Production and Cleaning of Freeze-Etch Replicas which Show Complementary Surfaces of Fractured Fungus Spores and Hyphae

London finder grids (H-2) and 3 mm copper discs were flared on opposite sides by use of a die and punch. The grids were registered on the discs and attached with vaseline. The specimen was squeezed through the grid apertures by the 2 copper discs. The complete sandwich was frozen in freon 22 and placed in a hinged specimen holder which fractured the layer of the specimen between the 2 finder grids. Platinum-carbon and carbon replicas were made immediately after fracturing. After drying the replicas, the vaseline was removed by chloroform, and replicas were cleaned with 70% H₂SO₄ for 1-18 hr at 52 C, followed by 6% commercial sodium hypochlorite (Chlorox) for 1-2 hr at 23-25 C.     

Freeze Fracture of Intact Plant Tissues

A procedure for dissolving and handling replicas of cutinized leaves and other plant tissues is described. This technique yields comparatively large replicas which can include vascular tissue, epidermal cells and the cuticle. Dissolution of the tissue involves the sequential use of alcoholic KOH, sulfuric acid and chromic acid. The use of clean, uncoated grids for transfer of the tissue and replicas results in rapid, easy handling with a minimum of breakage or loss. The procedure is more efficient than previous methods and produces large replicas. This allows tissue orientation and more positive identification of cell types for better correlation of freeze fracture results with thin sections of similar material.     

A Simple Method for Handling Grids

A simple method for handling electron microscope grids is described here. While assuring their identification and safety, this method provides improved handling, temporary storage, and identification of grids bearing ultra-thin sections, and in addition, provides a novel method for preparing bulk samples. Grids are attached at their edges to the weakly adhesive surface of a “Post-it” note pad which sits in a petri dish. The grids are safely immobilized on the pad, classified based on their location, and identified by convenient pad notation. Grid manipulation and identification are simplified using this device, which is easily assembled from readily available and inexpensive materials.     

A Disrupting Factor in Silver Staining Techniques

Single-stage surface replicas of treated or fresh pollen grains can be made ready for the electron microscope in 1.5 hr. The microspores are discharged into a drop of 50% acetone on a 1 cm square of cleaved mica and air dried. Carbon is evaporated to a film thickness of 35 mμ during rotation of the mica support. The carbon film and microspores are parted from the mica with water and heated in 2-aminoethanol at 145-155 C for 10 min to 3 hr. The replicas are then washed 5 min or longer on water at 90 C and picked up on electron microscope grids. The resulting self-shadowed surface replica can be immediately observed by electron microscopy.     

Single-Stage Carbon Replicas of Microspores

Single-stage surface replicas of treated or fresh pollen grains can be made ready for the electron microscope in 1.5 hr. The microspores are discharged into a drop of 50% acetone on a 1 cm square of cleaved mica and air dried. Carbon is evaporated to a film thickness of 35 mμ during rotation of the mica support. The carbon film and microspores are parted from the mica with water and heated in 2-aminoethanol at 145-155 C for 10 min to 3 hr. The replicas are then washed 5 min or longer on water at 90 C and picked up on electron microscope grids. The resulting self-shadowed surface replica can be immediately observed by electron microscopy.     

The Ultramicrotome Superstage: A Versatile Aid for Section Manipulation

The design and properties of a rigid, box-like device to be placed on the knife stage of a Porter-Blum MT-2 ultramicrotome are described. This “superstage” acts as a hand and tool rest, and a wind screen for the knife boat. The superstage easily permits tight rafts of ultrathin sections to be centrally located on grids. Additionally, it aids in placing 1 μm thick sections at the same relative location on glass slides to facilitate their examination in the light microscope.     

Rapid estimation of length distributions of microtubule preparations by quasi-elastic light scattering

A new method for determining the length distribution of microtubule preparations, using quasi-elastic light scattering (QELS) is described. The experimental electric field autocorrelation functions are analyzed using a closed-form expression recently described by Hallett, Craig, and Nickel [(1985) Biopolymers 24 , 947–960], which is incorporated into an exponential sampling procedure. The resulting length distributions are compared with those obtained for the same samples with electron microscopy (EM). If standard grid-preparation procedures were used, the EM results yielded shorter length distributions than QELS. When grids were prepared at lower microtubule number densities, less grid washing was required. In these cases, excellent agreement between the EM and QELS techniques were achieved.     

