Image analysis method for evaluation of specific and non-specific hand contamination

AIMS: To evaluate a quantifying image analysis method for assessing the degree of hand contamination and efficacy of hand washing procedures. METHODS AND RESULTS: Two types of experimental design were used. In one, different concentrations of pure cultures of Escherichia coli, Listeria innocua and Pseudomonas flourescens were applied to hands. In the other, hands were contaminated by handling various raw foods. Imprints of the contaminated palms were made on 24.5 x 24.5 cm agar plates using appropriate agars. After incubation, digital photographs of the plates were analysed using image analysis. In pure culture studies with selective agars, levels from 1 to 10(6) CFU cm(-2) palm could be monitored. For aerobic, mesophilic organisms from raw chicken, levels from 10(3) to 10(6) CFU cm(-2) palm were correlated linearly to image analysis data. CONCLUSIONS: The image analysis of palm imprints made on agar plates was suitable for assessing the degree of contamination from foods on the palms. Sensitivity and specificity depended on the agar used and the type of contamination encountered. SIGNIFICANCE AND IMPACT OF THE STUDY: Data capture by the image analysis method is simple and can be partly automated. Sampling time is short for the person to be tested, which makes it an attractive method for assessing hand hygiene status in larger field trials.     

Dense chromatin plates in metaphase chromosomes

In a previous work we observed multilayered plate-like structures surrounding partially denatured HeLa chromosomes at metaphase ionic conditions. This unexpected finding has led us to carry out an extensive investigation of these structures. Our results show that plates can also be found in metaphase chromosomes from chicken lymphocytes. We have used atomic force microscopy (AFM) to image and investigate the mechanical properties of plates in aqueous solution. Plates are thin (~6.5 nm each layer) but compact and resistant to penetration by the AFM tip: their Young’s modulus is ~0.2 GPa and the stress required for surface penetration is ~0.03 GPa in the presence of Mg²⁺ (5–20 mM). Low-ionic strength conditions produce emanation of chromatin fibers from the edges of uncrosslinked plates. These observations and AFM results obtained applying high forces indicate that the chromatin filament is tightly tethered inside the plates. Images of metal-shadowed plates and cryo-electron microscopy images of frozen-hydrated plates suggest that nucleosomes are tilted with respect to the plate surface to allow an interdigitation between the successive layers and a thickness reduction compatible with the observed plate height. The similarities between denatured plates from chicken chromosomes and aggregates of purified chromatin from chicken erythrocytes suggest that chromatin has intrinsic structural properties leading to plate formation. Scanning electron micrographs and images obtained with the 200-kV transmission microscope show that plates are the dominant component of compact chromatids. We propose that metaphase chromosomes are formed by many stacked plates perpendicular to the chromatid axis.     

Application of robotics and image processing to automated colony picking and arraying.

We describe a system that applies image processing and robotic techniques to automatically pick individual colonies from square petri dishes and array them in 96-well microtiter plates. Digital images of the colony distribution in the dishes are acquired using a video camera and frame buffer. Commercial image processing software is used to identify individual colonies and determine their locations. A Hewlett-Packard Microassay System robot reads the resulting coordinate file for each dish, picks cells from each identified colony and transfers the cells into a microtiter plate well. A disposable pipet tip is used as the sterile implement for colony picking. Custom holders position the dishes accurately and provide common coordinate systems for imaging and picking. The system is calibrated to account for the depth of agar in the dishes. The robot can process up to 10 dishes and 20 plates (1920 colonies) in a single run. It has successfully arrayed a cosmid library of the S. pombe genome consisting of approximately 6000 colonies in 30 petri dishes in about 40 hours of robot time. Future enhancements to the system are discussed.     

阳桃次生木质部导管分子及侵填体的研究

运用细胞图象分析系统和显微照相的方法对阳桃次生木质部导管分子进行了观察研究。次生木质部导管分子类型有两端具尾导管、一端具尾导管和无尾导管。导管分子穿孔板存在着2种类型:两端均为一个单穿孔板;一端为一个单穿孔板,另一端为具两个单穿孔的复穿孔板。48%的导管分子具有囊状侵填体;管间纹孔式为互列纹孔式。     

Specific absorption rate in electrically coupled biological samples between metal plates

