Assessing Confocal Microscopy Systems for Purchase

Confocal laser scanning microscopy now has so many functions and applications that choosing a new multiuser confocal laser microscopy system can be a daunting task, particularly for a first-time buyer and new users of confocal microscopy. How does one determine which features are most appropriate for any particular laboratory, application, or imaging environment? Each confocal microscopy system has its own individual advantages and limitations, which ultimately defines its market niche. Here, we describe the features that differentiate the four confocal microscopy systems we assessed. The decisive factors in choosing a confocal microscopy system for our anatomical laboratory were user-friendly software for on-line acquisition and off-line processing; the working distances of objective lenses; applicability for a multiuser environment; interactions with company representatives; and turnaround times for questions, service, and accessories.     

生物科学研究中的激光多光子显微技术

本文综述了生物科学中近红外（NIR）多光子激光扫描显微技术的进展,其中包括：多光子显微特点,激光光源,荧光寿命的测量,多光子多色实时杂化荧光（FISH）,非人侵性活体光学解剖,植物学中的多光子显微技术,细胞的多光子显微损伤、皮米非入侵和非接触性外科手术等。     

Nanoscale topography of the basement membrane underlying the corneal epithelium of the rhesus macaque

This paper quantitatively defines the nanoscale topography of the basement membrane underlying the anterior corneal epithelium of the macaque. Excised corneal buttons from macaques were placed in 2.5 mM ethylenediaminetetraacetate (EDTA) for 2.5 h, after which the epithelium was carefully removed to expose the underlying basement membrane. The integrity of the remaining basement membrane was verified using fluorescent microscopy in conjunction with antibody staining directed against laminin and collagen type IV as well as transmission electron microscopy. Characterization of the surface of the basement membrane was performed using transmission electron microscopy, high-resolution, low-voltage scanning electron microscopy, and atomic force microscopy. Quantitative data were obtained with all three imaging techniques and compared. The basement membrane has a complex topography consisting of tightly cross-linked fibers intermingled with pores. The mean elevation of features measured by transmission electron microscopy, scanning electron microscopy, and atomic force microscopy was 149 +/- 60 nm, 191 +/- 72 nm, and 147 +/- 73 nm, respectively. Mean fiber diameter as measured by SEM was 77 +/- 44 nm and pore diameter was 72 +/- 40 nm, with pores occupying approximately 15% of the total surface area. Similar feature types and dimensions were also found for Matrigel, a commercially available basement membrane-like complex, supporting that a minimum of artifact was introduced by corneal preparative procedures to remove the overlying epithelium. Topographic features amplified the surface area over which cell-substratum interactions occur by an estimated 400%. The three-dimensional structure of the basement membrane exhibits a rich complex topography of individual features, consisting of pores and fibers with dimensions ranging from 30 to 400 nm. These nanoscale substratum features may modulate fundamental cell behaviors such as adhesion, migration, proliferation, and differentiation.     

Use of nanoparticles for studying the conformations of submembrane hemoglobin

The advantages and specific features of integrated application of atomic force microscopy, laser interference microscopy, and Raman microscopy in the study of erythrocytes are discussed. For successful application of Raman microscopy in the surface-enhanced mode, use was made of silver colloids. The dependence of the enhancement of Raman signals on silver nanoparticle size is demonstrated. The use of developed methods in clinical diagnostics is discussed.     

The use of nanoparticles for the study of conformations submembrane hemoglobin

The advantages and features of the integrated application of methods of atomic force microscopy, laser interference microscopy and Raman microscopy in the study of erythrocytes was discussed. For the successful application of Raman microscopy in surface enhanced Raman spectroscopy mode the silver colloids was using. The dependence of the enhancement of Raman signals from silver colloids size was demonstrated. The using of developed methods for clinic diagnostic was discussed.     

生物胆汁液晶态的研究

本文报导了用偏光显微镜、电子显微镜研究人体胆汁中的液晶态.结果表明:胆汁中存在有双射折的微粒,它们的图象指出是具有片层结构的液晶态.胆汁中还存在有类脂球滴液晶.     

MORPHOLOGY OF THE IMMATURE STAGES OF DACUS TRYONI (FROGGATT) (DIPTERA: TEPHRITIDAE)

Abstract
External features of the puparium, larvae and egg of the Queensland fruit fly, Dacus (Bactrocera) tryoni (Froggatt) were studied by scanning electron microscopy and significant morphological characters are described and illustrated. Cephalopharyngeal skeletons are described from examination by light microscopy.     

