Examining cement-based materials by laser scanning confocal microscopy

While laser scanning confocal microscopy (LSCM) is widely used in a variety of fields, LSCM has not been broadly applied for the characterization of cement-based materials, despite the potential advantages of this technique. It is believed that the use of confocal microscopy for characterizing cement-based materials would be expanded by the further development of sample preparation and examination protocols. Three imaging methodologies: (1) “through-aggregate” examination, (2) wet-chemistry studies, and (3) surface characterization; and their potential applications to cement-based materials are described.     

激光扫描共聚焦显微镜在荧光原位杂交中的应用

简要介绍激光扫描共聚焦显微镜在荧光原位杂交中的应用方法及其优越性。     

Measurement of wet fiber flexibility by confocal laser scanning microscopy

A new method for fiber flexibility measurement has been developed using confocal laser scanning microscopy (CLSM), based on the fiber conformability method. Freespan length was measured directly from the transverse view of the fiber span, so the accuracy of the measurement was greatly improved. The collapsibility and moment of inertia can also be measured from the fiber cross section obtained from CLSM. Thus, it was possible to measure the elastic modulus of individual fibers. Therefore, all the basic properties of fiber that affect its conformability, including fiber wall thickness, moment of inertia, and elastic modulus, can be measured within a single test. Results show that both fiber collapsibility and the elastic modulus of the fiber wall affect the fiber flexibility. Collapsibility is controlled mainly by fiber wall thickness. A refining or bleaching process can increase fiber flexibility mainly by altering fiber elastic modulus.     

Localization of analyte molecules in MALDI preparations by confocal laser scanning microscopy

In this study, the incorporation of Texas Red-labeled avidin into crystals of 2,5-dihydroxybenzoic acid (2,5-DHB) and 2,6-DHB (used as matrixes for matrix-assisted laser desorption/ionization (MALDI)) was investigated by fluorescence spectrophotometry and confocal laser scanning microscopy (CLSM). The analyte distribution in crystals, grown slowly under controlled conditions, was compared to the analyte localization in different standard preparations (dried-droplet and thin-layer preparation). Texas Red turned out to be a useful fluorescence label in the acidic environments of typical matrixes. Earlier results by absorption spectrophotometry could be confirmed by fluorescence measurements; 2,5-DHB incorporates the analyte proportionally, while 2,6-DHB excludes the protein from its crystal lattice. It is found that the analyte distribution can be analyzed well in both single crystals and standard preparation, by CLSM using Texas Red-labeled analytes. The present study allows for a conclusive and consistent interpretation of analyte incorporation into MALDI preparations.     

Measuring diffusion of molecules into individual polymer particles by confocal Raman microscopy

Raman microscopy is a powerful method for providing spatially resolved, chemically selective information about the composition of materials. With confocal collection optics, the method is well suited to the analysis of small particles in contact with liquid solutions. In this work, the transport of an organic solvent component into small polystyrene particles is investigated. An inverted confocal Raman microscope is used to acquire spectra from individual 75-microm polystyrene particles in contact with acetonitrile/water mixtures. Monitoring the Raman scattering from the C[triple bond]N stretching mode of acetonitrile provides a measure of solvent uptake into the polymer material. The small collection volume defined by the confocal optics provides the micrometer spatial resolution needed to track solvent concentration at different locations within the particle with 30-s time resolution. The volume fraction of acetonitrile in water in the surrounding solution was varied in order to determine the concentration dependence of the diffusion kinetics. Modeling the transport of molecules into a particle was addressed by using finite element methods for the evaluation of the coupled space- and time-dependent differential equations that govern the molecular transport. The results indicate that the diffusion coefficient changes with the local solvent concentration in the polymer. At longer times, with the highest acetonitrile concentrations, an evolution of the solvent transport mechanism was observed, from a diffusive rate that depends on local concentration to a linear increase in concentration with time accompanied by measurable swelling of the particle volume.     

