Rapid detection of food pathogens using RNA aptamers-immobilized slide

The purpose of this study was to develop a simple and rapid detection system for foodborne bacteria, which consisted of an optical microscope and its slide chip with artificial antibodies, or RNA aptamers. From an RNA pool, three each RNA aptamers were built by the method of SELEX (systematic evolution of ligands by exponential enrichment) for components of cell wall, LPS (lipopolysaccharide) from E. coli O157:H7, teichoic acid from Staphylococcus aureus and a cell membrane protein of OmpC from Salmonella typhimurium, respectively. These aptamers were hybridized with thiol-conjugated 16 dT-linker molecules in order to be immobilized on silver surface which was, in advance, fabricated on glass slide, using a spin-coating method. To confirm that each aptamers retained its specific binding activities to their antigenic live bacteria, microscopic view of bound cells immobilized on silver film were observed. Furthermore, we observed the fluorescence-emitting bacteria-aptamer complex immobilized on silver film after adding RNA aptamers hybridized with fluorophore, FAM-conjugated 16 dT-linker molecules. As a result, the RNA aptamers-immobilized slide system developed in this study was a useful new tool to rapidly monitor individual food pathogens.     

A non-invasive method for in situ quantification of subpopulation behaviour in mixed cell culture.

Ongoing advances in quantitative molecular- and cellular-biology highlight the need for correspondingly quantitative methods in tissue-biology, in which the presence and activity of specific cell-subpopulations can be assessed in situ. However, many experimental techniques disturb the natural tissue balance, making it difficult to draw realistic conclusions concerning in situ cell behaviour. In this study, we present a widely applicable and minimally invasive method which combines fluorescence cell labelling, retrospective image analysis and mathematical data processing to detect the presence and activity of cell subpopulations, using adhesion patterns in STRO-1 immunoselected human mesenchymal populations and the homogeneous osteoblast-like MG63 continuous cell line as an illustration. Adhesion is considered on tissue culture plastic and fibronectin surfaces, using cell area as a readily obtainable and individual cell specific measure of spreading. The underlying statistical distributions of cell areas are investigated and mappings between distributions are examined using a combination of graphical and non-parametric statistical methods. We show that activity can be quantified in subpopulations as small as 1% by cell number, and outline behaviour of significant subpopulations in both STRO-1+/- fractions. This method has considerable potential to understand in situ cell behaviour and thus has wide applicability, for example in developmental biology and tissue engineering.     

Towards Nonspecific Detection of Malignant Cervical Cells with Fluorescent Silica Beads

To date, the methods for detection of cancer cells are mostly based on traditional techniques used in biology, such as visual identification of malignant changes, cell‐growth analysis, specific ligand–receptor labeling, or genetic tests. Despite being well developed, these methods are either insufficiently accurate or require a lengthy complicated analysis. A search for alternative methods for the detection of cancer cells may be a fruitful approach. Proposed here is a novel method for the detection of cancer cells in vitro, which is based on nonspecific adhesion of silica beads to cells. First, atomic force microscopy is used to study the adhesion of single silica beads to malignant and normal cells cultured from human cervix. It is found that adhesion depends on the time of contact, and can be statistically different for malignant and normal cells. Using these data, an optical method utilizing fluorescent silica beads is developed, which is based on detection of the difference in the number of adherent particles. The method is tested using primary cells cultured from cervical tissues of three healthy individuals and three patients with cervical cancer. The method shows sufficiently high sensitivity for cancer to make it interesting to perform further statistical tests.     

Light microscopic methods to study cells on non-transparent materials

Imaging of cells growing on non-transparent materials is important for the development of improved biomaterials. Based on methods mostly owing to current cellular physiology we have applied epifluorescence light microscopic methods with the aim to describe morphological and physiological parameters of cells, e. g., on metals. After loading cells with the cell permeant fluorescent dye calcein AM cells could be viewed at low as well as with high magnification. Semi-automated cell counting, tests for even distribution of the cells, visualization of their fine structural details, and dye transfer studies on intracellular communication were all feasible with this basic staining method. Continuous long term observation of cells was achieved with a special contrast enhanced epi-illumination, which was also useful to analyze dark (immuno)cytochemical stainings. Optical sectioning similar to Nomarski differential interference contrast allowed to view the material's surface as well as cellular details. Examples chosen to illustrate these methods encompass effects of bone morphogenetic protein-2 (BMP-2) on cells residing on stainless steel.     

