A simple,straightforward correlative live‐cell‐imaging‐structured‐illumination‐microscopy approach for studying organelle dynamics

Most cellular organelles are highly dynamic and continuously undergo membrane fission and fusion to mediate their function. Documenting organelle dynamics under physiological conditions, therefore, requires high temporal resolution of the recording system. Concurrently, these structures are relatively small and determining their substructural organization is often impossible using conventional microscopy. Structured Illumination Microscopy (SIM) is a super resolution technique providing a two‐fold increase in resolution. Importantly, SIM is versatile because it allows the use of any fluorescent dye or protein and, hence, is highly applicable for cell biology. However, similar to other SR techniques, the applicability of SIM to high‐speed live cell imaging is limited. Here we present an easy, straightforward methodology for coupling of high‐speed live cell recordings, using spinning disk (SD) microscopy, with SIM. Using this simple methodology, we are able to track individual mitochondrial membrane fission and fusion events in real time and to determine the network connectivity and substructural organization of the membrane at high resolution. Applying this methodology to other cellular organelles such as, ER, golgi, and cilia will no doubt contribute to our understanding of membrane dynamics in cells. Microsc. Res. Tech. 78:777–783, 2015. © 2015 Wiley Periodicals, Inc.     

Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope

Raster image correlation spectroscopy (RICS) is a new and novel technique for measuring molecular dynamics and concentrations from fluorescence confocal images. The RICS technique extracts information about molecular dynamics and concentrations from images of living cells taken on commercial confocal systems. Here we develop guidelines for performing the RICS analysis on an analogue commercial laser scanning confocal microscope. Guidelines for typical instrument settings, image acquisition settings and analogue detector characterization are presented. Using appropriate instrument/acquisition parameters, diffusion coefficients and concentrations can be determined, even for highly dynamic dye molecules in solution. Standard curves presented herein demonstrate the ability to detect protein concentrations as low as ～ 2 nM. Additionally, cellular measurements give accurate values for the diffusion of paxillin‐enhanced‐green fluorescent protein (EGFP), an adhesion adaptor molecule, in the cytosol of the cell and also show slower paxillin dynamics near adhesions where paxillin interacts with immobile adhesion components. Methods are presented to account for bright immobile structures within the cell that dominate spatial correlation functions; allowing the extraction of fast protein dynamics within and near these structures. A running average algorithm is also presented to address slow cellular movement or movement of cellular features such as adhesions. Finally, methods to determine protein concentration in the presence of immobile structures within the cell are presented. A table is presented giving guidelines for instrument and imaging setting when performing RICS on the Olympus FV300 confocal and these guidelines are a starting point for performing the analysis on other commercial confocal systems.     

Specific labeling of connexin43 in NRK cells using tyramide-based signal amplification and fluorescence photooxidation

Imaging of gap junction proteins, the connexins, has been performed in tissue culture cells both by labeling of connexins with immunocytochemical tags and by cloning and expressing chimeras of connexins and fluorescent proteins such as Green Fluorescent Protein. These two approaches have been used to gain information about protein localization or trafficking at light microscopic resolution. Electron microscopy provides higher resolution; however, analysis of electron micrographs of unlabeled connexins has been generally limited to recognition of gap junction structures. Immunolabeling of gap junction proteins in whole cells at the electron microscopic level has been difficult to achieve because of the fixation sensitivity of most gap junction antibodies. To obtain reasonable sensitivity, immunoperoxidase procedures are typically employed, and these suffer from relatively poor resolution. Here we describe the combination of tyramide signal amplification techniques and fluorescence photooxidation for higher resolution immunolocalization studies for correlative light and electron microscopic imaging. By using correlative microscopy, we can not only localize connexin pools or structures, but also discover what other cellular substructures interact with gap junction proteins. The use of tyramide signal amplification techniques is necessary to increase fluorescence levels that have decreased due to increased specimen fixation required to maintain cell ultrastructure. The fluorescence photooxidation technique provides a high-resolution method for staining of proteins in cells. Unlike colloidal gold-based methods, fluorescence photooxidation allows for three-dimensional localization using high-voltage electron microscopy.     