The specific absorption rate (SAR) in a biological sample irradiated by electromagnetic fields between the metal plates of a transmission line can be altered significantly by the spacing of the metal plates and the distance between neighboring samples. The SAR in spherical biological samples is calculated for a number of neighboring sample arrangements and metal-plate spacings by using the method of images and induced dipole coupling. For a decrease in metal-plate spacing, the derived equations predict an increase in SAR within a sample and a decrease in SAR with a decrease in neighboring-sample spacing. The calculations are compared with measurements made with the aid of an array of 1-in radius metal hemispheres on the lower plate of two parallel plates (thus forming an image system). The hemisphere on which measurements are taken is insulated from the metal plate and is connected via a coaxial center conductor to an HP 3582A spectrum analyzer that measures the voltage and hence the electric field intensity at the hemisphere. Measurements made at a frequency where wavelength is large compared with sample size (48 Hz) are in good agreement with calculations.     

Non-uniform strain distribution within rat cartilaginous growth plate under uniaxial compression

Growth plates are highly inhomogeneous in morphology and composition. Mechanical loading can modulate longitudinal bone growth, though the mechanisms underlying this mechanobiology are poorly understood. The proximal tibial growth plates of six rats were tested in vitro under uniaxial compression to 5% strain, and confocal microscopy was used to track and capture images of fluorescently labeled cell nuclei with increasing applied strains. The local strain patterns through the growth plate thickness were quantified using texture correlation analysis. The technique of texture correlation analysis was first validated by comparing theoretical simulated strain maps generated from numerically distorted images. The texture correlation algorithm was sensitive to the grid size superimposed on the original image, but remained insensitive to parameters related to the size of the final image mask, which was searched by the correlation algorithm for each grid point of the original image. Within the growth plate, experimental strain distributions were non-uniform in all six specimens. Growth plates were mostly under compression strains. The strain distributions differed among the histomorphological zones of the growth plate, which was most obvious in specimens with regular growth plate shape: higher compressive strains (4-8 times higher than the applied 5% strain) were located mainly in regions overlapping the reserve and hypertrophic zones with lower compressive strains in the proliferative zone. This study documents the non-uniform mechanical behavior of growth plate across its three histological zones when exposed to compression. Further investigation is required to establish the significance of non-uniform strain fields during growth in vivo.     

Instrumentation for diagnostics and control of laser-accelerated proton (ion) beams

Suitable instrumentation for laser-accelerated proton (ion) beams is critical for development of integrated, laser-driven ion accelerator systems. Instrumentation aimed at beam diagnostics and control must be applied to the driving laser pulse, the laser–plasma that forms at the target and the emergent proton (ion) bunch in a correlated way to develop these novel accelerators. This report is a brief overview of established diagnostic techniques and new developments based on material presented at the first workshop on ‘Instrumentation for Diagnostics and Control of Laser-accelerated Proton (Ion) Beams’ in Abingdon, UK. It includes radiochromic film (RCF), image plates (IP), micro-channel plates (MCP), Thomson spectrometers, prompt inline scintillators, time and space-resolved interferometry (TASRI) and nuclear activation schemes. Repetition-rated instrumentati on requirements for target metrology are also addressed.     

Retrofit implementation of Zernike phase plate imaging for cryo-TEM

In-focus phase-plate imaging is particularly beneficial for cryo-TEM because it offers a substantial overall increase in image contrast, without an electron dose penalty, and it simplifies image interpretation. We show how phase-plate cryo-TEM can be implemented with an appropriate existing TEM, and provide a basic practical introduction to use of thin-film (carbon) phase plates. We point out potential pitfalls of phase-plate operation, and discuss solutions. We provide information on evaluating a particular TEM for its suitability.     

水同木次生木质部导管分子观察研究

运用细胞图像分析系统及显微照相方法,观察研究水同木次生木质部导管分子。结果发现,水同木次生木质部导管分子中存在着许多不同的样式,大多数导管分子短而宽,多数无尾或仅一端具尾;导管均为单穿孔板,未发现有侵填体。     

