体内生物膜研究进展

生物膜是指由黏附于生命体或非生命体表面的微生物和其胞外聚合物所构成的复杂、多维的空间结构,在自然界中普遍存在。微生物在人类体内许多部位均可形成生物膜结构,如肺、心脏瓣膜、泌尿生殖道、肝胆系统、耳、鼻、皮肤等。越来越多的感染性疾病患者或动物模型体内均可检测出生物膜,如囊性纤维化肺炎、慢性中耳炎、细菌性心内膜炎、皮肤外伤后慢性感染、慢性鼻窦炎等。生物膜的形成与感染性疾病的关系及其在疾病的发生、发展、转归过程中的作用成为近年来研究的热点。目前体内生物膜检测技术主要有扫描电镜、激光共聚焦显微镜、荧光原位杂交技术等。就临床常见感染性疾病与生物膜的关系及体内生物膜检测方法展开综述。     

激光扫描共聚焦显微镜观察细菌生物膜形成的方法学研究

目的:建立激光扫描共聚焦显微镜观察生物膜形成过程的方法,为进一步研究生物膜的形成机制奠定基础。方法:以临床分离金葡菌X428为研究对象,在盖玻片上形成生物膜,分别于接种后的4、8、12、16、24和48h取出玻片,采用免疫荧光技术标记多糖和细菌,激光扫描共聚焦显微镜(CLSM)观察生物膜形成情况。结果:取得了生物膜形成过程的不同时间点的CLSM图像,4h时细菌在盖玻片上粘附形成小菌落,8h和12h细菌聚集成簇,多糖基质产生并逐渐增多,至16h形成成熟生物膜结构;24h和48h生物膜已经播散,其结构变小。结论:应用免疫荧光技术和激光扫描共聚焦显微镜技术研究生物膜形成过程是一种简便可行的方法。     

利用转盘共聚焦显微镜进行快速实验的新方法

转盘共聚焦显微镜是快速激光共聚焦显微镜的一种,与传统的激光共聚焦显微镜相比具有一些相同点,也有其特有的优势。本文主要介绍转盘共聚焦显微镜的基本原理及如何利用转盘共聚焦显微镜进行快速实验及应用实例,并与传统激光共聚焦显微镜进行比较。转盘共聚焦显微镜具有速度快、灵敏度高、对样品光损伤和光淬灭程度低、操作灵活简单,是随着实验技术发展使用越来越广泛的实验仪器。     

心肌细胞钙瞬变和细胞收缩的激光共聚焦成像研究

目的：游离钙离子参与机体的多种重要生理功能。本研究着重探讨如何利用激光共聚焦显微镜线扫描成像技术同时记录正常情况下心肌细胞的钙瞬变以及由此引起的细胞收缩过程。方法与结果：本研究以分离的心室肌细胞为对象,通过局部场刺激诱发细胞的钙瞬变和收缩,同时配合使用激光共聚焦显微镜成像系统,以线扫描方式记录实验结果。结果表明,钙瞬变先于细胞收缩发生(约早31ms),而收缩最大处远落后于钙瞬变峰值发生处(约慢346ms)。结论：激光共聚焦显微镜线扫描成像技术具有较好的时问分辨率和空间分辨率,其实验结果直观、明确、可靠,是较理想的研究钙瞬变和细胞收缩的光学记录方法。     

丹皮酚对变形链球菌生物膜影响的体外研究

目的测定丹皮酚对变形链球菌的抑菌作用;共聚焦显微镜观察丹皮酚对变形链球菌生物膜结构和活性的影响。方法梯度法测定丹皮酚对变形链球菌的MIC(最小抑菌浓度)、MBC(最小杀菌浓度);体外构建变形链球菌生物膜模型,共聚焦显微镜观察不同浓度丹皮酚对变形链球菌生物膜作用后形态结构的影响并进行红绿荧光定量分析其活性变化。结果丹皮酚对变形链球菌的MIC为6.25mg/mL,MBC为25mg/mL;激光共聚焦显微镜观察丹皮酚对变形链球菌生物膜作用后其生物膜结构变稀疏,细菌链变短,生物膜活性也随丹皮酚浓度的提高而逐渐降低。结论丹皮酚对变形链球菌和变形链球菌生物膜结构及其活性均具抑制作用。     

