重现的激光作用质粒DNA和小牛胸腺DNA的AFM图像,显示它们的特殊的表达结构,讨论了激光辐照导致DNA链断裂的作用机理。     

共聚焦激光扫描显微镜在发育生物学中的应用

共聚焦激光扫描显微镜以高空间分辨率、非介入无损伤性连续光学切片、实时动态观察等优越性,应用于生物医学众多领域中。本文主要论述共聚焦激光扫描显微镜在发育生物学中的应用。     

Contact-Mode High-Resolution High-Speed Atomic Force Microscopy Movies of the Purple Membrane

High-speed atomic force microscopy (HS-AFM) is becoming a reference tool for the study of dynamic biological processes. The spatial and time resolutions of HS-AFM are on the order of nanometers and milliseconds, respectively, and allow structural and functional characterization of biological processes at the single-molecule level. In this work we present contact-mode HS-AFM movies of purple membranes containing two-dimensional arrays of bacteriorhodopsin (bR). In high-resolution movies acquired at a 100 ms frame acquisition time, the substructure on individual bR trimers was visualized. In regions in between different bR arrays, dynamic topographies were observed and interpreted as motion of the bR trimers. Similarly, motion of bR monomers in the vicinity of lattice defects in the purple membrane was observed. Our findings indicate that the bR arrays are in a mobile association-dissociation equilibrium. HS-AFM on membranes provides novel perspectives for analyzing the membrane diffusion processes of nonlabeled molecules.     

中国对虾卵子活化及卵裂的激光扫描共聚焦显微镜研究(英文)

有报导,与其它动物不同,对虾的卵子一接触海水、不经过受精,就可以被活化。但是,只有受精卵才能卵裂发育。而且,各种对虾卵子内皮质棒及皮层颗粒的形态结构、活化过程各有不同。本文就中国对虾卵子的活化及卵裂过程进行了激光扫描共聚焦显微镜（ＬＳＣＭ）研究。该技术可以清晰地显示细胞的立体图像。采集中国对虾,人工诱导产卵,未受精卵取自产卵虾体内,受精卵分期分批取样,固定。供试样品经ＰＢＳ漂洗,ＡＯ、ＦＩＴＣ和ＰＩ染色,ＬＳＣＭ观察、图像处理。刚产出的、未活化卵子形态不规则,皮层内富含皮质棒和皮层颗粒（Ｆｉｇ１）。不论受精与否,产入海水的卵子都能被激活并恢复减数分裂。活化后的卵子释放出皮层囊泡内的皮质棒（Ｆｉｇｓ２＆３）。释放出的皮质棒转变为均质的胶质层（Ｆｉｇｓ４＆５）。随后,皮层颗粒释放并在卵子表面转化为均质化的孵化膜。卵子开始举起,与孵化膜之间形成卵周隙。卵周隙内具有絮状物质（Ｆｉｇｓ６,７＆８）。卵子产出后笫８～１０ｍｉｎ,经过减数分裂,第一极体排放,并随着孵化膜的举起而被推向卵外（Ｆｉｇ５）。笫２０ｍｉｎ,第二极体排放,并由卵子表面到达孵化膜的内缘（Ｆｉｇ６）。在２０℃水温条件下,受精卵产入海水中６０     

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.     

Atomic Force Microscopy in Imaging of Viruses and Virus-Infected Cells

Summary: Atomic force microscopy (AFM) can visualize almost everything pertinent to structural virology and at resolutions that approach those for electron microscopy (EM). Membranes have been identified, RNA and DNA have been visualized, and large protein assemblies have been resolved into component substructures. Capsids of icosahedral viruses and the icosahedral capsids of enveloped viruses have been seen at high resolution, in some cases sufficiently high to deduce the arrangement of proteins in the capsomeres as well as the triangulation number (T). Viruses have been recorded budding from infected cells and suffering the consequences of a variety of stresses. Mutant viruses have been examined and phenotypes described. Unusual structural features have appeared, and the unexpectedly great amount of structural nonconformity within populations of particles has been documented. Samples may be imaged in air or in fluids (including culture medium or buffer), in situ on cell surfaces, or after histological procedures. AFM is nonintrusive and nondestructive, and it can be applied to soft biological samples, particularly when the tapping mode is employed. In principle, only a single cell or virion need be imaged to learn of its structure, though normally images of as many as is practical are collected. While lateral resolution, limited by the width of the cantilever tip, is a few nanometers, height resolution is exceptional, at approximately 0.5 nm. AFM produces three-dimensional, topological images that accurately depict the surface features of the virus or cell under study. The images resemble common light photographic images and require little interpretation. The structures of viruses observed by AFM are consistent with models derived by X-ray crystallography and cryo-EM.     

应用激光扫描共聚焦显微镜研究卵母细胞和早期胚胎的细胞骨架

介绍了应用激光扫描共聚焦显微镜, 结合免疫荧光技术研究卵母细胞和早期胚胎细胞骨架的方法.观察了β-微管蛋白、γ-微管蛋白、微丝以及染色体在小鼠卵母细胞和早期胚胎中的分布和形态, 讨论了实验过程中的注意事项以及实验结果的分析和处理方法.     

激光扫描共聚焦显微镜在蛋白质晶体生长研究中的应用

激光扫描共聚焦显微镜与普通光学显微镜相比,其分辨率高,同时具有可对样品进行非侵入性无损伤断层扫描,以及对样品形貌进行三维成建等特点,因此,可作为研究晶体生长强有利的工具。本文介绍了其在定量测量晶体的个数,重组三维图像以获得晶体生长的过程信息及测定晶体生长台阶动态变化等方面的应用。还对激光扫描共聚焦显微镜在晶体生长研究的其它方面应用前景作了展望。     

Investigation of Adhesion and Mechanical Properties of Human Glioma Cells by Single Cell Force Spectroscopy and Atomic Force Microscopy

Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma -HG- and Gasc for low-grade glioma -LG-) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.     

利用激光扫描共聚焦显微术对同源四倍体水稻胚囊形成与发育的进一步观察

应用改进的整体染色透明激光扫描共聚焦显微术(WCLSM),对同源四倍体水稻PDER-2B-4x胚囊的形成与发育过程进行观察。发现其胚囊的形成发育过程与二倍体的一致,可以清楚地划分为8个发育时期,即孢原细胞形成期、大孢子母细胞形成期、大孢子母细胞减数分裂期、功能大孢子形成期、单核胚囊形成期、胚囊有丝分裂期、八核胚囊发育期和成熟胚囊期。除正常发育的过程外,大孢子发育的各个过程均出现一些异常现象,包括:细胞退化、核位置异常、核数目异常和细胞分化异常等。这些异常可能最终导致多种结构异常成熟胚囊的形成。