FitEM2EM--tools for low resolution study of macromolecular assembly and dynamics

Studies of the structure and dynamics of macromolecular assemblies often involve comparison of low resolution models obtained using different techniques such as electron microscopy or atomic force microscopy. We present new computational tools for comparing (matching) and docking of low resolution structures, based on shape complementarity. The matched or docked objects are represented by three dimensional grids where the value of each grid point depends on its position with regard to the interior, surface or exterior of the object. The grids are correlated using fast Fourier transformations producing either matches of related objects or docking models depending on the details of the grid representations. The procedures incorporate thickening and smoothing of the surfaces of the objects which effectively compensates for differences in the resolution of the matched/docked objects, circumventing the need for resolution modification. The presented matching tool FitEM2EMin successfully fitted electron microscopy structures obtained at different resolutions, different conformers of the same structure and partial structures, ranking correct matches at the top in every case. The differences between the grid representations of the matched objects can be used to study conformation differences or to characterize the size and shape of substructures. The presented low-to-low docking tool FitEM2EMout ranked the expected models at the top.     

A New Imprinting Material for Scanning Electron Microscopic Studies

A new imprinting material, “Ther-mocool”, has been used for preparing negative replicas of plant as well as metal surfaces for SEM study. The technique has wide application and gives very good results.     

Movement behaviour of woodland rodents: looking from beyond small trapping grids

Live-trapping of rodents was conducted over a 12-ha plot on unevenly spaced trap lines, with inner lines forming a 3.5-ha grid of closely spaced traps. This design was used to estimate the probability that bank volesClethrionomys glareolus (Schreber, 1780) and yellow-necked miceApodemus flavicollis (Melchior, 1834) trapped in small grids are true residents rather than visitors from the surrounding area. On average, 12% of the voles and 19% of the mice were trapped within and beyond the grid in the same trapping sessions. As these were mainly wide-ranging males moving over the plot, home ranges of males may be underestimated on small grids. In total, 36% of voles and 39% of mice that were marked in spring-summer were trapped at least once in their life beyond the grid. Typically, these were individuals shifting home ranges and migrating to or from the grid. The size of lifetime ranges of rodents was significantly larger than temporary home ranges and may therefore be underestimated on small grids. “Single--capture” individuals were mainly true transients rather than visitors. Only 12% of voles and 15% of mice resided on the plot for longer time than in the grid.     

Greedy replica exchange algorithm for heterogeneous computing grids

Replica exchange molecular dynamics (REMD) has become a valuable tool in studying complex biomolecular systems. However, its application on distributed computing grids is limited by the heterogeneity of this environment. In this study, we propose a REMD implementation referred to as greedy REMD (gREMD) suitable for computations on heterogeneous grids. To decentralize replica management, gREMD utilizes a precomputed schedule of exchange attempts between temperatures. Our comparison of gREMD against standard REMD suggests four main conclusions. First, gREMD accelerates grid REMD simulations by as much as 40 %. Second, gREMD increases CPU utilization rates in grid REMD by up to 60 %. Third, we argue that gREMD is expected to maintain approximately constant CPU utilization rates and simulation wall-clock times with the increase in the number of replicas. Finally, we show that gREMD correctly implements the REMD algorithm and reproduces the conformational ensemble of a short peptide sampled in our previous standard REMD simulations. We believe that gREMD can find its place in large-scale REMD simulations on heterogeneous computing grids.

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Graphical Abstract Standard replica exchange molecular dynamics (REMD) typically requires all replicas to complete prior to initiation of the replica exchange protocol. Greedy REMD decentralizes this process and therefore only requires a replica and its predetermined exchange partner to have finished simulations prior to initiating replica exchange. Because greedy REMD reduces the idle time associated with replica exchange tasks, it becomes particularly well suited for performing REMD on heterogeneous distributed computing environments.

     

基于卷积运算的城市公园绿地聚集度评价

为使公园绿地聚集度计算能够充分反映其辐射效应,提出基于卷积运算的局部网格单元和整个城市公园绿地聚集度评价方法。基于高分辨率遥感影像获取山东省37个主要园林城市公园绿地分布图,利用GIS技术,采用500 m网格将城区网格化;建立3×3绿地聚集度卷积模板,基于卷积运算计算城市网格单元公园绿地聚集度;选择评价因子,依据37个城区公园绿地网格单元聚集度分布现状确定其分级值,建立整个城市公园绿地聚集度评价模型,并对东营市和泰安市进行实例评价。研究结果显示:基于卷积运算的网格单元公园绿地聚集度计算方法能够有效量化相邻网格单元绿地的辐射效应,计算由网格内部及相邻区域绿地共同作用产生的绿地聚集度,其取值范围为0—4;整个城市公园绿地聚集度分为1级(极弱)、2级(弱)、3级(中等)、4级(强)、5级(极强)共5个等级,评价结果与研究区37个城市绿地现状相对应。实例评价结果显示,东营市网格单元公园绿地聚集度主要分布在0—0.2之间,整个城市公园绿地聚集度为2级;泰安市网格单元公园绿地聚集度以0为主,整个城市公园绿地聚集度为5级。     