Marker-free method for accurate alignment between correlated light,cryo-light,and electron cryo-microscopy data using sample support features

Combining fluorescence microscopy with electron cryo-tomography allows, in principle, spatial localization of tagged macromolecular assemblies and structural features within the cellular environment. To allow precise localization and scale integration between the two disparate imaging modalities, accurate alignment procedures are needed. Here, we describe a marker-free method for aligning images from light or cryo-light fluorescence microscopy and from electron cryo-microscopy that takes advantage of sample support features, namely the holes in the carbon film. We find that the accuracy of this method, as judged by prediction errors of the hole center coordinates, is better than 100?nm.     

28 S ribosomal RNA in vertebrates. Locations of large-scale features revealed by electron microscopy in relation to other features of the sequences.

The 28 S rRNA from several vertebrate species, when examined by electron microscopy, is seen to contain regions of extensive secondary structure, as first reported for HeLa-cell 28 S rRNA by Wellauer & Dawid [(1973) Proc. Natl. Acad. Sci. U.S.A. 70, 2827-2831]. Here we correlate the locations of these regions, determined from the electron-microscopic data, with the primary structure of 28 S rRNA from human, mouse and Xenopus laevis determined by sequence analysis of rDNA. The secondary-structure features observed by electron microscopy correspond closely to phylogenetically variable G + C-rich regions that largely comprise the eukaryotic expansion segments in these three species. In most if not all cases the features can be identified with long G + C-rich helices deduced from sequence data. Correlations are given between the locations of the secondary-structure features and several 'landmark' restriction sites in 28 S rDNA. By correlating the locations of the rRNA methyl groups reported elsewhere [Maden (1988) J. Mol. Biol. 201, 289-314] with the present findings it is concluded that the rRNA secondary-structure features revealed by electron microscopy are largely or wholly unmethylated.     

Properties of the initial stages of embryoidogenesis in vitro in wheat calli of various origin

The formation of embryoids obtained in vitro both in the culture of anthers and in the culture of the immature embryos of spring soft wheat was analyzed using light microscopy and transmission electron microscopy. The features of embryoid formation in the calli of both types are detected.     

Post-embryonic development of Psammopsyllus maricae Cottarelli,Saporito & Puccetti, 1983 (Copepoda,Harpacticoida)

The post-embryonic development of Psammopsyllus maricae Cottarelli, Saporito & Puccetti, 1983 has been described for the first time. Six morphologically distinct naupliar and five copepodid stages were discerned and studied using light microscopy. Some features were also examined by scanning electron microscopy.     

The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the calgary biofilm device

Microbes frequently live within multicellular, solid surface-attached assemblages termed biofilms. These microbial communities have architectural features that contribute to population heterogeneity and consequently to emergent cell functions. Therefore, three-dimensional (3D) features of biofilm structure are important for understanding the physiology and ecology of these microbial systems. This paper details several protocols for scanning electron microscopy and confocal laser scanning microscopy (CLSM) of biofilms grown on polystyrene pegs in the Calgary Biofilm Device (CBD). Furthermore, a procedure is described for image processing of CLSM data stacks using amira™, a virtual reality tool, to create surface and/or volume rendered 3D visualizations of biofilm microorganisms. The combination of microscopy with microbial cultivation in the CBD — an apparatus that was designed for highthroughput susceptibility testing — allows for structure-function analysis of biofilms under multivariate growth and exposure conditions.     

Scanning probe recognition microscopy investigation of tissue scaffold properties

Scanning probe recognition microscopy is a new scanning probe microscopy technique which enables selective scanning along individual nanofibers within a tissue scaffold. Statistically significant data for multiple properties can be collected by repetitively fine-scanning an identical region of interest. The results of a scanning probe recognition microscopy investigation of the surface roughness and elasticity of a series of tissue scaffolds are presented. Deconvolution and statistical methods were developed and used for data accuracy along curved nanofiber surfaces. Nanofiber features were also independently analyzed using transmission electron microscopy, with results that supported the scanning probe recognition microscopy-based analysis.     

秋茄次生木质部的生态解剖学研究

为了探讨秋茄(Kandelia obovata Sheue et al.)次生木质部的形态解剖和数量解剖特征变化对不同红树林生境的生态适应意义,采用光学显微镜(LM)、扫描电镜(SEM)、透射电镜(TEM)和激光共聚焦显微镜(LSCM)对深圳福田红树林自然保护区内7个秋茄种群的次生木质部解剖特征进行观测,并对种群样地的土壤盐分含量、pH值和土壤养分含量进行测定。结果显示,(1)7个秋茄种群的次生木质部具有一些共同形态解剖特征:具纤维状导管和环管管胞;许多导管壁的微观结构(如管壁附物、穿孔板附物和螺旋雕纹等)有利于提高水分输导的高效性和安全性,以适应潮间带生境;(2)应用逐步回归分析法对秋茄次生木质部数量解剖特征和土壤理化因子的关系进行分析发现,随着土壤Na+、土壤全盐量增高,秋茄次生木质部导管分子趋向于"大型化"。"大型化"导管有利于水分输导,但降低了安全性。在土壤盐离子含量越高、秋茄导管分子越大其水分输导安全性越低的情况下,推测可能有其它机制保证秋茄导管水分输导的安全性。     

