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1.
Phylogenetic analysis of peat bog methanogen populations 总被引:5,自引:0,他引:5
Debra Nercessian Mathew Upton David Lloyd Clive Edwards 《FEMS microbiology letters》1999,173(2):425-429
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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. 相似文献
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E. A. van Spronsen V. Sarafis G. J. Brakenhoff H. T. M. van der Voort N. Nanninga 《Protoplasma》1989,148(1):8-14
Summary The newly developed confocal scanning laser microscope, together with image processing by computer, has been used to obtain three-dimensional information on the organization of grana in chloroplasts in living plant tissue. Chloroplasts are ideally suited for such studies because their pigments show bright autofluorescence. The high-resolution stereo images bridge a gap between classic light microscopy and electron microscopy. Our preliminary observations on several plant species resemble most the early observations of Strugger (1951: Die Strukturordnung im Chloroplasten. Ber Deutsch Bot Ges 64: 69–83) and suggest that the 3-D technique might well be suitable to solve discrepancies in the interpretation of classical light microscopic and electron microscopic observations.Abbreviations 3-D
three dimensional
- CSLM
confocal scanning laser microscopy
- DCMU
3-(3,4-dichlorophenyl)-1,1-dimethyl urea
- DNA
deoxyribonucleic acid 相似文献
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Paul C DeLeo Philippe Baveye William C Ghiorse 《Journal of microbiological methods》1997,30(3):193-203
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. 相似文献
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目的建立体外表皮葡萄球菌(Staphylococcus epidermidis)生物膜(biofilm,BF)模型,观察和定量分析表皮葡萄球菌BF的动态形成过程。方法采用可形成BF的表皮葡萄球菌RP62A,平板法建立体外BF模型,四唑盐(tetrazolium salt,XTT)减低法定量检测BF形成过程中细菌活力的变化,激光共聚焦显微镜(confocal laser scan-ning microscopy,CLSM)结合图像结构分析软件(image structure analyer,ISA)对BF形成过程结构参数进行动态分析,扫描电镜(scanning electron microscope,SEM)观察BF形成过程中的形态结构。结果在12、24和48 h时,XTT减低法A450的值分别为2.39±0.48、3.41±0.18和3.92±0.27,P0.05;ISA软件定量分析显示在区域孔径(AP)的值分别为0.84±0.08、0.68±0.01和0.59±0.13,P0.05,平均扩散距离(ADD)的值分别为1.34±0.24、1.49±0.09和1.89±0.39,P0.05,结构熵(TE)的值分别为4.71±0.82、8.69±0.68和8.94±0.28,24 h、48 h与12 h相比,P0.05。结论表皮葡萄球菌BF的形成是个动态的过程,24 h时BF基本形成,48 h BF结构更加复杂。XTT减低法,CLSM结合ISA软件,SEM三种方法联合使用是观察和定量分析体外BF模型较理想的方法。 相似文献
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W. H. Reijnen M. M. A. van Herpen P. F. M. de Groot A. Olmedilla J. A. M. Schrauwen K. A. P. Weterings G. J. Wullems 《Sexual plant reproduction》1991,4(4):254-257
Summary The application of confocal laser scanning microscopy together with in situ hybridization experiments in tobacco pollen enabled a detailed localization of a pollen-specific mRNA. The three-dimensional distribution of this specific mRNA over the whole pollen grain was reconstructed by means of optical sections of one specimen. 相似文献
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激光扫描共聚焦显微镜在医学研究中的应用 总被引:1,自引:0,他引:1
激光扫描共聚焦显微镜(Confocal laser scanning microscope,CLSM)具有高分辨率、高灵敏度、三维重建、动态分析等优点,使图像更为精确清晰和数字化。该仪器现已广泛应用于细胞生物学、生理学、病理学、遗传学和药理学等研究领域中。本文简述了激光扫描共聚焦显微镜的结构、工作原理并归纳了其在医学各领域研究中的应用。 相似文献
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This study explored the feasibility of using immunofluorescence labelling in conjunction with confocal laser scanning microscopy (CLSM) for detection of common fungal colonisers of unseasoned radiata pine in New Zealand. Wood sections infected with Ophiostoma piceae were treated with monoclonal antibody IF3 (1), and then Oregon green 514 goat anti-mouse IgG, a fluorescent secondary antibody. Additional wood sections infected with other Ophiostoma spp., Sphaeropsis sapinea, Leptographium procerum, Trichoderma sp. and Phlebiopsis gigantea were treated similarly to determine whether the antibody was specific to O. piceae or was recognising other fungal species. Sections were examined using phase contrast and fluorescence light microscopy prior to CLSM. Immunolabelled fungal hyphae showed relatively weak fluorescence compared to the strong autofluorescence of wood cell walls and extractives. Labelled hyphae of O. piceae were detected in wood using CLSM but not with ordinary fluorescence microscopy. This is because CLSM has stronger illumination power and superior imaging ability compared with ordinary fluorescence microscopy. The monoclonal antibody did not cross-react with the other Ophiostoma species. However, non-specific antibody binding was observed with L. procerum and Trichoderma species. Furthermore, cell walls of L. procerum showed strong autofluorescence with optical properties similar to wood extractives when examined prior to incubation with the monoclonal and secondary antibody, therefore cross-reactivity tests were inconclusive for Leptographium and Trichoderma species. The current investigation demonstrated that CLSM provides possibilities for future investigations on in situ interactions of common radiata pine fungal colonisers, with one another and with wood. 相似文献
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The transport kinetics of lanthanide species in a single erythrocyte probed by confocal laser scanning microscopy 总被引:6,自引:0,他引:6
Y. Cheng Qihua Huo Jingfen Lu Rongchang Li K. Wang 《Journal of biological inorganic chemistry》1999,4(4):447-456
A novel method has been developed to visualize and follow the temporal course of lanthanide transport across the membrane
into a single living erythrocyte. By means of confocal scanning microscopy and the optical section technique, the entry of
lanthanide ions was followed by the fluorescence quenching of fluorescein isothiocyanate (FITC)-labeled membrane and cytosol.
