The use of a digitizing frame store in microscopy

A digitizing frame store giving real time read out of the image for TV or slow scan rates is now available. This equipment has a number of applications in both optical and electron microscopy. Examples of results obtained by linkage to a low light level TV system are given showing that use of the frame store yields image information not otherwise available. Other possibilities of the system are indicated.     

Image registration in electron microscopy: Application of a robust method

The geometric registration of two electron microscopic images generally is performed by maximizing the cross-correlation coefficient between them. We show that a new similarity measure (the number of sign changes) is useful for performing simultaneously geometric and gray-level registration. This method is robust, which means that it provides a good estimation of the parameters even in the presence of outliers that cannot be described by the registration model.     

Real time computer simulation of electron microscope images with tilted illumination: Grain boundary applications

Computer programs have been developed to simulate electron microscope images from digitized graphically represented model structures. Via a television rate image processing system, these programs allow real time, interactive modification of the microscope objective lens parameters, incident beam inclination, and incident beam energy. In addition to explaining the computational methods, the need for using tilted beam illumination is explored to extend microscope resolution. For this study, the subject of grain boundary imaging is analyzed for a copper σ = 5,36.9°,(310) tilt boundary with a [001] common rotation axis. The Cu {200} lattice spacings of ～1.8Å on both sides of the interface cannot be reliably resolved under axial illumination conditions in a 200 kV microscope. Therefore, either tilted beam modes or higher incident beam energies were explored and the types of image features correlated with atomic position data through the digital frame store system.     

影像诊断图象的实时硬件处理方法

本文介绍的实时硬件处理方法，能够实现逼真模拟心脏和血流动态过程的显示；消除背景噪声的影响；并能分辨人眼不能分辨的灰度细节。适用于小型或便携式影像诊断或探测设备     

Measurement of electron probe beam diameter by digital image processing

The present report illustrates a computerized method for precise measurement of the diameter of an electron beam. The value of this measurement extends beyond simply providing an accurate estimate of resolution. Other salient areas which will benefit include quantitative X-ray microanalysis, energy loss spectroscopy, diffraction studies, and electron beam lithography. The biological sciences as well as the material sciences will gain enormously from improved accuracy in measurement (control) of beam diameter. It is anticipated that most or all of the mathematical manipulations outlined in this paper will be incorporated into digital electronic packages which will perform the functions automatically for setting the electron beam diameter to the scientist's choice. The purpose of the present report is to indicate some of the principles involved so that as electron microscopy becomes more computerized and automated, the user will have some understanding of what the electronics are doing rather than simply depressing a button or two and ignoring the power of what resides within the walls of the instrument. The performance of a scanning electron microscope (SEM) and a scanning transmission electron microscope (STEM) is roughly determined by the incident electron probe beam size (diameter) involving a sufficient electron current. In the present paper, the diameter of an ultrafine electron beam is measured indirectly from the information given by the blurring of an edge in a STEM or a SEM image of a crystalline specimen with fine, sharp edges. The obtained data were processed by digital image processing methods which give an accurate value of the beam diameter. For confirming the validity of this method, a suitable simulation based on the convolution theorem was performed. By using this measurement, we could measure the diameter of an ultrafine electron beam down to 2 nm, which could not be measured easily by previous techniques.     

Evaluation of high-resolution electron microscopy as a method for studying Y-Ba-Cu-O superconductors

High-resolution transmission electron microscopes operating at 300 and 400 kV were used to investigate the crystallography and microstructure of the perovskitelike YBa₂Cu₃O_7−x. In this paper, we evaluate the performance attainable with these microscopes both empirically and by computer modelling. Based upon the assumption that oxygen may be a key to superconductivity properties, we have also investigated the visibility of the oxygen sites as well as the heavier yttrium and barium ion positions and the lighter Cu atom positions. We propose a scheme for observing different twin orientations in these structures and hence the oxygen atom positions seen in projection for the [100] and [010]. Our observations of both thick and thin regions of Y-Ba-Cu-O materials are reported as well as the problems of adjusting microscope parameters and specimen alignment to obtain interpretable images. We also give a preliminary report on the effects of heat treatment as seen in high-resolution micrographs to assess disorder of the heavy atoms and oxygen vacancies.     