Quantitative characterization of pores in transparent ceramics by coupling electron microscopy and confocal laser scanning microscopy

This work is dedicated to the implementation of a new characterization method of porosity in transparent ceramics. This quantitative method couples scanning electron microscopy with confocal laser scanning microscopy. From this original method, the volume fraction of pores has been determined for different sintered Nd:YAG specimens with an accuracy of about 10%. This technique appears to be promising because it leads to both the pore size distribution and the residual porosity for fully dense samples. For example, it becomes possible to reach porosity levels ranging from 0.09% to 0.0004% for transparent Nd:YAG specimens which could not be measured by using conventional techniques. Finally, correlations between the residual porosity of these full dense samples and their optical properties could be established.     

Imaging solute distribution in capillary electrochromatography with laser scanning confocal microscopy

A method for the direct observation of solute molecules interacting with a C18 stationary phase under real separation conditions in capillary electrochromatography (CEC) is investigated. The experiments were performed in a capillary electrochromatographic mode; however, the method and findings are useful both in CEC and revered-phase liquid chromatography. The distribution of solute molecules in the packed capillary is directly imaged with laser scanning confocal fluorescence microscopy. Conventional imaging techniques produce images where the C18 silica beads cannot be distinctively identified as a result of the deep depth of field. The optical sectioning capability of confocal imaging overcomes this problem to afford clearly defined images of the stationary-phase packing and the surrounding mobile phase. Fluorescein molecules are preferentially distributed in the mobile phase under reversed-phase chromatographic conditions. Nile Red and rhodamine 6G molecules prefer the environments of the porous C18 beads. Intensity distributions over time for areas within the stationary-phase beads differ from distributions of areas outside the beads in the mobile phase. Images taken at different depths into the capillary probe the internal structure of the C18 beads. While the internal structures of most beads are porous, confocal images show a small fraction (2%) of the silica beads have porous shells and nonporous cores. The capability of imaging the stationary phase distinctively from the mobile phase opens the possibilities of studying the quality of stationary phase, the structure of the column packing, and the mechanisms of separation.     

Transverse dimensions of wood pulp fibres by confocal laser scanning microscopy and image analysis

Cross-sectional images of wood-pulp fibres were generated using optical sectioning by a confocal laser scanning microscope. The technique has a distinct advantage over mechanical sectioning with a microtome, as it simplifies specimen preparation. Cross-sectional images were obtained for unbleached softwood kraft pulp fibres using epifluorescence mode. The accuracy of cross-sectional images was verified by imaging fluorescent microspheres. An image analysis procedure, in which the boundaries of fibre cross-sections were defined with a maximum-gradient edge-finding technique, was developed for measuring fibre cross-sectional area and wall thickness rapidly and accurately. The measurements were insensitive to the confocal microscope's asymmetric resolution, signal deterioration through the specimen thickness, overall image quality and operator bias.     

Studying multilayer langmuir film structure by confocal laser scanning and atomic force microscopy techniques

Features of extracting information on the surface structure of a multilayer organic film from data on its optical properties and surface relief are considered. The object of investigation was a multilayer Langmuir-Blodgett film based on a prepolymer (polyamic acid salt). It is suggested that the formation of the film volume is influenced by inhomogeneities in the structure of layers.     

Fluorescence confocal laser scanning microscopy as a probe of pH gradients in electrode reactions and surface activity

The application of fluorescence confocal laser scanning microscopy (CLSM) to quantify three-dimensional pH gradients near electrode surfaces is described. The methodology utilizes a trace quantity of a fluorescent dye, fluorescein, in solution, which fluoresces strongly above pH 6.5, to map the pH adjacent to various ultramicroelectrodes undergoing electrochemical processes that lead to pH changes. The experimental fluorescence profiles, determined by CLSM, have been compared to models by solving the underlying mass transport equations, including the effect of natural convection, using the finite element method. The methodology has been validated through studies of the galvanostatic reduction of water at both disk and ring ultramicroelectrodes. The fluorescence profiles were found to be highly sensitive to both the initial bulk solution pH and applied current in a predictable fashion. The potentiostatic reduction of oxygen has been investigated at 25- and 10-microm-diameter platinum electrodes to confirm the effective number of electrons transferred in the reaction. Finally, the application of this methodology to observe defects in microelectrode arrays, particularly those that cannot be seen by optical microscopy, is described.     