Tracking protein dynamics with photoconvertible Dendra2 on spinning disk confocal systems

Understanding the dynamic properties of cellular proteins in live cells and in real time is essential to delineate their function. In this context, we introduce the Fluorescence Recovery After Photobleaching‐Photoactivation unit (Andor) combined with the Nikon Eclipse Ti E Spinning Disk (Andor) confocal microscope as an advantageous and robust platform to exploit the properties of the Dendra2 photoconvertible fluorescent protein (Evrogen) and analyse protein subcellular trafficking in living cells. A major advantage of the spinning disk confocal is the rapid acquisition speed, enabling high temporal resolution of cellular processes. Furthermore, photoconversion and imaging are less invasive on the spinning disk confocal as the cell exposition to illumination power is reduced, thereby minimizing photobleaching and increasing cell viability. We have tested this commercially available platform using experimental settings adapted to track the migration of fast trafficking proteins such as UBC9, Fibrillarin and have successfully characterized their differential motion between subnuclear structures. We describe here step‐by‐step procedures, with emphasis on cellular imaging parameters, to successfully perform the dynamic imaging and photoconversion of Dendra2‐fused proteins at high spatial and temporal resolutions necessary to characterize the trafficking pathways of proteins.     

含自重载荷的功能梯度材料结构时域动力学拓扑优化设计

提出了一种含自重载荷的功能梯度材料（FGM）结构时域动力学拓扑优化设计方法。在固体各向同性材料惩罚（SIMP）框架下,提出了一种针对FGM-SIMP的结构自重载荷分布策略。以FGM结构动柔度最小为优化目标、以结构体积为约束,建立了动力学结构优化列式。基于伴随法,在时域内进行了灵敏度推导,并用移动渐近线方法进行求解。通过二维和三维典型数值算例系统研究了含自重载荷下FGM结构的拓扑优化设计问题,并深入探讨了自重载荷和材料梯度分布方向对结构优化结果的影响,发现自重载荷和材料梯度分布方向对FGM结构的优化构型和动刚度具有很大影响。最后,以均一材料（FGM的特例）为例,通过数值仿真和实验测试方法验证了所提方法的有效性,并证实了所提方法可有效提高结构的固有频率和结构动刚度。     

虚拟轴机床定位方法

提出一种基于改进的多传感器一致性数据融合的虚拟轴机床定位方法,在虚拟轴机床动平台的位姿测量中使用了视觉技术与电子罗盘,解决了虚拟轴机床动平台上的主轴定位问题。此方法将虚拟轴机床各根杆上的编码器信息、视觉信息与动态惯性的测量数据进行数据融合,经过动态计算获得置信距离的关系矩阵,虚拟轴机床动平台的位置与姿态的精确定位得到了保证。经过一个单轴单方向的运动测量简化试验,将虚拟轴机床动平台6自由度位姿的测量作为研究对象,对其进行仿真试验。试验结果也表明了该定位方法有效可行。     

NONLINEAR DYNAMICS OF LATERAL MICRO-RESONATOR INCLUDING VISCOUS AIR DAMPING

The nonlinear dynamics of the lateral micro-resonator including the air damping effect is researched. The air damping force is varied periodically during the resonator oscillating, and the air damp coefficient can not be fixed as a constant. Therefore the linear dynamic analysis which used the constant air damping coefficient can not describe the actual dynamic characteristics of the mi-cro-resonator. The nonlinear dynamic model including the air damping force is established. On the base of Navier-Stokes equation and nonlinear dynamical equation, a coupled fluid-solid numerical simulation method is developed and demonstrates that damping force is a vital factor in micro-comb structures. Compared with existing experimental result, the nonlinear numerical value has quite good agreement with it. The differences of the amplitudes (peak) between the experimental data and the results by the linear model and the nonlinear model are 74.5% and 6% respectively. Nonlinear nu-merical value is more exact than linear value and the method can be applied in other mi-cro-electro-mechanical systeme (MEMS) structures to simulate the dynamic performance.     

Spatial kinematics and virtual prototype modeling of Bucyrus shovel

In this study, a 3D model of the shovel motion based multi-body system dynamics in the Automatic Dynamic Analysis of Mechanical Systems (ADAMS) environment was developed. The kinematics model of the shovel was modeled as a multi-body system, which comprises the lower, upper, and the attachment mechanisms. The Euler–Lagrangian equations were employed as the computational framework to describe the dynamic behavior of the model. A virtual prototype of the shovel was built in the ADAMS software environment. This environment allows the visualization of a 3D motion of the general mechanical system and the prediction of interference of the moving components of the shovel. The numerical analysis and animation of digging, swinging, and dumping motions for the shovel were performed. The superimposed display of the deployment for the shovel in three phases allows a detailed examination of the dynamic interference among assembly components. This research provides a solid foundation for further shovel dynamics performance studies.     