Grid alignment in entorhinal cortex

The spatial responses of many of the cells recorded in all layers of rodent medial entorhinal cortex (mEC) show mutually aligned grid patterns. Recent experimental findings have shown that grids can often be better described as elliptical rather than purely circular and that, beyond the mutual alignment of their grid axes, ellipses tend to also orient their long axis along preferred directions. Are grid alignment and ellipse orientation aspects of the same phenomenon? Does the grid alignment result from single-unit mechanisms or does it require network interactions? We address these issues by refining a single-unit adaptation model of grid formation, to describe specifically the spontaneous emergence of conjunctive grid-by-head-direction cells in layers III, V, and VI of mEC. We find that tight alignment can be produced by recurrent collateral interactions, but this requires head-direction (HD) modulation. Through a competitive learning process driven by spatial inputs, grid fields then form already aligned, and with randomly distributed spatial phases. In addition, we find that the self-organization process is influenced by any anisotropy in the behavior of the simulated rat. The common grid alignment often orients along preferred running directions (RDs), as induced in a square environment. When speed anisotropy is present in exploration behavior, the shape of individual grids is distorted toward an ellipsoid arrangement. Speed anisotropy orients the long ellipse axis along the fast direction. Speed anisotropy on its own also tends to align grids, even without collaterals, but the alignment is seen to be loose. Finally, the alignment of spatial grid fields in multiple environments shows that the network expresses the same set of grid fields across environments, modulo a coherent rotation and translation. Thus, an efficient metric encoding of space may emerge through spontaneous pattern formation at the single-unit level, but it is coherent, hence context-invariant, if aided by collateral interactions.     

A simple and reliable method for the localization in cell culture of single identifiable cells for ultrastructural analyses

A simple method for relocating single cells in monolayer cultures for subsequent morphological or ultrastructural analysis is reported. This consists of producing, on the culture dish surface, a nontoxic carbon grid that is preserved during processing for either transmission (TEM) or scanning (SEM) electron microscopy. For TEM studies these grids are readily transferred along with the cells into the embedding plastic, and thus individual grid squares containing a cell(s) of interest can be quickly located, remounted, and sectioned. These grids may be useful for ultrastructural analyses of single cells previously studied electrophysiologically or after microinjection of macromolecules.     

Can demographic parameters be estimated from capture-recapture data on small grids? A test with forest rodents

This preliminary study proposes small trapping grids as an alternative to traditional large grids for the simultaneous monitoring of several rodent populations by capture--recapture. Monthly trapping sessions of wood mouse Apodemus sylvaticus (Linnaeus, 1758) and bank vole Clethrionomys glareolus (Schreber, 1780) were carried on over a small area (0.015 ha, 21 traps). The coherence of demographic parameter estimates on such small grids with those obtained on classical large grids was checked by performing two trapping sessions on a larger grid (0.9 ha, 110 traps, 10 m mesh) surrounding the small grid. We compared the two grid designs on the basis of sex ratio upon first capture, trap saturation rate, minimum number alive (MNA), monthly survival, and trappability. These demographic parameters proved to be non-biased by the trapped area, even though the precision was lower on the small grid. Small grids seem therefore to give the same picture of population dynamics as classical large grids except for parameters sensitive to an edge effect (eg density). By decreasing significantly the trapping effort, small grids will be of particular interest whenever the simultaneous operation of several trapping grids is needed (eg to compare different environmental conditions).     

Enclosure design and space utilization by Indian leopards (Panthera pardus) in four zoos in Southern India

Enclosure design and the use of enclosure space influence the activity budget of captive leopards. The study laid out in grids all enclosures on the base map and segregated these grids into 4 zones. Every 5 min, the study recorded the proportion of time spent in these zones with the leopards' behavior. Captive leopards most frequently used the “edge ”zone. Almost all leopards used the edge zone for stereotypic pacing, the “back ”zone for resting, and the “other ”zone for activity. The study positively correlated the proportion of time spent in the “enriched ”zone with activity levels exhibited by leopards housed in some enclosures and with resting in others. Thus, the study segregated structural objects in the enriched zone into activity-related features (e.g., logs) and rest-related features (e.g., trees and sleeping platforms). Compared with individuals housed in barren enclosures, leopards housed in structurally enriched on-exhibit enclosures exhibited higher levels of activity. Enclosure design was found to be an important factor influencing the welfare of leopards in captivity.     