Spatially resolved analysis of small particles by confocal Raman microscopy: depth profiling and optical trapping

Raman microscopy is a powerful method to provide spatially resolved information about the chemical composition of materials. With confocal collection optics, the method is well suited to the analysis of small particles, either resting on a surface or optically trapped at a laser focus, where the confocal collection volume optimizes the signal from the particle. In this work, the sensitivity and spatial selectivity of detecting Raman scattering from single particles was determined as a function of particle size. An inverted confocal Raman microscope was used to acquire spectra of individual surface-bound and optically trapped polystyrene particles with sizes ranging between 200 nm and 10 microm. The particles are in contact with aqueous solution containing perchlorate ion that served as a solution-phase Raman-active probe to detect interferences from the surrounding medium. The collection volume is scanned through single particles that are attached to the surface of the coverslip, and the sensitivity and selectivity of detection are measured versus particle size. The results compare favorably with a theoretical analysis of the excitation profile and confocal collection efficiency integrated over the volumes of the spherical particles and the surrounding solution. This analysis was also applied to the detection of particles that are optically trapped and levitated above the surface of the coverslip. The results are consistent with the optical trapping of particles at or near the excitation beam focus, which optimizes excitation and selective collection of Raman scattering from the particle.     

Reagentless identification of single bacterial spores in aqueous solution by confocal laser tweezers Raman spectroscopy

We demonstrate that optical trapping combined with confocal Raman spectroscopy using a single laser source is a powerful tool for the rapid identification of micrometer-sized particles in an aqueous environment. Optical trapping immobilizes the particle while maintaining it in the center of the laser beam path and within the laser focus, thus maximizing the collection of its Raman signals. The single particle is completely isolated from other particles and substrate surfaces, therefore eliminating any unwanted background signals and ensuring that information is collected only from the selected, individual particle. In this work, an inverted confocal Raman microscope is combined with optical trapping to probe and analyze bacterial spores in solution. Rapid, reagentless detection and identification of bacterial spores with no false positives from a complex mixed sample containing polystyrene and silica beads in aqueous suspension is demonstrated. In addition, the technique is used to analyze the relative concentration of each type of particle in the mixture. Our results show the feasibility for incorporating this technique in combination with a flow cytometric-type scheme in which the intrinsic Raman signatures of the particles are used instead of or in addition to fluorescent labels to identify cells, bacteria, and particles in a wide range of applications.     

Comparison of discrete and continuous motion in scanning probe microscopy monitored via confocal Raman microspectroscopy

Confocal Raman imaging is a relatively new analytical technique that combines the strengths of Raman microspectroscopy and confocal optics. The images collected by the microscope are obtained by monitoring specific bands in the Raman spectra that are collected at many points in a sample, with the number of spectra usually numbering in the hundreds or thousands. Some commercially available systems acquire data while the sample is continuously moving with respect to the microscope objective. The distance that the stage moves during a single acquisition is a parameter that can be set prior to data acquisition. Data in this report was acquired with both a static and continuously moving sample for comparison, utilizing the 520 cm(-1) Si phonon of a silicon wafer to monitor an edge. Scattering collected from each discrete step, i.e., no motion during spectral acquisition, showed excellent precision of location, but a loss in resolution was observed as the pixel size was increased beyond the maximum theoretical resolution of the instrument. A continuously moving stage contributed to erroneous position data as the pixel size was increased beyond the maximum theoretical resolution of the instrument.     

Low-cost, scalable laser scanning module for real-time reflectance and fluorescence confocal microscopy

We present a low-cost, high-speed, retrofitted laser scanning module for microscopy. The cage-mounted system, with various available fiber-coupled sources, offers a real-time imaging alternative to costly commercial systems with capabilities for conventional or confocal reflectance and fluorescence applications as well as advanced laser scanning microscopy implementations. Reflectance images of a resolution target and confocal images of fluorescent polystyrene beads are presented for system characterization. Confocal fluorescence image stacks of T84 epithelial cancer cells are presented to demonstrate application to biological studies. This laser scanning module is a flexible, scalable, high-speed alternative to commercial laser scanning systems suitable for applications requiring a simple imaging tool and for teaching laboratories.     

