Rapid FlAsH labelling in the budding yeast Saccharomyces cerevisiae

Live cell imaging of protein distributions is an essential tool in modern cell biology. It relies on the functional labelling of a host protein with a fluorophore, which may either be a genetically fused fluorescent protein or an organic dye binding to the host protein. The biarsenical-tetracysteine system or 'FlAsH-labelling', is based on the high affinity interaction between a biarsenical probe and a small protein tag. This approach has been successfully used for live cell imaging in the budding yeast Saccharomyces cerevisiae. However, the established labelling protocols require a lengthy overnight incubation of the cells with the dye under tightly controlled growth conditions, which severely limits the use of this approach. In this study, we characterize an efficient method for introducing FlAsH-EDT(2) into live budding yeast cells using standard electroporation. The labelling time is reduced from more than 12 h to less than 1 h without compromising the labelling efficiency or cell viability. This approach may be used for cells in different growth phases or grown under different conditions. It may be further extended to other small high affinity probes, thus opening up new possibilities for labelling in budding yeast.     

General 3D offsetting of a triangular net using an implicit function and the distance fields

A novel approach which uses the distance fields based on cell subdivision and an implicit surface interpolation based on the radial basis function is proposed in order to get an accurate and error-free offset model of arbitrary shapes composed of a large number of triangle meshes. In the method, the space bounding the original model is divided into smaller cells. For the efficient calculation of distance fields, valid cells which will generate a portion of offset model are selected previously by the proposed detection algorithm. These valid cells are divided again into much smaller voxels which assure required accuracy. At each voxel, the distance fields are created by calculating the minimum distances between the corner points of voxels and the triangle meshes. For the more efficient calculation of distance fields, valid vertices among the triangle meshes which will generate minimum distances with current cell are selected by checking the intersection between current cell and a cone prism generated at each vertex of triangle meshes. In addition, a new approach based on an implicit surface interpolation scheme is proposed to perform two types of offsetting operations including uniform and non-uniform offsetting in the same framework. In the method, a smooth implicit surface is generated from the discrete offset distance values given by the user. After generating the whole distance fields, the offset surface was constructed by using the conventional marching cube algorithm together with mesh smoothing scheme. The effectiveness and validity of this new offset method was demonstrated by performing numerical experiments for the various types of triangle meshes.     

基于球面并联机构的换档机械手换档力分析

对基于二自由度非对称直线内副驱动的球面并联机构的换档机械手进行了静力学分析。在建立机构输出力与驱动力之间的映射关系模型的基础上,通过仿真计算得出驱动杆在各档位之间变换时驱动力的变化曲线,分析最大驱动力和外部载荷之间的关系,为实际操作中换档力的控制提供理论方法。     

Combining ocFLIM and FIDSAM reveals fast and dynamic physiological responses at subcellular resolution in living plant cells

For a deeper understanding of molecular mechanisms within cells and for the realization of predictive biology for intracellular processes at subcellular level, quantitative biology is required. Therefore, novel optical and spectroscopic technologies with quantitative and dynamic output are needed in cell biology. Here, we present a combined approach of novel one-chromophore fluorescence lifetime imaging microscopy to probe the local environment of fluorescent fusion proteins and fluorescence intensity decay shape analysis microscopy to suppress interfering autofluorescence. By applying these techniques, we are able to analyse the subcellular localization and partitioning of a green fluorescence protein fusion of the salt stress-induced protein low temperature induced (LTI)6b in great detail with high spatial and temporal resolution in living cells of Arabidopsis plants.     

基于KCCA虚假邻点判别的非线性变量选择

特征变量选择技术是非线性系统建模过程中降低信息冗余和提高精度的有效方法。提出一种结合核典型相关法(kernel canonical correlation analysis,KCCA)与虚假最近邻法的变量选择法。首先引入核方法,将非线性原始数据映射到线性空间,再采用典型相关法有效合理地消除因子之间的多重共线性,受混沌相空间虚假最近邻点法的启示,通过计算原始数据在KCCA子空间中投影的距离,判断其对主导变量的解释能力,由此进行变量的选择。该方法用氢氰酸生产工艺工程中的非线性模型验证,并与全参数模型进行比较,结果显示该方法有良好的变量选择能力。因此,该研究为非线性系统建模的变量选择方法提供了一种新方法。     

A robust forward-displacement analysis of spherical parallel robots

The forward-displacement analysis of spherical parallel robots (SPRs) is revisited. A robust approach, based on the input–output (I/O) equation of spherical four-bar linkages, is proposed. In this approach, the closed-loop kinematic chain of a SPR is partitioned into two four-bar spherical chains, whose I/O equations are at the core of the analysis reported here. These equations lead to a trigonometric equation in the joint angles, which is solved semigraphically to obtain the joint variables for the determination of the moving plate orientation. Examples are included to demonstrate the application of the method.     

