Simple procedures for the transfer of grid images onto glass coverslips for the rapid relocation of cultured cells

Procedures are described whereby electron microscope-grid images may be transferred onto the surface of glass coverslips. By using coordinate grids, patterns are obtained that are ideal for the rapid relocation of cultured cells. Two basic procedures are presented. In the first, metal is evaporated, in a vacuum unit, onto glass coverslips carrying electron microscope grids to produce an exposed, glass replica of the grid pattern. The grid pattern is then etched in the glass surface with hydrofluoric acid and the metal subsequently dissolved away. In the second method silicone monoxide is evaporated onto coverslips carrying a ‘negative grid’ or a negative grid image, to produce a pattern directly visible in the phase contrast or interference microscope. Modifications for scanning electron microscopy are also described.     

Parameters affecting specimen flatness of two-dimensional crystals for electron crystallography

The flatness of two-dimensional (2D) crystals on the support film is a critical factor in protein electron crystallography. The influence of the carbon support film and of different grid makes and materials on flatness was investigated, using as a criterion the sharpness of diffraction spots perpendicular to the tilt axis of electron diffraction patterns of purple membrane tilted in the microscope at 45 degrees. In a quantitative test, carbon film that had been evaporated without sparks forming gave a much larger proportion of flat crystals than "sparked" carbon. Titanium grids were superior to copper, probably because they introduce less cryo-crinkling of the carbon film when the sample is cooled to liquid nitrogen temperature, as their thermal expansion coefficient is closer to that of carbon. While the molybdenum grids from Plano were unsuitable for data collection because of their tendency of break the carbon, molybdenum grids from Pacific GridTech gave a much larger yield of flat crystals than the titanium grids. Scanning electron microscope images of the grids as supplied by the manufacturer showed that the Plano grids had very narrow and irregular grid bars, while the Pacific GridTech grids were very smooth with a large surface-to-hole ratio.     

A technique for studying one and the same section of a cell in sequence with the light and electron microscope

Methods for mounting and staining relatively thin sections on electron microscope grids, in order that one and the same section of a cell can be photographed in sequence with the light and electron microscope are described. Toluidine blue is used as a stain and hexachlorabuta-1,3 diene as a medium which enables the grid carrying the stained sections to be temporarily mounted under a coverslip and examined with an oil-immersion lens. Results obtained with pollen mother cells of Fritillaria lanceolata at zygotene are illustrated.     

An improved hole grid pattern for the relocation of specimens in the electron microscope

An electron-microscope grid with circular holes arranged in a pattern indicating the orientation of the central area to the edge is described. The grids allow regions observed in a transmission electron microscope to be relocated easily or identified in lower resolution instruments.     

Behaviour of TEM metal grids during in-situ heating experiments

The stability of Ni, Cu, Mo and Au transmission electron microscope (TEM) grids coated with ultra-thin amorphous carbon (α-C) or silicon monoxide film is examined by in-situ heating up to a temperature in the range 500–850 °C in a transmission electron microscope. It is demonstrated that some grids can generate nano-particles either due to the surface diffusion of metal atoms on amorphous film or due to the metal evaporation/redeposition. The emergence of nano-particles can complicate experimental observations, particularly in in-situ heating studies of dynamic behaviours of nano-materials in TEM. The most widely used Cu grid covered with amorphous carbon is unstable, and numerous Cu nano-particles start to form once the heating temperature reaches 600 °C. In the case of Ni grid covered with α-C film, a large number of Ni nano-crystals occur immediately when the temperature approaches 600 °C, accompanied by the graphitization of amorphous carbon. In contrast, both Mo and Au grids covered with α-C film exhibit good stability at elevated temperature, for instance, up to 680 and 850 °C for Mo and Au, respectively, and any other metal nano-particles are detected. Cu grid covered Si monoxide thin film is stable up to 550 °C, but Si nano-crystals appear under intensive electron beam. The generated nano-particles are well characterized by spectroscopic techniques (EDXS/EELS) and high-resolution TEM. The mechanism of nano-particle formation is addressed based on the interactions between the metal grid and the amorphous carbon film and on the sublimation of metal.     