Investigations of dye-sensitised titania solar cell electrode using confocal laser scanning microscopy

We have characterised a dye-sensitised nanoporous nanocrystalline titania film used in prototype photoelectrochemical solar cell production. The film was sensitised with two types of ruthenium (II) based dyes for different times, between 0.5 and 24 hours. The penetration and coverage for each type of dye was studied using Confocal Laser Scanning Microscopy (CLSM). Varying laser powers were applied and fluorescence images from the film have been analysed. Both dyes were found to percolate through the whole of the nanoporous film irrespective of the dyeing time but the amount of dye increased with dyeing time. The CLSM is shown to be a valuable tool for the investigation of dye penetration and coverage in porous films.     

高分辨率大视场线扫描共焦显微镜的设计与研制

商用共聚焦显微镜使用二维光点扫描对样品成像,成像帧频被限制在30 Hz以内,扫描速度大多在10帧/秒(f/s)左右。为了提高共聚焦系统成像速度,满足生物细胞在体成像的需求,本文使用线光束对样品进行一维扫描照明,成像速度大大提高,同时根据共聚焦成像原理,在线阵CCD前使用狭缝滤除非聚焦平面杂散光以提高成像质量。实验表明,系统光学放大倍率为55倍,横向分辨率高于2.2 μm,当线阵CCD以28 kHz行频扫描成像时,帧频可达50 f/s,通过对动植物细胞成像证明,本系统可用于生物细胞的在体成像。     

Multivariate analysis applied to the study of spatial distributions found in drug-eluting stent coatings by confocal Raman microscopy

Multivariate data analysis was applied to confocal Raman measurements on stents coated with the polymers and drug used in the CYPHER Sirolimus-eluting Coronary Stents. Partial least-squares (PLS) regression was used to establish three independent calibration curves for the coating constituents: sirolimus, poly(n-butyl methacrylate) [PBMA], and poly(ethylene-co-vinyl acetate) [PEVA]. The PLS calibrations were based on average spectra generated from each spatial location profiled. The PLS models were tested on six unknown stent samples to assess accuracy and precision. The wt % difference between PLS predictions and laboratory assay values for sirolimus was less than 1 wt % for the composite of the six unknowns, while the polymer models were estimated to be less than 0.5 wt % difference for the combined samples. The linearity and specificity of the three PLS models were also demonstrated with the three PLS models. In contrast to earlier univariate models, the PLS models achieved mass balance with better accuracy. This analysis was extended to evaluate the spatial distribution of the three constituents. Quantitative bitmap images of drug-eluting stent coatings are presented for the first time to assess the local distribution of components.     

Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy

We report on a rapid method for reagentless identification and discrimination of single bacterial cells in aqueous solutions using a combination of laser tweezers and confocal Raman spectroscopy (LTRS). The optical trapping enables capturing of individual bacteria in aqueous solution in the focus of the laser beam and levitating the captured cell well off the cover plate, thus maximizing the excitation and collection of Raman scattering from the cell and minimizing the unwanted background from the cover plate and environment. Raman spectral patterns excited by a near-infrared laser beam provide intrinsic molecular information for reagentless analysis of the optically isolated bacterium. In our experiments, six species of bacteria were used to demonstrate the capability of the confocal LTRS in the identification and discrimination between the diverse bacterial species at various growth conditions. We show that synchronized bacterial cells can be well-discriminated among the six species using principal component analyses (PCA). Unsynchronized bacterial cells that are cultured at stationary phases can also be well-discriminated by the PCA, as well as by a hierarchical cluster analysis (HCA) of their Raman spectra. We also show that unsynchronized bacteria selected from random growth phases can be classified with the help of a generalized discriminant analysis (GDA). These findings demonstrate that the LTRS may find valuable applications in rapid sensing of microbial cells in diverse aqueous media.