Quantification of Bone Microarchitecture with the Structure Model Index

The deterioration of cancellous bone structure due to aging and disease is characterized by a conversion from plate elements to rod elements. Consequently the terms “rod-like” and “plate-like” are frequently used for a subjective classification of cancellous bone. In this work a new morphometric parameter called Structure Model Index (SMI) is introduced, which makes it possible to quantify the characteristic form of a three-dimensionally described structure in terms of the amount of plates and rod composing the structure. The SMI is calculated by means of three-dimensional image analysis based on a differential analysis of the triangulated bone surface. For an ideal plate and rod structure the SMI value is 0 and 3, respectively, independent of the physical dimensions. For a structure with both plates and rods of equal thickness the value lies between 0 and 3, depending on the volume ratio of rods and plates. The SMI parameter is evaluated by examining bone biopsies from different skeletal sites. The bone samples were measured three-dimensionally with a micro-CT system. Samples with the same volume density but varying trabecular architecture can uniquely be characterized with the SMI. Furthermore the SMI values were found to correspond well with the perceived structure type.     

Rat liver endothelial cells isolated by anti-CD31 immunomagnetic separation lack fenestrae and sieve plates

The gold standard for the identification of sinusoidal endothelial cells (SEC) is the presence of fenestrae organized in sieve plates, which is characteristic of SEC in vivo. One of the methods currently in use to isolate SEC is immunomagnetic sorting for CD31. However, there is evidence to suggest that CD31 is not present on the surface of differentiated SEC. The present study used scanning electron microscopy to image rat hepatic endothelial cells isolated by anti-CD31 and immunomagnetic sorting and cells isolated by gradient centrifugation and centrifugal elutriation. Cells isolated by elutriation had well-developed fenestrae and sieve plates, whereas cells isolated by anti-CD31 and immunomagnetic sorting had significantly fewer fenestrae organized in sieve plates. In conclusion, cells isolated by anti-CD31 and immunomagnetic sorting lacked the hallmark features of SEC.     

洋蒲桃次生木质部中导管分子的解剖学

运用细胞图像分析系统和显微照相的方法对洋蒲桃(Syzygium samarangense)次生木质部导管分子进行了观察研究。次生木质部导管分子类型有: 两端具尾导管、一端具尾导管和无尾导管。导管分子穿孔板存在着4种类型: 两端均为具2个单穿孔的复穿孔板;一端为1个单穿孔板, 另1端为具2个单穿孔的复穿孔板;两端均为单穿孔板;两单穿孔板位于同一端壁两侧相互对应以及一些过渡类型穿孔板。根据观察结果, 分析了各类型穿孔板之间的演化关系。     

Development of automated imaging and analysis for zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens ¹. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates ². In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling ³. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.     

洋蒲桃次生木质部中导管分子的解剖学

运用细胞图像分析系统和显微照相的方法对洋蒲桃（Syzygium samarangense）次生木质部导管分子进行了观察研究。次生木质部导管分子类型有：两端具尾导管、一端具尾导管和无尾导管。导管分子穿孔板存在着4种类型：两端均为具2个单穿孔的复穿孔板;一端为1个单穿孔板,另1端为具2个单穿孔的复穿孔板;两端均为单穿孔板：两单穿孔板位于同一端壁两侧相互对应以及一些过渡类型穿孔板。根据观察结果,分析了各类型穿孔板之间的演化关系。     

Quantification of Bone Microarchitecture with the Structure Model Index

The deterioration of cancellous bone structure due to aging and disease is characterized by a conversion from plate elements to rod elements. Consequently the terms "rod-like" and "plate-like" are frequently used for a subjective classification of cancellous bone. In this work a new morphometric parameter called Structure Model Index (SMI) is introduced, which makes it possible to quantify the characteristic form of a three-dimensionally described structure in terms of the amount of plates and rod composing the structure. The SMI is calculated by means of three-dimensional image analysis based on a differential analysis of the triangulated bone surface. For an ideal plate and rod structure the SMI value is 0 and 3, respectively, independent of the physical dimensions. For a structure with both plates and rods of equal thickness the value lies between 0 and 3, depending on the volume ratio of rods and plates. The SMI parameter is evaluated by examining bone biopsies from different skeletal sites. The bone samples were measured three-dimensionally with a micro-CT system. Samples with the same volume density but varying trabecular architecture can uniquely be characterized with the SMI. Furthermore the SMI values were found to correspond well with the perceived structure type.     