Dynamic responses of structures to moving bodies using combined finite element and analytical methods

This paper presents a technique using combined finite element and analytical methods for determining the dynamic responses of structures to moving bodies. In previous work (Comput. Struct., submitted), moving masses were treated as moving loads, ignoring inertia effects. This is not always reasonable and the technique described here allows inertia effects to be included in the analysis. In order to illustrate the methodology, and for validation purposes, the technique is first applied to a clamped–clamped beam subjected to a single mass moving along the beam. Finally, it is applied to the problem that initiated the work: to predict the dynamic response of an experimental mobile gantry crane structure due to the two-dimensional motion of the trolley.     

Fracture analysis of composites by time independent moving-crack orthotropic XFEM

Time-independent orthotropic enrichment functions are introduced for dynamic propagation analysis of moving cracks in composites by the extended finite element method (XFEM). The proposed enrichment functions are derived from the analytical solutions for a moving/propagating crack in orthotropic media, and can be considered as a new extension to the available XFEM techniques for dynamic analysis of stationary and moving cracks in orthotropic materials. They are included within the framework of partition of unity and XFEM to enhance the accuracy of basic FEM solution near a moving crack tip in orthotropic media. The method allows for analysis of the whole crack propagation pattern on an unaltered finite element mesh, which is independently defined from the existence of any predefined crack or its propagation path. A combination of dynamic crack initiation toughness and crack orientation along the maximum circumferential stress is used to design a relatively simple and efficient formulation. Dynamic stress intensity factors (DSIFs) are evaluated by means of the domain separation integral method and the dynamic energy release rate. The time dependent XFEM equations are constructed by discretizing the standard weak formulation of the governing elastodynamics equation. They are solved by the unconditionally stable Newmark time integration scheme. A number of benchmark and test problems are simulated and the results are compared with the available reference results to illustrate the accuracy and efficiency of the proposed scheme.     

Algorithms for automated characterization of cell populations in thick specimens from 3-D confocal fluorescence microscopy data

Methods are presented for the automated, quantitative and three-dimensional (3-D) analysis of cell populations in thick, essentially intact tissue sections while maintaining intercell spatial relationships. This analysis replaces current manual methods which are tedious and subjective. The thick sample is imaged in three dimensions using a confocal scanning laser microscope. The stack of optical slices is processed by a 3-D segmentation algorithm that separates touching and overlapping structures using localization constraints. Adaptive data reduction is used to achieve computational efficiency. A hierarchical cluster analysis algorithm is used automatically to characterize the cell population by a variety of cell features. It allows automatic detection and characterization of patterns such as the 3-D spatial clustering of cells, and the relative distributions of cells of various sizes. It also permits the detection of structures that are much smaller, larger, brighter, darker, or differently shaped than the rest of the population. The overall method is demonstrated for a set of rat brain tissue sections that were labelled for tyrosine hydroxylase using fluorescein-conjugated antibodies. The automated system was verified by comparison with computer-assisted manual counts from the same image fields.     

An integrated CAE system for dynamic stress and fatigue life prediction of mechanical systems

This paper presents computer simulation methodology for dynamic stress time history computation to predict the fatigue life of machine components using flexible multi-body dynamics. A hybrid method which employes stress superposition as a function of constraint loads and component accelerations that are predicted by flexible body dynamic simulation is utilized and implemented using established codes. A system integration methodology for dynamic stress computation of mechanical system components is described to provide a usable environment for an engineer. It uses a database management system such as the IAC and the established dynamics and finite element analysis codes.     

移动质量简支梁耦合时变系统建模与实验设计

建立了移动质量简支梁耦合时变系统的动力学模型,通过数值仿真分析了移动质量速度及加速度对耦合时变系统模态参数的影响,得到移动质量诱导产生的附加阻尼。设计并搭建移动质量简支梁实验系统,通过参考实验得到实验系统的初始阻尼,并分别采用频域和时域模态参数辨识方法对质量块不同移动速度下的实验系统进行辨识。结果表明,所建立动力学模型能够对移动质量问题进行准确描述,实验系统可为时变结构动力学分析的理论研究提供实验支持,特别是对时变结构模态参数辨识方法进行实验验证。     

The plant endoplasmic reticulum: an organized chaos of tubules and sheets with multiple functions

The endoplasmic reticulum is a fascinating organelle at the core of the secretory pathway. It is responsible for the synthesis of one third of the cellular proteome and, in plant cells, it produces receptors and transporters of hormones as well as the proteins responsible for the biosynthesis of critical components of a cellulosic cell wall. The endoplasmic reticulum structure resembles a spider-web network of interconnected tubules and cisternae that pervades the cell. The study of the dynamics and interaction of this organelles with other cellular structures such as the plasma membrane, the Golgi apparatus and the cytoskeleton, have been permitted by the implementation of fluorescent protein and advanced confocal imaging. In this review, we report on the findings that contributed towards the understanding of the endoplasmic reticulum morphology and function with the aid of fluorescent proteins, focusing on the contributions provided by pioneering work from the lab of the late Professor Chris Hawes.     