An equilibrium method for continuous-flow cell sorting using dielectrophoresis

Separations represent a fundamental unit operation in biology and biotechnology. Commensurate with their importance is the diversity of methods that have been developed for performing them. One important class of separations are equilibrium gradient methods, wherein a medium with some type of spatial nonuniformity is combined with a force field to focus particles to equilibrium positions related to those particles' intrinsic properties. A second class of techniques that is nonequilibrium exploits labels to sort particles based upon their extrinsic properties. While equilibrium techniques such as iso-electric focusing (IEF) have become instrumental within analytical chemistry and proteomics, cell separations predominantly rely upon the second, label-based class of techniques, exemplified by fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). To extend the equilibrium techniques available for separating cells, we demonstrate the first implementation of a new microfluidic equilibrium separation method, which we call isodielectric separation (IDS), for sorting cells based upon electrically distinguishable phenotypes. IDS is analogous to isoelectric focusing, except instead of separating amphoteric molecules in a pH gradient using electrophoresis, we separate cells and particles in an electrical conductivity gradient using dielectrophoresis. IDS leverages many of the advantages of microfluidics and equilibrium gradient separation methods to create a device that is continuous-flow, capable of parallel separations of multiple (>2) subpopulations from a heterogeneous background, and label-free. We demonstrate the separation of polystyrene beads based upon surface conductance as well as sorting nonviable from viable cells of the budding yeast Saccharomyces cerevisiae.     

New methods to identify and rank high pedestrian crash zones: an illustration

Identifying and ranking high pedestrian crash zones plays a key role in developing efficient and effective strategies to enhance pedestrian safety. This paper presents (1) a Geographical Information Systems (GIS) methodology to study the spatial patterns of pedestrian crashes in order to identify high pedestrian crash zones, and (2) an evaluation of methods to rank these high pedestrian crash zones. The GIS based methodology to identify high pedestrian crash zones includes geocoding crash data, creating crash concentration maps, and then identifying high pedestrian crash zones. Two methods generally used to create crash concentration maps based on density values are the Simple Method and the Kernel Method. Ranking methods such as crash frequency, crash density, and crash rate, as well as composite methods such as the sum-of-the-ranks and the crash score methods are used to rank the selected high pedestrian crash zones. The use of this methodology and ranking methods for high pedestrian crash zones are illustrated using the Las Vegas metropolitan area as the study area. Crash data collected for a 5-year period (1998-2002) were address matched using the street name/reference street name intersection location reference system. A crash concentration map was then created using the Kernel Method as it facilitates the creation of a smooth density surface when compared to the Simple Method. Twenty-two linear high crash zones and seven circular high crash zones were then identified. The GIS based methodology reduced the subjectivity in the analysis process. Results obtained from the evaluation of methods to rank high pedestrian crash zones show a significant variation in ranking when individual methods were considered. However, rankings of high pedestrian crash zones were relatively consistent with little to no variation when the sum-of-the-ranks method and the crash score method were used. Thus, these composite methods are recommended for use in ranking high pedestrian crash zones instead of individual methods.     

射流电子水表原理及流量测量特性研究

介绍了基于恒磁励磁电磁检测方法的射流电子水表,研究了其结构原理、主要技术特征、流量测量特性、以及传感与信号处理技术。针对射流电子水表小流量测量特性,提出了降低射流计量腔的起振阈值,改进了电磁传感原理检测射流振荡信号的方法。经射流电子水表批量生产验证,证明了改进小流量特性思路和方法的有效性。     

Spectroscopic evaluation of the effect of a red microalgal polysaccharide on herpes-infected Vero cells