Investigation of cholesterol,bilirubin, and protein distribution in human gallstones by color cathodoluminescence scanning electron microscopy and transmission electron microscopy

The application of color cathodoluminescent scanning electron microscopy (CCL-SEM) for qualitative luminescence analysis of cholesterol, bilirubin, and protein in human gallstones was demonstrated. Images of these deposits (cholesterol, bilirubin, and protein) were formed in real colors (blue—cholesterol, red, orange—bilirubin, yellow, green—protein) in accordance with the cathodoluminescent spectrum for each control material. The other method described for transmission electron microscopy (TEM) of ultra-thin sections provides more detailed characterization of the ultrastructure of cholesterol-containing regions and their spatial interrelations with bilirubin-containing regions. Using CCL-SEM combined with TEM permits the receipt of more complete information about the chemical composition and ultrastructure of gallstones and may lead to more effective understanding of the pathogenesis of cholesterol cholelithiasis.     

A general model for resolution of digital holographic microscopy

For digital holographic microscopic imaging, the resolution in the reconstructed image is one of the most important parameters. To optimize the lateral resolution, a general model for the resolution of digital holographic microscopy (DHM) is proposed in this work, in which the effects of the sizes of each pixel, total area of the charge coupled device (CCD) and the microscopic objective lens are taken into account. Comparison between our model and others was carried out by calculating the point spread function (PSF) of DHM at different reconstruction distances and with different fill factors. It is shown that the effect of fill factors on the resolution of DHM becomes significant when the reconstruction distance is long. For high resolution DHM imaging the influence of fill factors must be taken into account when estimating the resolution of the reconstructed image. Furthermore, It is also demonstrated that the sidelobe of PSF can be cut effectively choosing appropriate values of the fill factors. Finally, the reconstructions of polyethylene microspheres have been implemented to demonstrate the theoretical analysis. These results obtained are helpful for estimation of the resolution and design of the DHM systems.     

A Stopped-flow/rapid-freezing machine with millisecond time resolution to prepare intermediates in biochemical reactions for electron microscopy

We have developed an instrument capable of freezing transient intermediates in rapid biochemical reactions for subsequent freeze-fracturing, replication, and viewing by transmission electron microscopy. The machine combines a rapid mixing unit similar to one widely used in chemical kinetics (Johnson, 1986) with a propane jet freezing unit previously used to prepare static samples for freeze-fracturing (Gilkey and Staehelin, 1986). The key element in the system is a unique thin-walled flow cell of copper that allows for injection and aging of the sample, followed by rapid freezing. During freeze-fracturing, a tangential cut is made along the wall of the flow cell to expose the sample for etching and replication. The dead time required for mixing and injection of the reactants into the flow cell is less than 5 ms. Electronic controls allow one to specify, on a millisecond time scale, any time above 5 ms between initiation of the reaction and quenching by rapid freezing.     

半导体制冷型高帧频CMOS数字摄像机研究与成像噪声分析

为了提高高速CMOS图像传感器的成像质量,降低图像传感器暗电流和随机噪声,文章中介绍了采用半导体制冷的方法来设计高帧频CMOS数字摄像机成像系统,针对该相机系统设计出结构一体化的多级半导体制冷制冷器,实现制冷量可控。图像传感器制冷后的温度与环境温度的温差最大可达40度,暗电流固定模式噪声降低50%,瞬态随机噪声减少90%,图像信噪比提高6dB。该相机系统具有集成度高,结构紧凑,高分辨率高帧频,高信噪比,宽动态范围,成像质量好的特点。     