From the difference of the quenching kinetics of the whole section and the central area, the time for diffusion through the
membrane and the diffusion in the extracellular and intracellular media can be deduced. To clarify the mechanism of lanthanide-induced
fluorescence quenching of FITC-labeled erythrocytes and to ensure that this reaction can be used in this method, the reaction
was investigated by steady-state fluorescence techniques. The results showed that the lanthanides strongly quenched the florescence
emitted by FITC covalently bound to membrane proteins and cytosolic proteins. The static quenching mechanism is responsible
for the fluorescence quenching of FITC-labeled proteins by Ln species. The quenching mechanism is discussed on the basis of
complex formation. The dependence of fluorescence quenching on both ion size and the total orbital angular momentum L supports the complexation mechanism. The transport time across the membrane is strikingly correlated with Ln species and
extracellular concentration. For a given concentration, the transport time of [Ln(cit)2]3– is much shorter than that of Ln3+, since they enter the cells via the anion channel. This is supported by the inhibition effect of 4,4′-diisothiocyanato-2,2′-stilbenendisulfonate
on the transport of [Ln(cit)2]3–. On the other hand, the transport of free Ln3+ might be attributed to the enhanced permeability of erythrocytes owing to Ln3+ binding. These findings strongly demonstrate the existence of the non-internalization mechanism of Ln species uptake by erythrocytes.
Received: 7 January 1999 / Accepted: 7 May 1999 相似文献
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Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb-Cephalotaxus hainanensis Li, can induce apoptosis in promyelocytic leukemia HL-60 cells. With both two-photon laser scanning microscopy and confocal laser scanning microscopy in combination with the fluorescent probe Hoechst 33342, tetramethyrhodamine ethyl ester (TMRE) and Fluo 3-AM, we simultaneously observed HT-induced changes in nuclear morphology, mitochondrial membrane potential and intracellular calcium concentration ([Ca2+]i) in HL-60 cells, and developed a real-time, sensitive and invasive method for simultaneous multi-parameter observation of drug- treating living cells at the level of single cell. 相似文献
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Jones CW Smolinski D Keogh A Kirk TB Zheng MH 《Progress in histochemistry and cytochemistry》2005,40(1):1-71
Confocal laser scanning microscopy (CLSM) is a type of high-resolution fluorescence microscopy that overcomes the limitations of conventional widefield microscopy and facilitates the generation of high-resolution 3D images from relatively thick sections of tissue. As a comparatively non-destructive imaging technique, CLSM facilitates the in situ characterization of tissue microstructure. Images generated by CLSM have been utilized for the study of articular cartilage, bone, muscle, tendon, ligament and menisci by the foremost research groups in the field of orthopaedics including those teams headed by Bush, Errington, Guilak, Hall, Hunziker, Knight, Mow, Poole, Ratcliffe and White. Recent evolutions in techniques and technologies have facilitated a relatively widespread adoption of this imaging modality, with increased "user friendliness" and flexibility. Applications of CLSM also exist in the rapidly advancing field of orthopaedic implants and in the investigation of joint lubrication. 相似文献
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Visualization of alginate-poly-L-lysine-alginate microcapsules by confocal laser scanning microscopy 总被引:2,自引:0,他引:2
Confocal laser scanning microscopy (CLSM) was used to study the distribution of polymers and cross-linking ions in alginate-poly-L-lysine (PLL) -alginate microcapsules made by fluorescent-labeled polymers. CLSM studies of Ca-alginate gel beads made in the presence and absence of non-gelling sodium ions revealed a more inhomogeneous distribution of alginate in beads formed in the absence of non-gelling ions. In the formation of alginate-PLL capsules, the polymer gradients in the preformed gel core were destabilized by the presence of non-gelling ions in the washing step and in the PLL solution. Ca-alginate gels preserved the inhomogeneous structure by exposure to ion-free solution in contrast to exposure to non-gelling ions (Na(+)). By exchanging Ca(2+) with Ba(2+) (10 mM), extremely inhomogeneous gel beads were formed that preserved their