The sulfated polysaccharide obtained from a species of red microalga has proved to be a potent antiviral agent against various members of the herpes family. In the present study, we used microscopic Fourier transform infrared spectroscopy (FT-IR) to investigate differences between normal cells, those infected with herpes viruses, and infected cells treated with red microalgal polysaccharide. FT-IR enables the characterization of cell or tissue pathology based on characteristic molecular vibrational spectra of the cells. The advantage of microscopic FT-IR spectroscopy over conventional FT-IR spectroscopy is that it facilitates inspection of restricted regions of cell cultures or tissue. Our results showed significant spectral differences at early stages of infection between infected and noninfected cells, and between infected cells treated with the polysaccharide and those not treated. In infected cells, there was an impressive decrease in sugar content and a considerable increase in phosphate levels in conjunction with the infection progress. Our results also proved that sugars penetrated and accumulated inside cells treated with the red microalgal polysaccharide. These could have been sugar fragments of low molecular weight present in the polysaccharide solution, despite purification by dialysis. Such sugar accumulation might be responsible for a breakdown in the internal steps of the viral replication cycle.     

Highly sensitive method for assay of drug-induced apoptosis using fluorescence correlation spectroscopy

Apoptosis plays a crucial role in many biological processes and pathogenesis of various malignancies and diseases of the immune system. In this paper, we described a novel method for sensitive detection of drug-induced apoptosis by using fluorescence correlation spectroscopy (FCS). The principle of this method is based on the assay of DNA fragmentation in the process of the drug-induced apoptosis. FCS is a single molecule method, and it can be used for sensitive and selective assay of DNA fragmentation without separation. We first developed a highly sensitive method for characterization of DNA fragments using a home-built FCS system and SYBR Green I as fluorescent DNA-intercalating dye, and then established a model of drug-induced apoptosis using human pancreatic cancer cells and a drug lidamycin. Furthermore, FCS method established was used to directly detect the fragmentation of DNA extracted from apoptotic cells or in the apoptotic cell lysate. In FCS assay, the single-component model and the multiple-components model were used to fit raw FCS data. The characteristic diffusion time of DNA fragments was used as an important parameter to distinguish the apoptotic status of cells. The obtained data documented that the characteristic diffusion time of DNA fragments from apoptotic cells significantly decreased with an increase of lidamycin concentration, which implied that DNA fragmentation occurred in lidamycin-induced apoptosis. The FCS results are well in line with the data obtained from flow cytometer and gel electrophoresis. Compared to current methods, the method described here is sensitive and simple, and more importantly, our detection volume is less than 1 fL, and the sample requirement can easily be reduced to nL level using a droplets array technology. Therefore, our method probably becomes a high throughput detection platform for early detection of cell apoptosis and screening of apoptosis-based anticancer drugs.     

Dielectrophoresis-activated multiwell plate for label-free high-throughput drug assessment

Dielectrophoresis (DEP) offers many advantages over conventional cell assays such as flow cytometry and patch clamp techniques for assessing cell electrophysiology as a marker for cancer studies and drug interaction assessment. However, despite the advantages offered by DEP analysis, uptake has been low, remaining largely in the academic arena, due to the process of analysis being time-consuming, laborious, and ultimately allowing only serial analysis on small numbers of cells. In this paper we describe a new method of performing DEP analysis based on laminate manufacturing methods. These use a three-dimensional "well" structure, similar in size and pitch to conventional microtiter well plates, but offer electrodes along the inner surface to allow easy measurement of cell properties through the whole population. The result can then be determined rapidly using a conventional well-plate reader. The nature of the device means that many electrodes, each containing a separate sample, can be tested in parallel, while the mode of observation means that analysis can be combined with simultaneous measurement of conventional fluorimetric well-based assays. Here we benchmark the device against standard DEP assays, then show how such a device can be used to (a) rapidly determine the effects both of ion channel blockers on cancer cells and antibiotics on bacteria and (b) determine the properties of multiple subpopulations of cells within a well simultaneously.     