On a test bench for studying lubrication in a spherical bearing: simulations and experimental validation

In this paper a test bench dedicated to the analysis of a lubricated spherical pair is presented. The experimental set‐up has been used to validate a numerical code that has been developed by the authors in order to simulate a complex lubricated spherical pair that affects the reliability of a novel continuous variable transmission (CVT). The test bench is modular and can be adapted to other lubricated pairs, either cylindrical or spherical. The stand is equipped with an I/O control card and allows the on‐line acquisition of some important parameters. As for the numerical code, the finite‐difference method has been used to solve the indirect problem in the lubricated spherical pad, under the condition of hydrodynamic regime, with externally pressurized inlets. Two simulators are needed since the physical properties of the test bench and the real system are different. The one that simulates the behavior of the spherical pair in the test stand has been used to ascertain the numeric approach prediction capability. Indeed, the experimental tests gave results which are in very good correspondence with the numerical predictions. Hence, the second simulator, which has been tailored on the particular spherical pair the CVT is equipped with, gained reliability in predicting the behavior of the real system under different load and speed conditions. These predictions have been useful in achieving new insights into the novel CVT, which, under this new light, has been criticized by making the point that its range of application in automotive is, at the actual state of the art, rather limited, in spite of a high intrinsic complexity. Copyright © 2006 John Wiley & Sons, Ltd.     

Six-color segmentation of multicolor images in the infection studies of Listeria monocytogenes

Multiple immunofluorescent staining is a powerful strategy for visualizing the spatial and temporal relationship between antigens, cell populations, and tissue components in histological sections. To segment different cell populations from the multicolor image generated by immunostaining based on color addition theory, a systems approach is proposed for automatic segmentation of six colors. After image acquisition and processing, images are automatically segmented with the proposed approach and six-pseudo channels for individual or colocalized fluorescent dye are generated to distinguish different cell types. The principle of this approach is the classification of each pixel into one of six colors (red, green, blue, yellow, magenta, and cyan) by choosing the minimal angular deviation between the RGB vector of the given pixel and six classically defined edge vectors. In the present infection studies of Listeria monocytogenes, the new multicolor staining methods based on the color addition were applied and the proposed color segmentation was performed for multicolor analysis. Multicolor analysis was accomplished to study the migration and interaction of Listeria and different cell subpopulations such as CD4CD25 double positive T regulatory cells; we also visualized simultaneously the B cells, T cells, dendritic cells, macrophages, and Listeria in another experiment. After Listeria infection, ERTR9 macrophages and dendritic cells formed cluster with Listeria in the infection loci. The principle of color addition and the systems approach for segmentation may be widely applicable in infection and immunity studies requiring multicolor imaging and analysis. This approach can also be applied for image analysis in the multicolor in vivo imaging, multicolor FISH or karyotyping or other studies requiring multicolor analysis.     

一个基于球面纹理映射的车身虚拟原型系统

采用图像镶嵌技术实现三维重建的原理，提出了用于球面纹理映射的矩阵和公式，阐述了系统实现的编程方法，采用新颖的贴图算法，实现了一个基于球面纹理是映射的汽车车身虚拟原型系统，使设计者能依据用户的反馈及时修改设计，提高了设计效率。     

Pushing the resolution limits in cryo electron tomography of biological structures

Cryo electron tomography is a three-dimensional imaging technique that is suitable for imaging snapshots of the structural arrangements of biomolecular complexes and macromolecules, both in vitro and in the context of the cell. In terms of attainable resolution, cryo electron tomographic reconstructions now show resolvable details in the 5-10 nm range, connecting optical microscopy with molecular imaging techniques. In view of the current developments in super-resolution light microscopy and correlative light and electron microscopy, cryo electron tomography will be increasingly important in structural biology as a tool to bridge light microscopy with molecular imaging techniques like NMR, X-ray diffraction and single particle electron microscopy. In cell biology, one goal, often referred to as visual proteomics, is the molecular mapping of whole cells. To achieve this goal and link cryo electron tomography to these high-resolution techniques, increasing the attainable resolution to 2-5 nm is vital. Here, we provide an overview of technical factors that limit the resolution in cryo electron tomography and discuss how during data acquisition and image processing these can be optimized to attain the highest possible resolution. Also, existing resolution measurement approaches and current technological developments that potentially increase the resolution in cryo electron tomography are discussed.     

