Cytoskeletal architecture of neuromuscular junction: Localization of vinculin

Cytoskeletons underneath the postsynaptic membrane of neuromuscular junctions were studied by using a quick-freeze deep-etched method and immunoelectron microscopy of ultrathin frozen sections. In a quick-freeze deep-etched replica of fresh, unfixed muscles, 8.9 ± 1.5-nm particles were present on the true postsynaptic membrane surface. Underneath this receptor-rich postsynaptic membrane, networks of fine filaments were observed. These cytoskeletal networks were more clearly observed in extracted samples. In these samples, diameters of the filaments which formed networks were measured. In the platinum replica, three kinds of filament were recognized—12 nm, 9 nm, and 7 nm in diameter. The 12-nm filament seemed to correspond to the intermediate filament. The other two filaments formed meshworks between intermediate filaments and plasma membrane. In ultrathin frozen sections vinculin label was localized just beneath the plasma membrane. Thirty-six percent of the label was within 18 nm from the cytoplasmic side of the plasma membrane and 50% was within 30 nm. Taking the size of the vinculin molecule into account, it was concluded that vinculin is localized just beneath the plasma membrane and might play some role in anchoring filaments which formed meshworks underneath the plasma membrane.     

Ultrahigh vacuum system for advanced tribology studies: Design principles and applications

The main difficulty in designing of an ultrahigh vacuum (UHV) tribometer combined with tribophysical and tribochemical characterization techniques is to find the critical compromise between the scientific requirements and technical or technological limitations from different subsystems and components. The principal conflicts, their possible solutions and the recommended tribometer configurations are analyzed. The developed methodological principles were applied for designing and construction of two UHV experimental tribological systems: TriDes-2 and Ca³UHV. The advances in the design and development of the vacuum system as well as the UHV force sensor and sample holder are presented and discussed.     

Drosophila larval neuromuscular junction: molecular components and mechanisms underlying synaptic plasticity

Understanding the mechanisms that mediate synaptic plasticity is a primary goal of molecular neuroscience. The Drosophila larval neuromuscular junction provides a particularly useful model for investigating the roles of synaptic components in both structural and functional plasticity. The powerful molecular genetics of this system makes it possible to uncover new synaptic components and signaling molecules, as well as their function in the intact organism. Together with the mouse hippocampus and Aplysia dissociated cell culture, the Drosophila larval neuromuscular junction has been among the most valuable model systems for examining the molecular and cellular basis of neuronal plasticity.     

Structural and functional correlates of synaptic transmission in the vertebrate neuromuscular junction

Because vertebrate neuromuscular junctions are readily accessible for experimental manipulation, they have provided a superb model in which to examine and test functional correlates of chemical synaptic transmission. In the neuromuscular synapse, acetylcholine receptors have been localized to the crests of the junctional folds and visualized by a variety of ultrastructural techniques. By using ultrarapid freezing techniques with a temporal resolution of less than 1 msec, quantal transmitter release has been correlated with synaptic vesicle exocytosis at discrete sites called “active zones.” Mechanisms for synaptic vesicle membrane retrieval and recycling have been identified by using immunological approaches and correlated with endocytosis via coated pits and coated vesicles. In this review, available ultrastructural, physiological, immunological, and biochemical data have been used to construct an ultrastructural model of neuromuscular synaptic transmission that correlates structure and function at the molecular level.     

An automatic system for in vitro cell migration studies

This paper describes a system for in vitro cell migration analysis. Adult neural stem/progenitor cells are studied using time-lapse bright-field microscopy and thereafter stained immunohistochemically to find and distinguish undifferentiated glial progenitor cells and cells having differentiated into type-1 or type-2 astrocytes. The cells are automatically segmented and tracked through the time-lapse sequence. An extension to the Chan-Vese Level Set segmentation algorithm, including two new terms for specialized growing and pruning, made it possible to resolve clustered cells, and reduced the tracking error by 65%. We used a custom-built manual correction module to form a ground truth used as a reference for tracked cells that could be identified from the fluorescence staining. On average, the tracks were correct 95% of the time, using our new segmentation. The tracking, or association of segmented cells, was performed using a 2-state Hidden Markov Model describing the random behaviour of the cells. By re-estimating the motion model to conform with the segmented data we managed to reduce the number of tracking parameters to essentially only one. Upon characterization of the cell migration by the HMM state occupation function, it was found that glial progenitor cells were moving randomly 2/3 of the time, while the type-2 astrocytes showed a directed movement 2/3 of the time. This finding indicates possibilities for cell-type specific identification and cell sorting of live cells based on specific movement patterns in individual cell populations, which would have valuable applications in neurobiological research.     