Numerical algorithms for dynamic traffic demand estimation between zones in a network

This paper presents numerical methods for dynamic traffic demand estimation between N zones in a network, where the zones are disjoint subsets of nodes of the network. Traffic is assumed to be generated or absorbed only in the zones and nowhere else in the network. Traffic volumes between zones over a fixed period of time are modeled as independent random variables with unknown means which it is desired to estimate. For each zone, the volume of all incoming and outgoing traffic is counted on a regular basis but no information about the origin or destination of the observed traffic is used. Procedures are suggested for a regular update of estimates of the N(N - 1) mean traffic demands between the zones on the basis of an incoming stream of the 2N traffic counts. The procedures are based on an exponential smoothing scheme and are reminiscent of the expectation maximization (EM) algorithm if smoothing is removed. Fast and reliable numerical algorithms, based on the conjugate gradient method, are presented for normal as well as for Poisson traffic demands. The Poisson case is linked with entropy maximization. Computational tests based on simulated data demonstrate both the numerical and statistical efficiency of the procedures.     

Determination of zonal boundaries in articular cartilage using infrared dichroism

To determine the sub-tissue structural zonal boundaries in articular cartilage, a novel infrared (IR) microscopic imaging method based on the dichroic nature of the amide components has been developed and is discussed in this article. Thin canine cartilage-bone sections embedded in paraffin as well as in poly(methyl methyacrylate) (PMMA) were imaged under two orthogonal polarization states at 6.25 mum pixel size. The depth-dependent anisotropy of the amide components at perpendicular polarization states attributed by the collagen constituent in cartilage was analyzed. Since the transitional zone fibers are randomly arranged and the dichroic ratio value reaches unity in this zone, it is possible to identify the transitional zone boundaries, thus dividing the whole-depth tissue into three structural zones (superficial, transitional and radial). The zone division results from the infrared method agree well with the results from the established polarized light microscopy (PLM) method, which promises the potential of infrared imaging as an independent technique for the zonal boundary determination. The advantages of this dichroic ratio method are (1) it is independent of mode of operation (transmission/reflection), (2) it is independent of sample thickness, (3) either a polarizer or an analyzer can be used in experiments to determine zonal boundaries, and (4) it is sample orientation independent.     

Development of a method for integrating time-dependent constitutive equations with large,small or negative strain rate sensitivity

A unified numerical method to integrate stiff time-dependent constitutive equations has been developed. This method is a stable, non-iterative and self-correcting solution procedure which works successfully over a wide range in strain rate sensitivity. Time steps are automatically controlled during integration to achieve a user-specified accuracy. This method is implemented in the program package NONSS whose dual purpose is examination of the behaviour of unified constitutive models by themselves (‘one-element behaviour’) as well as providing a computationally efficient subroutine for utilizing such models with existing finite element programs for non-linear structural and metal forming analyses. This paper first reviews the relation between the numerical characteristic of constitutive equations and the choice of integration methods. Then the paper presents the derivation of the governing basic equations in the new method, and also derives a special algorithm which permits large integration steps within the negative strain rate sensitivity (‘serrated yielding’) regime. Examples of the program's performance are given, including plasticity at high and low temperatures, cyclic deformation and multiaxial straining.     

流动颗粒显微图像测量技术研究

针对显微环境下流动颗粒测量的需要,构建了测量平台,研制了基于MEMS工艺的测量器件。在分析流动颗粒显微图像特征的基础上,提出了基于颗粒运动特性的图像测量流程,其中提出了基于基准桢差分的目标提取算法,解决了流动颗粒目标分割的问题,根据图像运动模糊的退化模型,研究了图像模糊恢复的问题。最后以润滑油中的磨损颗粒为例,验证了该文的分析方法对流动颗粒检测的有效性。     

Validation of a new pertinent packing coefficient to estimate flow properties of pharmaceutical powders at a very early development stage, by comparison with mercury intrusion and classical flowability methods

The present study compares four characterisation techniques, such as packing and rearrangement under pressure methods or shear cell measurement methods, used to evaluate powder flow properties. The reduction of the powder bed volume under low pressures is analysed using mercury porosimetry and two compressibility methods (uniaxial press and volumenometer). Flow functions, deduced from shear cell measurements, are determined using a Johanson Indicizer^TM Tester. The examination of the reduction of the powder bed volume leads to new parameters such as the packing coefficient (C _t) and the volume of mercury intruded (V _hg). The packing coefficient appears to be a reliable approximation of powder flow properties, whatever cohesive or free flowing : it is actually well correlated with shear cell measurements and it is more accurate than classical flowability tests recommended by the European Pharmacopoeia. Furthermore, this method is easy to use and consumes a small amount of powders (<1 g). All together, this method is able to give—very early in the development—a quite accurate estimation of powder flow properties of new drug substances. This may be very helpful for an early determination of the optimum particle granulometry or for a rapid development of a feasible industrial process.     