用气溶胶法和摇床法建立铜绿假单胞菌生物膜体外模型

目的模拟体内环境,体外建立细菌生物膜模型,为进一步深入研究细菌生物膜生物学特点提供基础。方法将粘附载体置于气溶胶法和摇床法模拟体内细菌生物膜形成的微环境中,将铜绿假单胞菌株培养3d后,取出标本分别进行通过FITC—ConA染色及SYT09／PI染色,然后分别进行荧光显微镜检测及激光共聚焦检测,观察细菌生物膜的形成情况;进行电子显微镜扫描观察形成的细菌生物膜的形态特点。结果在气溶胶的微环境下,FITC—ConA染色后在荧光显微镜观察到明亮成片状的细菌生物膜;SYT09／PI染色后在激光共聚焦检测,观察到片状,层叠如积云状,棉絮样的细菌生物膜;在电子显微镜扫描观察到大量细菌成团聚集,团状丛生突出表面,具有立体结构的细菌生物膜。在摇床法的微环境下,用3种检测方法都观察到成流线状的细菌生物膜。结论运用气溶胶法、摇床法可成功建立分别模拟体内呼吸系统及循环、泌尿系统的微环境下生物膜形成模型。     

厚朴酚对变形链球菌生物膜致龋毒力因子作用的研究

目的 通过激光共聚焦显微镜观察厚朴酚对变形链球菌生物膜的抑菌效果,并初步了解厚朴酚对变形链球菌生物膜的产酸、耐酸、胞外多糖形成及生物膜形成能力等相关致龋毒力因子的转录表达的影响,为进一步研究厚朴酚防龋的药理作用机制奠定基础.方法 建立变形链球菌生物膜体外模型,激光共聚焦显微镜观察不同药物浓度作用后效果,并进行红绿荧光定量分析;根据GenBank基因库查询ffh、gtfD、pdp等基因序列并设计引物,进行RT-PCR.结果 CLSM观察厚朴酚作用变形链球菌生物膜后可使膜内活菌比例明显下降;RT-PCR结果表明毒力因子ffh、gtfD、pdp的表达水平受到抑制.结论 厚朴酚对变形链球菌生物膜的致龋毒力因子ffh、gtfD、pdp的转录表达有明显的抑制作用.     

FISH技术在废水处理中的应用

该文介绍了FISH（Fluorescence in situ hybridization）技术与DGGE／TGGE、PER扩增技术、共聚焦激光扫描显微镜以及生物传感器等结合使用,可直观形象的研究微生物在废水处理系统中的形态、生理变化,以及微生物种群的演替的规律;为筛选和驯化活性污泥或生物膜中的对废水处理起主导作用的微生物,提供更加有效、快速、可视的信息,促进废水处理技术的发展。     

三种显微技术对人毛囊蠕形螨的观察和研究

目的 采用荧光显微镜、扫描电镜和激光共聚焦显微镜对人体毛囊形蠕形螨进行形态学观察.方法 选用5 μg/mL碘化丙啶(propidine iodide,PI)对虫体进行荧光染色,避光染色15 min,分别置于荧光显微镜和激光共聚焦显微镜下观察;2.5%戊二醛固定虫体标本,梯度酒精和叔丁醇脱水,金喷镀后扫描电镜观察.结果 荧光显微镜下碘化丙啶对虫体有很强的结合力,虫体荧光信号均匀展示于细胞表面,充分展现虫体形态,扫描电镜更加清楚、细致地展示人体毛囊型蠕形螨的超微结构,激光共聚焦显微镜将虫体分层扫描图片进行三维重建,真实、完全、直观地展露了虫体.结论 三种显微技术均可展示蠕形螨超微形态.PI对虫体有很好的荧光染色作用,使激光扫描共聚焦显微镜能够获得更加精准的超微形态结构,结合三维重建技术有着广泛的应用价值.     