An image processing system for cell behaviour studies in subconfluent cultures

DRIMAPS (digitally recorded interference microscopy with automatic phase-shifting) is a system that we have developed for quantifying the behaviour of cells in subconfluent cultures. The primary data generated by the system consist of phase-shifted interference (PSI) images which are accurate density maps of the distribution of dry mass (non-aqueous material) inside cells. Time-lapse sequences of PSI images may be viewed as movie sequences or processed in various ways to reveal many different aspects of the dynamics of cell growth and motile behaviour. Here we describe the image processing routines that are an integral part of the system and are required for four main functions: 1 initial calculation of the PSI images; 2 compensation of these images for instrumental distortion and instability; 3 identification and tracking of individual cells in a time-lapse sequence of PSI images; 4 extraction of cell behavioural data from a time-lapse sequence of PSI images. The first function converts standard interference microscope images into an image that accurately represents the optical phase-difference introduced by the specimen. The second function recalibrates a sequence of images by taking the cell-free region in each image as a reference plane of zero phase-difference. This is particularly necessary to compensate for the long-term instability of the Horn type of double-beam interference microscope, which has several advantages over other types of interference microscope for studying cell behaviour. The third function compares consecutive images in a sequence in order to trace the identity of individual cells throughout the sequence. Semi-automatic tracking, which allows close interaction with a human operator, is less prone to error than fully automatic tracking. The fourth function automatically extracts dynamic data from the identified cells. These data may include the true mass centroids of cells for translocation analysis and robust morphometric parameters for cell morphology examination. The integrated intensity of a cell is an accurate measure of cell mass and allows the growth (increase in dry mass) of individual cells to be studied. These data may be entered into a relational database of cell behaviour and a rule-based system allows efficient data access and analysis. Experiments with phase-contrast microscopy have revealed that many of these image processing methods are generally useful for cell behaviour studies using more conventional forms of microscopy.     

One-axis on-the-fly laser system development for wide-area fabrication using cell decomposition

We have developed a one-axis on-the-fly system that can continuously process a large area by using a laser scanner. Existing laser processing using scanner systems can be used for precision processing, but have a limitation in the area of processing. To supplement this shortcoming, a step and scanning technique using a stage has been developed. This technique can process a large area in such a way that, after laser processing, the stage moves repetitively; however, this method has a demerit in that processing quality in the part between the scan areas is not even. To solve this problem, there is a method of synchronizing the scanner and the stage. This study developed an on-the-fly system that synchronizes the one-axis stage and the two-axis scanner. Also, in synchronized processing, the velocity of the stage due to the velocity of fabrication is very important. If the velocity of the stage is not proper, the laser processing deviates from the scanner work area. We proposed an algorithm in which, using a cell decomposition method, the velocity of the stage is automatically calculated by compensating for the acceleration/deceleration time occurring in the stage rotation and the reverse rotation due to the velocity of the fabrication. We compared the step and scanning method to the on-the-fly method through a low-frequency experiment and proved that the latter has better precision for the boundary of the scan area; we further analyzed the precision by using a large area marking and an overlapping experiment. In addition, by measuring encoder output signals, a compensation algorithm for the stage acceleration/deceleration time was verified.     

Optimization of culture conditions for an efficient xeno‐feeder free limbal cell culture system towards ocular surface regeneration

Ex vivo expansion of limbal stem cells from a small biopsy and its subsequent transplantation is the golden choice of treatment for limbal stem cell deficiency. Use of murine 3T3 feeder layer is a prerequisite for this ex vivo expansion. There is an ever‐increasing demand for feeder free cultures to avoid xenotoxicity and transmission of xeno‐diseases to human system. This study was aimed to establish an efficient xeno‐feeder free limbal culture system towards ocular surface regeneration. To study the effect of initial dispase treatment and culture system used, migratory distance of cells from explants was analyzed from phase contrast images using “interactive measurements” of Qwin software (Leica). Expression of p63 in different culture systems was studied by immunofluorescent staining, followed by quantitative confocal microscopy (Carl Zeiss). Results showed dispase treatment was not necessary for establishing limbal explant culture. A combination of Iscove's modified Dulbecco's medium and Panserin 801 resulted in formation of autofeeder layer with maintenance of progenitor characteristics, thus mimicking natural tissue architecture. Further analysis of this culture system showed that cells could be cultured till confluency. Immunofluorescent staining of ABCG2 revealed presence of stem cell marker in the confluent cell layer. Scanning Electron Micrographs demonstrated homogenous population of tightly packed cells in this culture system. Replacement of bovine serum with autologous serum did not affect morphology or growth of cells in this culture system. This study will be a major step in the development of xeno‐feeder free epithelial equivalents towards ocular surface reconstruction. Microsc. Res. Tech. 73:1045–1052, 2010. © 2010 Wiley‐Liss, Inc.     