作大范围运动柔性梁的一种碰撞动力学求解方法

研究作大范围运动柔性梁的碰撞动力学问题。针对梁碰撞前、碰撞过程、碰撞后三个阶段,分别建立各阶段的动力学方程。基于柔性多体系统刚柔耦合动力学理论,建立梁无碰撞时的刚柔耦合动力学方程。结合冲量动量法和接触约束法提出一种碰撞动力学求解方法,先以冲量动量法实现运动转换得到协调的碰撞初始条件,再以接触约束法求解碰撞过程。导出系统的刚柔耦合碰撞动力学方程,给出了接触、分离判据,实现三个不同阶段的转换与动力学求解。编制仿真软件,对实例进行动力学仿真,得到系统不同阶段的动力学响应。研究表明,所提碰撞求解方法可以较方便地用于计算柔性多体系统的碰撞问题。梁的柔性、大范围运动及碰撞效应相互耦合,碰撞行为对于柔性多体系统碰撞过程和碰撞后的全局动力学性态均产生较大的影响。     

Morphological filter improve the efficiency of automated tracking of secretory vesicles with various dynamic properties

Membrane trafficking is a very important physiological process involved in protein transport, endocytosis, and exocytosis. The functions of vesicles are strongly correlated with various spatial dynamic properties of vesicles, including their types of movements and morphology. Several methods are used to quantify such dynamic properties, but most of them are specific to particular populations of vesicles. We previously developed the so-called PTrack system for quantifying the dynamics of secretory vesicles near the cell surface, which are small and move slowly. To improve the system performance in quantifying large and fast-moving vesicles, we firstly combined morphological filter with two-threshold image processing techniques to locate granules of various sizes. Next, Kalman filtering was used to improve the performance in tracking fast-moving and large granules. Performance evaluation by using simulation image sequences shown that the new system, called PTrack II, yields better tracking accuracy. The tracking system was validated using time-lapse images of insulin granules in βTC3 cells, which revealed that PTrack II could track better than PTrack, averaged accuracy up to 56%. The overall tracking results indicate that PTrack II is better at tracking vesicles with various dynamic properties, which will facilitate the acquisition of more-complete information on vesicle dynamics. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc.     

Motion-enhanced, differential interference contrast (MEDIC) microscopy of moving vesicles in live cells: VE-DIC updated

Video-enhanced differential interference contrast microscopy with background subtraction has made visible many structures and processes in living cells. In video-enhanced differential interference contrast, the background image is stored manually by defocusing the microscope before images are acquired. We have updated and improved video-enhanced differential interference contrast by adding automatic generation of the background image as a rolling average of the incoming image stream. Subtraction of this continuously updated 12-bit background image from the incoming 12-bit image stream provides a flat background which allows the contrast of moving objects, such as vesicles, to be strongly enhanced while suppressing stationary features such as the overall cell shape. We call our method MEDIC, for motion-enhanced differential interference contrast. By carrying out background subtraction with 12-bit images, the number of grey levels in the moving vesicles can be maximized and a single look-up table can be applied to the entire image, enhancing the contrast of all vesicles simultaneously. Contrast is increased by as much as a factor of 13. The method is illustrated with raw, background and motion-enhanced differential interference contrast images of moving vesicles within a neurite of a live PC12 cell and a live chick motorneuron.     

Dynamics analysis of novel parallel manipulator with one central rotational actuator and four translational actuators

A dynamics analysis of a novel parallel manipulator with one central rotational actuator and four translational actuators is conducted. A 3D model of the parallel manipulator is constructed and its characteristics and DoF are analyzed. The kinematics formulae are derived for solving the displacement, velocity and acceleration of the moving links. The dynamics formulae are derived for solving the inertial wrench of the moving links, the dynamic active forces along the active limbs, the dynamic active torque applied on a central active leg, and the dynamic constrained force exerted on the central active leg. A theoretical numerical example is given to solve the kinematics and dynamics solutions, and the theoretical solutions are verified by the simulation mechanism in Matlab. Finally, a reachable workspace of the novel parallel manipulator is constructed using CAD variation geometry.