利用激光共聚焦扫描显微镜对溃疡病菌侵染柑橘叶片的实时观察

柑橘溃疡病对柑橘产业造成了巨大损失,而研究柑橘与溃疡病菌的互作关系以及柑橘的感病和抗病性均需要观察溃疡病菌在柑橘寄主中的侵染和定殖过程。激光共聚焦扫描显微镜不仅可以观察活细胞,活组织的动态代谢过程,而且可以获得三维图像,对于病原菌在柑橘植物组织内的繁殖和致病机制研究具有重要意义。但是,选择适宜的植物材料和制片方法对激光共聚焦扫描显微镜的观察效果影响很大。本文对激光共聚焦扫描显微镜所观察的材料在其处理和观察方法上加以改进,获得了质量更好的图片和实验结果,也使得实验更为方便快捷。激光共聚焦扫描显微观察还在瞬时表达分析中得到应用,提高了柑橘瞬时表达分析的效果。通过将切片和压片相结合观察到溃疡病菌在不同时间点对柑橘叶片的侵染情况,而通过3D建模能观察到柑橘叶片不同组织层面中的病菌数量和病菌位置,为研究溃疡病菌在叶片中的定殖方式和入侵数量提供了前期基础。     

Structural analysis of microparticles by confocal laser scanning microscopy

This study demonstrates the potential of conforcal laser scanning microscopy (CLSM) as a characterization tool for different types of microparticles. Microparticles were prepared by various methods including complex coacervation, spray drying, double emulsion solvent evaporation technique, and ionotropic gelation. Protein drugs and particle wall polymers were covalently labeled with a fluorescent marker prior to particle preparation, while low molecular weight drugs were labeled by mixing with a fluorescent marker of similar solubility properties. As was demonstrated in several examples, CLSM allowed visualization of the polymeric particle wall composition and detection of heterogeneous polymer distribution or changes in polymer matrix composition under the influence of the drug. Furthermore, CLSM provides a method for three-dimensional reconstruction and image analysis of the microparticles by imaging several coplanar sections throughout the object. In conclusion, CLSM allows the inspection of internal particle structures without prior sample destruction. It can be used to localize the encapsulated compounds and to detect special structural details of the particle wall composition.     

Use of confocal laser scanning microscopy on soil thin-sections for improved characterization of microbial growth in unconsolidated soils and aquifer materials

Confocal laser scanning microscopy (CLSM) was utilized to examine samples from an aquifer microcosm that was used to investigate microbially mediated losses in hydraulic conductivity. Samples were fixed, dehydrated and dried to prepare the biological material in a fashion similar to that used previously for viewing under the scanning electron microscope. Then, samples were prepared as thin-sections by employing soil micromorphological techniques. Serial images generated by the CLSM technique were visualized using computer three-dimensional rendering software. Results from the CLSM technique were compared with simple fluorescence microscopy of thin-sections and scanning electron microscopy (SEM) of samples from the microcosm. Computer visualization of serial sections with the CLSM technique provided images on a submicron scale in three dimensions. SEM has a much higher resolution, on a nanometer scale, but the results are not three dimensional. Artifacts associated with thin-section preparation are minimal for natural porous media composed mostly of sand, such as aquifer materials. Also, CLSM images are affected minimally by changes to biological material due to sample preparation, whereas artifacts associated with SEM images are very prominent, due to the higher magnification and resolution. CLSM of thin-sections and SEM are very powerful methods for viewing microbial growth in natural porous media, but CLSM is preferable because it allows three-dimensional visualization and measurements of cells and aggregates with few artifacts.     

Software controlling algorithms for the system performance optimization of confocal laser scanning microscope

The relative slow scanning speed of a galvanometer commonly used in a confocal laser scanning microscopy system can dramatically limit the system performance in scanning speed and image quality, if the data collection is simply synchronized with the galvanometric scanning. Several algorithms for the optimization of the galvanometric CLSM system performance are discussed in this work, with various hardware controlling techniques for the image distortion correction such as pixel delay and interlace line switching; increasing signal-to-noise ratio with data binning; or enhancing the imaging speed with region of interest imaging. Moreover, the pixel number can be effectively increased with Acquire-On-Fly scan, which can be used for the imaging of a large field-of-view with a high resolution.     