Simple Penning ion source for laboratory research and development applications

A simple Penning ion generator (PIG) that can be easily fabricated with simple machining skills and standard laboratory accessories is described. The PIG source uses an iron cathode body, samarium cobalt permanent magnet, stainless steel anode, and iron cathode faceplate to generate a plasma discharge that yields a continuous 1 mA beam of positively charged hydrogen ions at 1 mTorr of pressure. This operating condition requires 5.4 kV and 32.4 W of power. Operation with helium is similar to hydrogen. The ion source is being designed and investigated for use in a sealed-tube neutron generator; however, this ion source is thoroughly described so that it can be easily implemented by other researchers for other laboratory research and development applications.     

Research and development of VUV optical coatings for micro mirror applications

1Introduction Duringthelastthreedecades,thedevelop mentofpowerfulUVlightsourcessuchasexci merlasers,frequencymultipliedsolidstatela sersorstoragefreeelectronlasershavegathered increasingresearcheffortsinthefieldsofUV photoninteractionwithmatteraswellasrising industrialapplicationssuchasintegratedcircuit manufacturing,microandnano materialpro cessingormedicine.Adominantdrivingforce downtotheshortestwavelengthsinthevacuum ultraviolet(VUV)spectralregionisthesemi conductormanufacturingwiththeopt…     

光谱仪器及其应用的发展和展望

本文以使用最多、覆盖面最广的常规光谱仪器(紫外可见分光光度计-UVS、原子吸收分光光度计-AAS)为代表,综述了光谱仪器及其应用的发展概况;指出了常规光谱仪器的发展方向;提出了光谱仪器发展中应该打破误区、注重可靠性等重要问题。     

Laser interferometer system for metrology and machine tool applications

This paper describes a recently introduced laser measurement system based on a new HeNe laser tube. Design principles of laser tube cavity length control, frequency stability measurements, and the limits to measurement resolution extension will be discussed.     

物理性质测量系统(PPMS)的原理及其应用

本文介绍美国QD公司的PPMS设备的基本原理、测量选项、性能指标、应用和使用中需要注意的问题     

磁性测量仪器(MPMS-XL)的原理及其应用

本文介绍美国QD公司的MPMS设备的基本原理、测量选项、实际应用和使用中需要注意的问题。     

A new method for cell culture on an electron-transparent melamine foil suitable for successive LM,TEM and SEM studies of whole cells

A new cell culture technique is described which is based on the observation that foils cast from the melamine resin hexamethylol-melamine-ether are suitable for the cultivation of beating heart muscle cells and fibroblasts of the rat. This foil can be flamed for sterilization, is about 80 nm in thickness, homogeneous and smooth, withstands dehydration and critical point-drying, can be removed from glass and permits the imaging of whole cells successively by light microscopy, transmission and scanning electron microscopy. The method is capable of narrowing the gap between light and electron microscopy, yielding excellent whole cell preparations in various kinds of microscopic studies to be performed on one and the same cell.     

直动式电液伺服阀的开发与应用

直动式电液伺服阀利用直线力马达直接驱动滑阀工作，提高了抗污染能力和工作可靠性，其性能指标达到喷嘴挡板式电液伺服阀的水平，拓宽了电液伺服阀的应用领域。     

Fibre optic distributed scattering sensing system: perspectives and challenges for high performance applications

As fiber optic distributed scattering sensing systems are providing innovative solutions for the monitoring of large structures, the comparison of different techniques and solutions is difficult because of the lack of standardized specifications and the difficulty associated to the characterization of such systems. The article presents a tentative definition of performance specifications and qualification aiming at providing clear guidelines for their design, procedures applicable to fiber optic distributed sensing systems specifications, qualification, application and selection.     

圆盘压榨机的国产化研制及其应用

针对国内设计的锥盘直径1200mm的圆盘式压榨机,对其结构特点、参数计算及液压系统等问题进行论证,随后对圆盘压榨机这种较为先进的压榨过滤设备的国产化的意义及其应用前景作了分析。对该设备今后几年在PVA行业的需求情况进行了预测,并指出了圆盘压榨机系列化、小型化的研制方向。