土鳖虫与其混淆品的微性状鉴别研究

为对目前药材市场流通的中药材土鳖虫进行微性状特征研究,探究土鳖虫与其混淆品的区别。实验通过查阅资料,对市场土鳖虫品种进行调查,运用中药微性状鉴定法对土鳖虫的不同部位采用体视显微镜、生物显微镜、扫描仪等仪器进行图像采集,并利用Photoshop CS5软件景深合成高清晰度微性状特征图片来对其进行鉴别研究。实验结果表明,土鳖虫口器、腹背板边缘、肛上板、生殖板、触角、单眼间距与复眼间距比例、单眼、复眼、前足胫节、跗节、爪、尾须、背甲及腹甲刚毛等方面与混淆品有比较明显的区别。     

ClearPEM,un TEP dédié au sein

Conventional breast imaging (mammography, ultrasound, MRI) relies on the analysis of anatomical characteristics. Whole-body positron emission tomography (PET) allows for the acquisition of a metabolic image, yet is limited by its poor spatial resolution. The Crystal Clear Collaboration developed a PET dedicated for breast imaging, the ClearPEM, in order to offer a high-resolution nuclear imaging technique. The patient is installed in the prone position on the exam bed, with two detector plates rotating around to breast to acquire a 3-dimensional image. Two prototypes were built and installed in hospitals. We summarize the technical solutions necessary for the development of this system and present a summary of its performances as well as an outlook on preclinical and clinical tests.     

Bacterial Biofilm Development on Hydroxyapatite-Coated Glass

Glass plates are frequently used as the substratum in flow cell experiments to allow continuous non-destructive observations of biofilm development via microscopy. The aim of this study was to evaluate hydroxyapatite-coated glass as a substratum for flow cell experiments, in comparison to plain glass, for modelling primary colonization of the tooth surface by Streptococcus sanguis. Glass plates were magnetron sputter coated with hydroxyapatite, producing a thin transparent layer. Biofilm development in the flow cell was recorded using image capture from a microscope, and images were analyzed to determine percentage coverage of the substratum over 24 h. Removal of biofilm by increasing the flow rate was also assessed. No statistically significant differences were detected between S. sanguis biofilms grown on the two different substratum materials. Hence, this work supports the proposal that the conditioning film reduces the influence of substratum surface properties.     

Three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea chloroplasts

Two forms of three-dimensional crystals of the light-harvesting chlorophyll a/b protein complex from pea have been obtained. Crystals of one form grew as hexagonal plates measuring up to 150 micron across and 2 to 3 micron in thickness. Electron diffraction patterns of thin hexagonal plates showed sharp reflections to a resolution of 3.7 A on a hexagonal reciprocal lattice. The unit cell in projection (a = 127.0 A) and the symmetry of the diffraction pattern (6 mm) suggested that the hexagonal plates were highly ordered stacks of two-dimensional crystals suitable for structure analysis by electron microscopy and image processing. Crystals of a second form grew as dark green octahedra measuring roughly 0.5 mm across. Low-resolution X-ray diffraction patterns suggested a large cubic unit cell (a = 390 A). SDS/polyacrylamide gel electrophoresis of single octahedral crystals showed the same polypeptide composition as the starting solution, one major band at 24,000 apparent molecular weight and two satellite bands of 23,000 and 23,500 apparent molecular weight.     

Investigation of the effect of combined variations in temperature, pH, and NaCl concentration on nisin inhibition of Listeria monocytogenes and Staphylococcus aureus.

Gradient plates were used to investigate the effects of varying temperature, pH, and sodium chloride (NaCl) concentration on nisin inhibition of Staphylococcus aureus and Listeria monocytogenes, Nisin was incorporated into the plates of 0, 50, 100, 250, and 500 IU ml -1. Gradients of pH (3.7 to 7.92) at right angles to NaCl concentration (2.1 to 7% [wt/vol]) were used for the plates, which were incubated at 20, 25, 30 and 35 degrees C. Growth on the plates were recorded by eye and by image analysis. The presence of viable but nongrowing cells was revealed by transfer to nongradient plates. Lower temperatures and greater NaCl concentrations increased the nisin inhibition of S. aureus synergistically. Increasing the NaCl concentration potentiated the nisin action against L. monocytogenes; the effect of temperature difference was not so apparent. Between pH 7.92 and ca. pH 5, a fall pH appeared to increase nisin's effectiveness against both organisms. At more acid pH values (ca. pH 4.5 to 5), the organisms showed resistance to both nisin and NaCl at 20 and 25 degrees C. Similar results were obtained with one-dimensional liquid cultures.