Fabrication of a versatile substrate for finding samples on the nanometer scale

With increasing interest in nanometer scale studies, a common research issue is the need to use different analytical systems with a universal substrate to relocate objects on the nanometer scale. Our paper addresses this need. Using the delicate milling capability of a focused ion beam (FIB) system, a region of interest (ROI) on a sample is labelled via a milled reference grid. FIB technology allows for milling and deposition of material at the sub 20-nm level, in a similar user environment as a standard scanning electron microscope (SEM). Presently commercially available transmission electron microscope (TEM) grids have spacings on the order 100 μm on average; this technique can extend this dimension down to the submicrometre level. With a grid on the order of a few micrometres optical, FIBs, TEMs, scanning electron microscopes (SEMs), and atomic force microscopes (AFM) are able to image the ROI, without special chemical processes or conductive coatings required. To demonstrate, Au nanoparticles of ∼ 25 nm in size were placed on a commercial Formvar^®- and carbon-coated TEM grid and later milled with a grid pattern. Demonstration of this technique is also extended to bulk glass substrates for the purpose of sample location. This process is explained and demonstrated using all of the aforementioned analytical techniques.     

SPARK,a kernel of software programs for spatial reconstruction in electron microscopy

SPARK, an acronym for ‘SPAtial Reconstruction Kernel’, is the nucleus of a software library being developed for the three-dimensional (3-D) reconstruction of objects observed by the electron microscope. A unifying concept is used: the Fourier transform, known in several central sections, is resampled to obtain a 3-D Cartesian grid, which is inverted by a fast Fourier transform. This technique is used for both single-axis tilting (of 2-D periodic layers or of isolated objects) and for the random conical-tilt technique. The principles that make it possible to recover Cartesian grids in the two different geometries are illustrated and some preliminary results are reported. SPARK resamples the Cartesian grids with the use of a fast and efficient algorithm of Shannon interpolation developed by the authors. Compared to back-projection techniques the method shows a considerable improvement in execution time with no sacrifice in accuracy; it therefore allows the effects of a variety of parameters in a given reconstruction to be scrutinized in a reasonable time. Some new possibilities and future extensions of the library are briefly outlined.     

Coating sections for electron microscopic autoradiography: a stripping technique using liquid emulsion.

A coating technique for electron microscope autoradiography is described which combines the advantage of forming an emulsion film by a dipping method with the ease of coating sections already on grids. Sections are coated so that a formvar support film separates the section and the emulsion crystals. This intermediate layer of formvar ensures a random distribution of the emulsion crystals. Using light gold sections, Ilford L-4 emulsion and Microdol-X development, the resolution of this technique, as determined by the half distance method, was 150 nm. The additional layer of formvar slightly reduced the image quality with biological samples in the electron microscope. This technique has a minimal loss of resolution and image quality for moderate resolution electron microscope autoradiography.     

A simple method for whole-cell preparation in electron microscopy

A simple method for whole-cell preparation without using gold or platinum grids as substrata for culture is described. Cells were cultured on formvar film over round pores, each 3 mm in diameter, of Thermonox coverslips. The cells on the formvar coated coverslip are fixed, stained, dehydrated in situ, and introduced into a critical point drying apparatus. A small quantity of 0.2% mesh-cement is applied to slot grids, and they are laid onto the formvar film over the pores of the coverslip. After the grids are removed from the plastic substratum, they are ready for observation under the electron microscope.     

A method for the rapid removal of sugars and salts from virus preparations on electron microscope grids

An elution method for the rapid removal of solutes from virus preparations mounted on carbon coated electron microscope grids. Other uses of the method, including negative staining, are noted.     

A method for providing comparative counts of small particles in electron microscopy

A method for estimating the relative concentrations of particles by electron microscopy is described. The method involves the determination of the area of the field of view in the microscope binoculars, relative to a standard area, and is independent of grid type, magnification, operator or microscope. Particle counts using this method are in close agreement with counts made from electron micrographs. The method is simple, rapid and reproducible, and, for low numbers of particles, more accurate than conventional methods.     

A simple method for holding electron microscope grids during autoradiography of serial sections

A simple, reliable, and inexpensive method is described for holding electron microscope grids during the application of photographic emulsion and during subsequent storage and processing. This method has proved to be especially useful in autoradiographic studies using serial sections.     

A porous carbon film for the collection of atmospheric aerosol for transmission electron microscopy

This paper describes a method of direct sampling of fine urban aerosol using porous carbon films, to increase collection efficiency and retain the structural integrity of the aerosol. A nuclepore membrane filter was coated with carbon and washed with chloroform to leave a clean porous carbon film on a 200-mesh transmission electron microscope copper grid. The grid was attached to a clean nuclepore filter using carbon cement. Sampling was conducted for 1 h at 1.9 L min⁻¹ flow rate. The porous carbon grid with sample was then assayed using transmission electron microscopy, and negatives were suitable for image analysis. The collection of fine airborne particles using this method is appropriate for electron microscopy, with a minimum of interference to the structure of collected particles.     