Microwave cavities for vapor cell frequency standards

In this paper, we report an analysis of the design criteria of microwave cavities for vapor cell frequency standards. Two main geometries exploited in those devices are considered: the cylindrical cavity, used, for example, in the coherent population trapping maser and in the pulsed optically pumped (POP) clock, and the spherical cavity used in the isotropically laser cooled clock. The cavity behavior is described through a lumped equivalent circuit in which the input coupling loop, the dielectric cell containing the atoms and the diodes for frequency tuning or Q control are taken into account. In particular, the effect of the cell on the cavity resonance frequency is analytically evaluated via a first-order perturbation approach. The theory is found in good agreement with the experiments performed with two different cylindrical cavities used for the POP clock; the model here developed can then be helpful in the design of the cavity system. The general principles here reported can be adapted to other standards, such as atomic fountains and hydrogen masers, and to other modes and/or geometries.     

Soft X‐ray spectromicroscopy for speciation,quantitation and nano‐eco‐toxicology of nanomaterials

There is a critical need for methods that provide simultaneous detection, identification, quantitation and visualization of nanomaterials at their interface with biological and environmental systems. The approach should allow speciation as well as elemental analysis. Using the intrinsic X‐ray absorption properties, soft X‐ray scanning transmission X‐ray spectromicroscopy (STXM) allows characterization and imaging of a broad range of nanomaterials, including metals, oxides and organic materials, and at the same time is able to provide detailed mapping of biological components. Thus, STXM offers considerable potential for application to research on nanomaterials in biology and the environment. The potential and limitations of STXM in this context are discussed using a range of examples, focusing on the interaction of nanomaterials with microbial cells, biofilms and extracellular polymers. The studies outlined include speciation and mapping of metal‐containing nanomaterials (Ti, Ni, Cu) and carbon‐based nanomaterials (multiwalled carbon nanotubes, C₆₀ fullerene). The benefits of X‐ray fluorescence detection in soft X‐ray STXM are illustrated with a study of low levels of Ni in a natural river biofilm.     

一种新的红细胞沉降率测定方法

血红细胞沉降率是临床诊断中需测试的一项重要参数 ,传统的魏氏法是一种手工测量方法且测试周期长 ,不适应大批量测试。运用映射函数和回归分析原理 ,提出了一种新的测试方法 ,该方法测试时间是魏氏法的一半、与魏氏法的相关性好。使用该方法构成的测试仪器经临床使用 ,测试稳定 ,效果良好。     

Formation of machine groups and part families in cellular manufacturing systems using a correlation analysis approach

The important step in the design of a cellular manufacturing (CM) system is to identify the part families and machine groups and consequently to form manufacturing cells. The scope of this article is to formulate a multivariate approach based on a correlation analysis for solving cell formation problem. The proposed approach is carried out in three phases. In the first phase, the correlation matrix is used as similarity coefficient matrix. In the second phase, principal component analysis (PCA) is applied to find the eigenvalues and eigenvectors on the correlation similarity matrix. A scatter plot analysis as a cluster analysis is applied to make simultaneously machine groups and part families while maximizing correlation between elements. In the third stage, an algorithm is improved to assign exceptional machines and exceptional parts using respectively angle measure and Euclidian distance. The proposed approach is also applied to the general group technology (GT) problem in which exceptional machines and part are considered. Furthermore, the proposed approach has the flexibility to consider the number of cells as a dependent or independent variable. Two numerical examples for the design of cell structures are provided in order to illustrate the three phases of proposed approach. The results of a comparative study based on multiple performance criteria show that the present approach is very effective, efficient and practical.     

A modified phasor approach for analyzing time-gated fluorescence lifetime images

Fluorescence lifetime imaging is a versatile tool that permits mapping the biochemical environment in the cell. Among various fluorescence lifetime imaging techniques, time-correlated single photon counting and time-gating methods have been demonstrated to be very efficient and robust for the imaging of biological specimens. Recently, the phasor representation of lifetime images became popular because it provides an intuitive graphical view of the fluorescence lifetime content of the images and, when used for global analysis, significantly improves the overall S/N of lifetime analysis. Compared to time-correlated single photon counting, time gating methods can provide higher count rates (～10 MHz) but at the cost of truncating and under sampling the decay curve due to the limited number of gates commonly used. These limitations also complicate the implementation of the phasor analysis for time-gated data. In this work, we propose and validate a theoretical framework that overcomes these problems. This modified approach is tested on both simulated lifetime images and on cells. We demonstrate that this method is able to retrieve two lifetimes from time gating data that cannot be resolved using standard (non-global) fitting techniques. The new approach increases the information that can be obtained from typical measurements and simplifies the analysis of fluorescence lifetime imaging data.     