In situ imaging of mitochondrial outer-membrane pores using atomic force microscopy

Here we describe a technique for imaging of the outer contours of the mitochondrial membrane using atomic force microscopy, subsequent to or during a toxic or metabolic challenge. Pore formation in both glucose-challenged and 1,3-dinitrobenzene (DNB)-challenged mitochondria was observed using this technique. Our approach enables quantification of individual mitochondrial membrane pore formations. With this work, we have produced some of the highest resolution images of the outer contours of the in situ mitochondrial membrane published to date. These are potentially the first images of the component protein clusters at the time of formation of the mitochondrial membrane transition pore in situ. With the current work, we have extended the application of atomic force microscopy of mitochondrial membranes to fluid imaging. We have also begun to correlate 3-D surface features of mitochondria dotted with open membrane pores with features previously viewed with electron microscopy (EM) of fixed sections.     

Confocal fluorescence microscopy in modern cell biology

Confocal fluorescence microscopy has become a major tool in modern cell biology. The paper explains the basic principles and especially the depth discrimination properties of confocal microscopy. An important application is described briefly and outlined with some figures. The paper concludes with remarks on features to be expected in the near future.     

Structural investigation of polysaccharides and nanocompositions based on them

The structural features of natural polysaccharides-cellulose and chitin and their derivates-are considered. Literature data on electron microscopy of natural polysaccharides are analyzed and summarized. The influence of the supramolecular structure on the characteristics of materials determining many of its features is demonstrated. Interesting data on sizes of crystallites whose homogeneity in size and shape significantly influences the physicochemical features of the materials are presented.     

A fluorescence microscopy based genetic screen to identify mutants altered for interactions with host cells

The study of microbial intracellular pathogenesis has benefited from the application of immunofluorescence microscopy to characterize interactions of the pathogen with host cells. Unfortunately, immunofluorescence microscopy is impractical for screening the large number of bacterial mutants necessary to represent the entire genome of the pathogen. Screening has been limited due to the lack of materials suitable for high-throughput processing (e.g. 96-well plates) that also possess the optical features needed for high resolution fluorescence microscopy. Recently marketed 96-well Special Optics (SO) plates provide both the 96-well template ideal for high-throughput analysis and optical features suitable for fluorescence microscopy. Until this work, mutants needed for the study of a fluorescence-based virulence phenotype could not be obtained by direct screening approaches. In this study, SO plates were used to examine 11520 individual Salmonella typhimurium MudJ mutants for the loss of the ability to disrupt host cell endocytic compartments. The direct application of the fluorescence phenotype for screening allowed us to obtain a set of mutants to characterize the formation of lysosomal membrane glycoprotein (lgp) containing tubules upon Salmonella infection of HeLa epithelial cells. This approach will facilitate the characterization of a wide range of microbial phenotypes detectable by fluorescence microscopy.     

Placentation in watersnakes II: Placental ultrastructure in Nerodia erythrogaster (Colubridae: Natricinae)

Ultrastructure of the placental tissues from redbelly watersnakes (Nerodia erythrogaster ) was analyzed during late pregnancy to provide insight into placental development and function. Examination of the chorioallantoic placenta with transmission electron microscopy reveals that chorionic and uterine epithelia are extremely attenuated but intact and that the eggshell membrane is vestigial and lacks a calcareous layer. These features minimize the interhemal diffusion distance across the placenta. Scanning electron microscopy reveals that fetal and maternal components of the placentas are richly vascularized by dense networks of capillaries. Although the yolk sac omphalopleure has largely been replaced by chorioallantois by late gestation, it retains patches of yolk droplets and regions of absorptive cells with microvilli and abundant mitochondria. Transmission electron microscopy reveals that yolk material is taken up for digestion by endodermal cells. As yolk is removed, allantoic capillaries invade to occupy positions just beneath the epithelium, forming regions of chorioallantoic placentation. Ultrastructural features indicate that the chorioallantoic placenta is specialized for gas exchange, while the omphalallantoic (“yolk sac”) placenta shows evidence of functions in yolk digestion and maternal‐fetal nutrient transfer. Placental features of this species are consistent with those of other thamnophines, and are evolutionarily convergent on snakes of other viviparous clades.