A versatile multi-specimen holder for processing and critical point drying of materials for examination in the scanning electron microscope (SEM)

A specimen holder has been designed specifically for use in the Polaron E.3000 critical point drier (CPD) and is capable of drying up to twenty different specimens within a size range of 4.5 mm to 30 μm by utilization of a variable grid system. The principle, however, could be employed in designs for most other critical point dryers. A large number of designs have been produced for handling small specimens during preparation and CPD procedures prior to observation in the scanning electron microscope (SEM). In a great number of cases these have been specific to one particular organism or preparation method. Marchant (1973) suggested culturing organisms on Millipore filters which could then be used in conjunction with a plain filter as a sieve mechanism to contain the specimens, the complete unit being housed in a BEEM embedding capsule. Scott et al. (1973) produced another design, again utilizing the BEEM capsule together with 100 grade copper mesh as the specimen retaining section. Both these designs were flexible in that different grade filters and meshes could be employed. The disadvantage of using BEEM capsules was noted by Taylor (1975) in that the chemical plasticizers present in the capsule material could leak out in the presence of substitution solvents. To overcome this he produced an all metal design for a container employing a similar type of grid system as in this multispecimen holder. His design was for a single chamber specimen holder with a maximum specimen thickness of 1 mm and was such that the complete chamber could be placed directly into the SEM. The need for a multispecimen holder arose when large numbers of specimens, each specific in its preparation required critical point drying without tedious repetition of critical point drying runs. It was necessary to consider the inflexible features of the design. These were, the external dimensions of the container, which had to fit within the existing aluminium specimen-holder of the Polaron E.3000 CPD, and the dimensions of the transmission electron microscope (TEM) grids used as the specimen retainers. Transmission electron microscope grids are available in two sizes, 3.01 mm and 2.3 mm, with a large range in patterns and materials to choose from to suit most types of specimen preparation. From these fixed dimensions a design was drawn up which allowed specimens of up to 4.5 mm across to be prepared and yet still gave the large number of individual chambers required. The construction of the holder can be seen, from the drawing (Fig. 1) and the photographs (Fig. 2) to be comprised of large dimension chambers with a TEM grid housed at each end to contain the specimens and yet still allow the free flow of dehydrating and substitution agents. The complex arrangement of screws was necessary to facilitate the assembly and use of a container which has a separate lid section that can be dismantled to allow different grids to be inserted depending on the dimensions of the material under preparation. The specimens are supported on the lower grid system which can also be varied and for the ease of removal of larger specimens the chamber section divides leaving the specimens readily accessible in the lower half. Where the specimens are very small, the chamber section can be completely removed by carefully removing all screws and then screwing a stud down the centre thread and extracting the chamber section leaving all the specimens supported on their grids on the base plate. These can easily be transferred directly onto the SEM stub and secured either by double-sided sellotape or careful application of an adhesive such as Durafix. It has even been found that discs punched out of Visking tubing can be used in place of the TEM grids to provide a finer sieve mechanism. It was noted, however, that the tubing was hardened by substitution solvents but this still did not seem to impair the results as satisfactory preparations of Penicillium expansum sporangiophores and pollen of Dactylis glomeratus have been achieved (Medi-Cine, 1976). The container has been made out of high quality brass because of its good machining properties necessary when such fine work has to be carried out. The metric design utilizes standard milling cutters with the inclusion of the ?th cutter to produce satisfactory grid housings allowing free movement to ensure that they always settle on the base plate. The versatility of the design can be further increased with the production of two accessory structures (see Fig. 1). The first, simply produced by cutting brass tubing, is for the preparation of small numbers of specimens per chamber, and ensures that the specimens are deposited onto the grid. The holes drilled through the tube wall allow easy removal with fine forceps. The second structure simply partitions the large chambers increasing the capacity to eighty specimens where such a large specific separation is required. The production of artifacts resulting from specimen handling is reduced considerably with this specimen holder. Once the specimens have been loaded, all the processing stages can be carried out on all the material at once and the specimens are ready for mounting prior to observation.     