Methods to calibrate and scale axial distances in confocal microscopy as a function of refractive index

Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive‐index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal microscopy remains cumbersome, although several high‐end methods exist. In this paper we present two methods to calibrate axial distances in 3D confocal microscopy that are both accurate and easily implemented. With these methods, we measured axial scaling factors as a function of refractive‐index mismatch for high‐aperture confocal microscopy imaging. We found that our scaling factors are almost completely linearly dependent on refractive index and that they were in good agreement with theoretical predictions that take the full vectorial properties of light into account. There was however a strong deviation with the theoretical predictions using (high‐angle) geometrical optics, which predict much lower scaling factors. As an illustration, we measured the PSF of a correctly calibrated point‐scanning confocal microscope and showed that a nearly index‐matched, micron‐sized spherical object is still significantly elongated due to this PSF, which signifies that care has to be taken when determining axial calibration or axial scaling using such particles.     

Zur konstruktion der sphärischen wendekurve

It is a well-known result of instantaneous spherical kinematics that the locus of those points of the moving sphere, whose paths have a vanishing geodesic curvature, is a curve w on a cubic cone Ω with vertex in the center O of the sphere. In this paper we give a simple construction of the inflection curve w using the following theorem: The intersecting curve l of the inflection cone Ω with a sphere κ, which is centered on the pole-axis p and contains the point O, lies on a cylinder of revolution. This cylinder contains the inflection circle of that planar motion in the tangent plane τ of κ in the pole P = p ∩ κ (P ≠ O), whose relationship between the points and centers of curvature of their paths is induced in τ by the spherical motion. Furthermore we use this result to draw some geometrical conclusions on the set of the ∞¹ inflection curves belonging to a given canonical frame. In a special case the inflection curve is a spherical trochoid.     

Low-energy foil aberration corrector

A spherical and chromatic aberration corrector for electron microscopes is proposed, consisting of a thin foil sandwiched between two apertures. The electrons are retarded at the foil to almost zero energy, so that they can travel ballistically through the foil. It is shown that such a low-voltage corrector has a negative spherical aberration for not too large distances between aperture and foil, as well as a negative chromatic aberration. For various distances the third- and fifth-order spherical aberration coefficients and the first- and second-order chromatic aberration coefficients are calculated using ray tracing. Provided that the foils have sufficient electron transmission the corrector is able to correct the third-order spherical aberration and the first-order chromatic aberration of a typical low-voltage scanning electron microscope. Preliminary results show that the fifth-order spherical aberration and the second-order chromatic aberration can be kept sufficiently low.     

A guided tour into subcellular colocalization analysis in light microscopy

It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well‐characterized markers. However, image‐analysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object‐based approach.     

From 2D slices to 3D volumes: image based reconstruction and morphological characterization of hippocampal cells on charged and uncharged surfaces using FIB/SEM serial sectioning

3D imaging at a subcellular resolution is a powerful tool in the life sciences to investigate cells and their interactions with native tissues or artificial objects. While a tomographic experimental setup achieving a sufficient structural resolution can be established with either X-rays or electrons, the use of electrons is usually limited to very thin samples in transmission electron microscopy due to the poor penetration depths of electrons. The combination of a serial sectioning approach and scanning electron microscopy in state of the art dual beam experimental setups therefore offers a means to image highly resolved spatial details using a focused ion beam for slicing and an electron beam for imaging. The advantage of this technique over X-ray μCT or X-ray microscopy attributes to the fact that absorption is not a limiting factor in imaging and therefore even strong absorbing structures can be spatially reconstructed with a much higher possible resolution. This approach was used in this study to elucidate the effect of an electric potential on the morphology of cells from a hippocampal cell line (HT22) deposited on gold microelectrodes. While cells cultivated on two different controls (gold and polymer substrates) did show the expected stretched morphology, cells on both the anode and the cathode differed significantly. Cells deposited on the anode part of the electrode exhibited the most extreme deviation, being almost spherical and showed signs of chromatin condensation possibly indicating cell death. Furthermore, EDX was used as supplemental methodology for combined chemical and structural analyses.