SERS microscopic imaging as novel tool for assessing viability and enumerating yeast cells at various stages of cell cycle in lag,log, exponential and stationary phases of growth in culture

Surface enhanced Raman scattering (SERS) microscopic imaging was employed to enumerate the yeast cells in culture. We found this imaging method as an efficient tool for easily differentiating and quantitatively enumerating yeast cell at different stages of cell-division cycle (G1, S, G2 and M phase) at various stages of growth phases namely lag, log, exponential and stationary phases in culture. Apart from enumerating the cells at different stages of cell cycle under lag, log, exponential and stationary phases, it was possible using SERS microscopy to differentiate the live cells from dead ones. The dead cells were SERS inactive and gave enhanced autofluorescence compared with the live cells, which were SERS active. The results from the present investigation suggest that SERS microscopic imaging, using silver nanoparticles (AgNPs) as a sensitive tool to enumerate the yeast cells in culture.     

我国螺杆冷水机组的能量调节亟待推广使用VSD法

螺杆冷水机广泛应用于中央空调中 ,鉴于其长期工作在部分负荷下的特点 ,本文在分析了滑阀及变速调节螺杆制冷压缩机负荷的基础上 ,提出借变频调速装置来实现滑阀、压缩机转速及内容积比的优化匹配控制的VSD法 ,并进行了实测 ,分析了其节能效果。     

Molecularly imprinted TiO2-matrix-assisted laser desorption/ionization mass spectrometry for selectively detecting alpha-cyclodextrin

This study describes a new means to conduct molecular recognition-based analysis using mass spectrometry. Taking advantage of the unique characteristic of the absorption capacity of the TiO(2) sol-gel material in the UV region, a TiO(2) sol-gel-deposited thin film was employed as the sample substrate to assist in UV laser desorption/ionization of analytes. Sol-gels are polymeric materials that are easy to prepare and modify at low temperatures. Molecularly imprinted TiO(2) sol-gels were generated for molecular recognition-based analysis. alpha-Cyclodextrin (CD) was selected as the template molecule and doped into TiO(2) in a sol-gel reaction. The molecularly imprinted TiO(2) sol was spin-coated on a glass slide, and appropriate template cavities in the TiO(2) sol-gel material were formed after the template molecules were removed. We demonstrate that this modified glass slide can be used to select alpha-CD from a sample solution containing equal amounts of alpha-, beta-, and gamma-CD (50 ppb each, 18 mL); alpha-CD was directly detected from the modified glass slide by matrix-assisted laser desorption/ionization mass spectrometry without the addition of extra matrix. This approach provides a new detection method for molecular recognition-based analysis.     

Optical measurement of microscopic torques

Abstract

In recent years there has been an explosive development of interest in the measurement of forces at the microscopic level, such as within living cells, as well as the properties of fluids and suspensions on this scale, using optically trapped particles as probes. The next step would be to measure torques and associated rotational motion. This would allow measurement on very small scales since no translational motion is needed. It could also provide an absolute measurement of the forces holding a stationary non-rotating particle in place. The laser-induced torque acting on an optically trapped microscopic birefringent particle can be used for these measurements. Here we present a new method for simple, robust, accurate, simultaneous measurement of the rotation speed of a laser trapped birefringent particle, and the optical torque acting on it, by measuring the change in angular momentum of the light from passing through the particle. This method does not depend on the size or shape of the particle or the laser beam geometry, nor does it depend on the properties of the surrounding medium. This could allow accurate measurement of viscosity on a microscopic scale.