The grid-cell-culture technique: the direct examination of virus-infected cells and progeny viruses

We describe a method for the structural analysis and identification of viruses, without purification or concentration steps which could alter virus morphology. Virus-infected cells grown on carbon-Parlodion-coated electron microscope grids release large numbers of progeny viruses which adsorb to the surface of the grid and are revealed by negative staining. The technique is rapid, sensitive and can be used at three levels. (1) Negative staining of whole cell preparations revealed both extracellular and intracellular viruses or nucleocapsids beneath the plasma membrane; (2) non-ionic detergent extraction of cells infected with certain viruses reveals cytoskeleton-associated, virus-specific structures normally only observed after thin sectioning; (3) cultures prepared by either procedure are suitable for colloidal gold immunological studies. Extracellular and cytoskeletal-associated viruses were heavily and specifically labelled with gold. The results indicate that the technique may be used to rapidly identify unknown viruses on the basis of size, topography, morphology and mode of maturation from the infected cell, as well as the presence of characteristic intracellular cytoskeletal-associated structures. The technique also has potential use in the sero-grouping and sero-typing of viruses with specific monoclonal antibodies.     

Indirect measurement of high grid strip densities over Nyquist sampling rate based on the moiré pattern analysis for quality assurance in grid manufacturing

We have recently developed very precise carbon-interspaced antiscatter grids having a wide range of grid strip densities (i.e., 85–234 lines/in.) by adopting the sawing process, depending on the demands of specific imaging modalities. However, because grid strips of the recently developed grids are mostly invisible through X-ray nondestructive testing due to their high strip densities, quality assurance in the manufacturing has not yet satisfactorily made. In this work, we proposed a pragmatic method to measure actual grid strip densities over the Nyquist sampling rate based on the moiré pattern analysis. We prepared several samples of the carbon-interspaced grids having nominal grid strip densities of 85, 103, 130, 150, 178, 203, 215, and 234 lines/in. and measured their strip densities by using the proposed method to demonstrate its viability assessment. The differences between the nominal and the measured grid strip densities were less than about 1.8%, indicating the proposed method is useful for the quality assurance in grid manufacturing.     

Topography and thickness of air-dried human platelets measured by correlative transmission and scanning electron microscopy.

Human platelets rapidly air-dried on carbon-coated grids were examined by transmission and scanning electron microscopy. Whole cell mounts were photographed in a transmission electron microscope (TEM), coated with gold, and then examined in a scanning electron microscope (SEM). The thickness of the cytoplasm towards the centre of the cells was estimated to be 20-40 nm, and the rim of dense material surrounding the cells was 40 nm thick. Some dense bodies stood out as much as 100 nm above the dried cytoplasm. These measurements are important for evaluating cytoplasmic volume during microprobe analyses of air-dried platelet preparations.     

轴流风扇扇叶网格的自动生成

利用著名CFD软件STAR-CD的PROSTAR网格产生规则,在选定扇叶中心线的基础上,以翼厚、攻角、投影半径、前倾角、安装角为参数,提出一种快速生成轴流风扇扇叶六面体结构网格的方法,大大降低了数值分析所用的时间。     

基于网格包络的工业机器人仿真碰撞检测算法

为提高工业机器人在复杂作业环境下的碰撞检测效率,提出了一种网格包络的碰撞检测算法,以大量等尺寸的立方体网格来包络模型本身,并在网格内部建立网格子模型的AABB树结构。该算法在建模过程中将网格的空间坐标进行有序存储,在遍历阶段可快速搜索到相交的网格,之后遍历网格内部的树结构来进一步判断模型是否碰撞。该算法网格内部的子模型几何数据量远小于整体模型几何数据量,其网格内的检测速度远快于以整体模型建模的传统层次包围盒方法的检测速度。实验结果表明,在大型复杂模型碰撞检测仿真中,该算法在不同网格数量下的检测效率比传统的Solid算法的检测效率快数倍到数十倍。     

Nanoparticle embedded chitosan film for agglomeration free TEM images

Transmission electron microscopy (TEM) is a very useful and commonly used microscopy technique, used especially for the characterization of nanoparticles. However, the identification of the magnetic nanoparticle could be thought problematic in TEM analysis, due to the fact that the magnetic nanoparticles are usually form aggregates on the TEM grid to form bigger particles generating higher stability. This prevents to see exact shape and size of each nanoparticle. In order to overcome this problem, a simple process for the formation of well‐dispersed nanoparticles was conducted, by covering chitosan film on the unmodified copper grid, it was said to result in aggregation‐free TEM images. It is also important to fix the magnetic nanoparticles on the TEM grids, due to possible contamination of TEM filament which is operated under high vacuum conditions. The chitosan film matrix also helps to protect the TEM filament from contact with magnetic nanoparticles during the imaging process. The proposed procedure offers a quick method to fix the nanoparticles in a conventional copper TEM grid and chitosan matrix prevents agglomeration of nanoparticles, and thus getting TEM images showing well‐dispersed individual nanoparticles.