Automatic registration of microarray images. II. Hexagonal grid

MOTIVATION: In the first part of this paper the author presented an efficient, robust and completely automated algorithm for spot and block indexing in microarray images with rectangular grids. Although the rectangular grid is currently the most common type of grouping the probes on microarray slides, there is another microarray technology based on bundles of optical fibers where the probes are packed in hexagonal grids. The hexagonal grid provides both advantages and drawbacks over the standard rectangular packing and of course requires adaptation and/or modification of the algorithm of spot indexing presented in the first part of the paper. RESULTS: In the second part of the paper the author presents a version of the spot indexing algorithm adapted for microarray images with spots packed in hexagonal structures. The algorithm is completely automated, works with hexagonal grids of different types and with different parameters of grid spacing and rotation as well as spot sizes. It can successfully trace the local and global distortions of the grid, including non-orthogonal transformations. Similar to the algorithm from part I, it scales linearly with the grid size, the time complexity is O(M), where M is total number of grid points in hexagonal grid. The algorithm has been tested both on CCD and scanned images with spot expression rates as low as 2%. The processing time of an image with about 50 000 hex grid points was less than a second. For images with high expression rates ( approximately 90%) the registration time is even smaller, around a quarter of a second. Supplementary information: http://fleece.ucsd.edu/~vit/Registration_Supplement.pdf     

Demography of the western harvest mouse,Reithrodontomys megalotis,in eastern Kansas

Summary Reithrodontomys megalotis was live-trapped on three open field grids in eastern Kansas from August 1979 to August 1982. One grid was a control on which normal demography was monitored, and two were experimental grids where periodic removal of residents allowed the investigation of the demographic and fitness consequences of emigration. Popullations on the control grid showed an annual cycle in numbers, reaching peaks in density during the winter of each year, falling to low densities during the summer. Low summer densities were attributed, at least partially, to low trappability of R. megalotis during periods of high resource abundance. Reproduction was initiated in the spring of each year at approximately the same time as the emergence of new vegetative growth, and ceased in late fall of each year. The trappable population was composed almost entirely of adults, and the sex ratio was skewed significantly toward females. A statistically significant negative association between the number of M. ochrogaster residents and the reproductive activity of female R. megalotis residents was found. A canonical correlation analysis revealed a common seasonal component to the demography of the two species, and possible interspecific affects. Emigrating R. megalotis were a nonrandom sample of the population, with emigrants more likely to be subadult and juvenile males when compared to residents. No association was detected between the numbers of M. ochrogaster colonizing the removal grid and the numbers of R. megalotis colonizing the same removal grid, or between the number of M. ochrogaster residents on the control grid and the numbers of R. megalotis colonizing the removal grids. However, the number of R. megalotis residents on the control grid is positively correlated with the number of R. megalotis on the removal grids.     

A grid‐based method for sampling and analysing spatially ambiguous plants

Abstract. Spatial data can provide much information about the interrelations of plants and the relationship between individuals and the environment. Spatially ambiguous plants, i.e. plants without readily identifiable loci, and plants that are profusely abundant, present non‐trivial impediments to the collection and analysis of vegetation data derived from standard spatial sampling techniques. Sampling with grids of presence/absence quadrats can ameliorate much of this difficulty. Our analysis of 10 fully‐mapped grassland plots demonstrates the applicability of the grid‐based approach which revealed spatial dependence at a much lower sampling effort than mapping each plant. Ripley's K‐function, a test commonly used for point patterns, was effective for pattern analysis on the grids and the gridded quadrat technique was an effective tool for quantifying spatial patterns. The addition of spatial pattern measures should allow for better comparisons of vegetation structure between sites, instead of sole reliance on species composition data.     

Signal-to-noise ratio improvements using anti-scatter grids with different object thicknesses and tube voltages

PurposeThe appropriate object thickness to start using anti-scatter grids (grids) has not sufficiently investigated in previous studies, and thus we rigorously investigated the effectiveness of two generally used grids with grid ratios of 6 and 10 (G6 and G10) for different 50–200 mm thicknesses at tube voltages of 60–100 kV.MethodsAcrylic phantoms with 30 × 30 cm² and different thicknesses were used to measure the signal-to-noise ratio improvement factors (SIFs) of grids. To evaluate the infants’ conditions, field sizes of 225, 400, and 625 cm² were also evaluated at 60–80 kV. In addition, the signal difference-to-noise ratio (SDNR) was used to evaluate tube voltage dependencies of grids for each thickness.ResultsSIF values exceeded 1.0 for ≥70 mm thicknesses and mostly exceeded 1.07 for the 100 mm thickness with 400 cm² field size corresponding to a 1-year-old infant abdomen. The estimated dose reduction capabilities for a 1-year-old infant were approximately 15% using G10 at 70 and 80 kV. The tube voltage dependencies for grid use was almost not prominent for all conditions tested, except for some conditions that are not clinically realistic.ConclusionsG6 and G10 can improve SNR for  ≥100 mm thickness. The results from this work demonstrate approximately 15% dose reduction or image quality improvements at the same dose level for the use of G6 and G10 grids for 100 mm thickness, traditionally excluded from the recommended grid use conditions.