structure during the washing and exposure to PLL in saline. PLL was shown to bind at the very surface of the alginate core, forming a shell-like membrane. The thickness of the PLL-layer increased about 100% after 2 weeks of storage, but no further increase was seen after 2 years of storage. The coating alginate was shown to overlap the PLL layer. No difference in binding could be observed among coating alginates of different composition. This paper shows an easy and novel method to study the distribution of alginate and PLL in intact microcapsules. As the labeling procedures are easy to perform, the method can also be used for a variety of other polymers in other microencapsulation systems. 相似文献
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目的 探讨使用激光共聚焦扫描显微镜 (Laser scanning confocal microscope,LSCM)观察大鼠纹状体内谷氨酸能突触连接的方法的可行性.方法 12只正常大鼠分为两组,6只大鼠进行纹状体中等棘刺神经元的CM-DiI 单细胞标记,然后Ⅰ型囊泡膜谷氨酸转运体(vesicular glutamate transporter 1,VGluT1 )免疫荧光标记,LSCM层扫后三维重建,观察VGluT1阳性位点在中等棘刺神经元树突上的分布.另外6只大鼠用TEM观察不对称性突触在纹状体神经元树突上的分布.对两种方法的结果进行比较.结果 用LSCM 和TEM方法观察到的纹状体神经元上谷氨酸能突触连接分布情况一致,没有统计学差异.但LSCM更具优越性的是,可以对图像进行三维重构,从而有利于对神经元之间突触连接的空间分布观察和定量分析.结论 神经细胞荧光标记技术结合LSCM观察是考察纹状体神经元上谷氨酸能突触连接的有效方法. 相似文献
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In recent years, confocal laser scanning microscopy has been developed into a non-invasive tool to probe intra-particle profiles
of protein in chromatographic adsorbents. A necessary prerequisite when using this technique lies in the labeling of proteins
with fluorescent probes. The quality of the obtained results is thus strongly dependent on the probes used, its sensitivity
on experimental parameters and the change of protein characteristics upon binding. In this review, the fundamental issues
when using fluorescent probes are described, before giving a critical evaluation on published literature in the field of confocal
laser scanning microscopy for the analysis of chromatographic principles. 相似文献
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The monogonont rotifer Bryceella stylata was investigated with light, electron and confocal laser scanning (CLSM) microscopy to provide detailed insights into its anatomy and new information for future phylogenetic analyses of the group. Results from CLSM and phalloidin staining revealed a total of six paired longitudinal muscles (musculi longitudinales I-VI) and eight circular muscles (musculi circulares I-VIII) as well a complex network of mostly fine visceral muscles. In comparison with other rotifer species that have been investigated so far, B. stylata shares the presence of the circular and longitudinal muscles: musculus longitudinalis ventralis, musculus longitudinalis lateralis inferior, musculus longitudinalis dorsalis, musculus longitudinalis capitis and musculus circumpedalis. However, the species lacks lateral and dorsolateral longitudinal muscles and some circular muscles (e.g., corona sphincter, musculus pars coronalis). With light and electron microscopy, we were able to document the precise number of pseudosegments and the arrangement of the chambers comprising the trophi elements. Furthermore, our observations revealed several new morphological characteristics, including a shield-like epidermal projection covering the dorsal antenna, an epidermal projection restricting the corona caudally and an unpaired hypopharynx with distinct shovel-like structures. 相似文献
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Apoplastic pH in corn root gravitropism: a laser scanning confocal microscopy measurement 总被引:1,自引:0,他引:1
The ability to measure the pH of the apoplast in situ is of special interest as a test of the cell wall acidification theory. Optical sectioning of living seedlings of corn roots using the laser scanning confocal microscope (LSCM) permits us to make pH measurements in living tissue. The pH of the apoplast of corn roots was measured by this method after infiltration with CI-NERF, a pH-sensitive dye, along with Texas Red Dextran 3000, a pH-insensitive dye, as an internal standard. In the elongation zone of corn roots, the mean apoplastic pH was 4.9. Upon gravitropic stimulation, the pH on the convex side of actively bending roots was 4.5. The lowering of the apoplastic pH by 0.4 units appears to be sufficient to account for the increased growth on that side. This technique provides site-specific evidence for the acid growth theory of cell elongation. The LSCM permits measurements of the pH of living tissues, and has a sensitivity of approximately 0.2 pH units. 相似文献