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。     

Evaluation of confocal microscopy system performance

BACKGROUND: The confocal laser scanning microscope (CLSM) has been used by scientists to visualize three-dimensional (3D) biological samples. Although this system involves lasers, electronics, optics, and microscopes, there are few published tests that can be used to assess the performance of this equipment. Usually the CLSM is assessed by subjectively evaluating a biological/histological test slide for image quality. Although there is a use for the test slide, there are many other components in the CLSM that need to be assessed. It would be useful if tests existed that produced reference values for machine performance. The aim of this research was to develop quality assurance tests to ensure that the CLSM was stable while delivering reproducible intensity measurements with excellent image quality. METHODS: Our ultimate research objective was to quantify fluorescence using a CLSM. To achieve this goal, it is essential that the CLSM be stable while delivering known parameters of performance. Using Leica TCS-SP1 and TCS-4D systems, a number of tests have been devised to evaluate equipment performance. Tests measuring dichroic reflectivity, field illumination, lens performance, laser power output, spectral registration, axial resolution, laser stability, photomultiplier tube (PMT) reliability, and system noise were either incorporated from the literature or derived in our laboratory to measure performance. These tests are also applicable to other manufacturer's systems with minor modifications. RESULTS: A preliminary report from our laboratory has addressed a number of the QA issues necessary to achieve CLSM performance. This report extends our initial work on the evaluation of CLSM system performance. Tests that were described previously have been modified and new tests involved in laser stability and sensitivity are described. The QA tests on the CLSM measured laser power, PMT function, dichroic reflection, spectral registration, axial registration, system noise and sensitivity, lens performance, and laser stability. Laser power stability varied between 3% and 30% due to various factors, which may include incompatibility of the fiber-optic polarization with laser polarization, thermal instability of the acoustical optical transmission filter (AOTF), and laser noise. The sensitivity of the system was measured using a 10-microm Spherotech bead and the PMTs were assessed with the CV concept (image noise). The maximum sensitivity obtainable on our TCS-SP1 system measured on the 10-microm Spherotech beads was approximately 4% for 488 nm, 2.5% for 568 nm, 20% for 647 nm, and 19% for 365 nm laser light. The values serve as a comparison to test machine sensitivity from the same or different manufacturers. CONCLUSIONS: QA tests are described on the CLSM to assess performance and ensure that reproducing data are obtained. It is suggested strongly that these tests be used in place of a biological/histological sample to evaluate system performance. The tests are more specific and can recognize instrument functionality and problems better than a biological/histological sample. Utilization of this testing approach will eliminate the subjective assessment of the CLSM and may allow the data from different machines to be compared. These tests are essential if one is interested in making intensity measurements on experimental samples as well as obtaining the best signal detection and image resolution from a CLSM. Published 2001 Wiley-Liss, Inc.     

Quantification of protein A-gold staining for peroxisomal enzymes by confocal laser scanning microscopy.

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific beta-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.(J Histochem Cytochem 47:1343-1349, 1999)     

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。     

Novel methodology utilizing confocal laser scanning microscopy for systematic analysis in arthropods (Insecta)

The use of confocal laser scanning microscopy (CLSM) for imagingarthropod structures has the potential to profoundly impactthe systematics of this group. Three-dimensional visualizationof CLSM data provides high-fidelity, detailed images of minusculestructures unobtainable by traditional methods (for example,hand illustration, bright-field light microscopy, scanning electronmicroscopy). A CLSM data set consists of a stack of 2-D images("optical slices") collected from a transparent, fluorescentspecimen of suitable thickness. Small arthropod structures areparticularly well suited for CLSM imaging owing to the autofluorescentnature of their tissues. Here, we document the practical aspectsof a methodology developed for obtaining image stacks via CLSMfrom autofluorescent insect cuticular structures.     

Tissue localization of phenolic compounds in plants by confocal laser scanning microscopy

Phenolic compounds are involved in many interactions of plants with their biotic and abiotic environment. These substances accumulate in different plant tissues and cells during ontogenesis and under the influence of various environmental stimuli, respectively. Studies on the tissue localization of phenolic compounds provide a fundamental prerequisite for understanding the ecological functions of these compounds. The present work shows the localization of various phenolics in cell walls, vacuoles, and associated with cell nuclei, in leaves of a monocotyledonous and a dicotyledonous plant, in a gymnosperm as well as in rhizomes of a horsetail by confocal laser scanning microscopy (CLSM). Using fresh plant material, it compares in detail the tissue localization of autofluorescent styrylpyrones and hydroxycinnamic acids and the visualization of epidermal flavonoid compounds using shift reagents like ammonia, and fluorescence-inducing reagents like Naturstoffreagenz A (diphenyl-boric acid 2-aminoethyl ester). The comparison of microscopic data obtained from different plant species shows the advantages and limitations of confocal laser scanning microscopy in ecological biochemistry of phenolic plant metabolites.