Iso-butanol saturated water: a simple procedure for increasing staining intensity of resin sections for light and electron microscopy

The addition of the alcohol iso‐butanol (2‐methylpropan‐1‐ol) to water was found to improve the post‐staining procedures for semi‐thin and ultrathin resin sections, for both light and electron microscopy. Stain penetration was enhanced with samples embedded in both acrylic and epoxy resins and provided structural information not previously seen. These improvements were found with general (non‐specific) stains and a fluorescence stain for light microscopy, as well as for a range of heavy metal stains for electron microscopy. The use of this water/solvent medium also gave improved results when used in a variety of immunogold labelling procedures, resulting in a greater specific label density without affecting background gold levels. The use of this solvent/water medium may have wider applications for other types of staining.     

Ultrathin formvar support films for transmission electron microscopy

We have found that ultrathin Formvar films are easily and reliably made at an air-water interface by the drop method. By varying the concentration of Formvar in the drop, films of different characteristics can be obtained. Concentrations of 0.25–0.4% in ethylene dichloride produce extremely flat, ultrathin, and stable films that are especially suited for shadowed and negatively stained preparations. Low concentrations (? 0.1%) produce nets consisting of many tiny holes which, after carbon stabilization, are ideal for supporting high-resolution samples. Above 0.5%, films made by the drop method develop bubbles, and this bubble defect makes them unsuitable for section support. For section support, Formvar films made by the stripping method off mica are far superior to those made off glass. The films are more uniform in surface contour and thickness. They are less readily attacked by alcohols. Consequently, they are more resistant to staining procedures involving organic solvents and continue to be strong and uniform for section support.     

利用VB与VC混合编程实现数字印花的图像处理

织物吸收墨水能力的差异、经向和纬向的属性不同,造成数字印花过程中出现偏色和不期望条纹的现象,达不到理想的印花效果.针对这种问题提出在印花之前对图像进行合理的处理方案,即调整图像各种颜色的灰度值,将调整后的误差按不同的比例分配到经向和纬向的点上,使得在经向和纬向的点得到不同程度的补偿.介绍用VC编写动态连接库(DLL)、在VB中调用的方法来开发对数字印花的图像处理软件,以适应不同的织物,从而提高印花质量.     

Microcomputer system for ion microscopy digital imaging and processing

Analytical ion microscopy is a powerful tool for biological tissue analysis as it allows direct chemical distribution imaging, even at low element concentrations. A microcomputer based digital imaging system achieving acquisition at low light level is presented. It includes a high sensitivity video camera connected to a specialized image processor subsystem. Acquired images consist of 512 times 512 pixels with 8 bits accuracy. Real-time image processing software has been implemented so that image processing may be performed on-line. Image processing software allows off-line image manipulation and correlation for biological interpretation of elemental mapping images. System capabilities are illustrated by a study of stable and radio iodine mapping in rat thyroid tissue.     

Application of an array processor to image processing in electron microscopy

It is now possible to obtain a number of so-called array processors to attach to existing computer systems, to increase the computing power and speed available. In this paper, the application of one such array processor to image processing in electron microscopy is considered, and some of the practical experience thus gained is reported.     

Polylysine as an adhesive for the attachment of nanoplankton to substrates for electron microscopy

Nanoplankton can be readily attached to polylysine-coated substrates for examination by transmission and scanning electron microscopy. This simple technique gives high yields of organisms which are consistently better preserved and less obscured by detritus than samples dried directly on to substrates.     

Natural color scanning electron microscopy based on the frequency characteristics of the human visual system

The principles of image formation in natural color scanning electron microscopy (NC-SEM) are discussed in detail. This method is based on the frequency characteristic of the human visual system. It is shown that the Mach effect and masking effect are important in the characteristics. The former, which can enhance structural details, is visually similar to the edge effect in secondary electron (SE) images, and the latter is required for proper representation of very degraded color information obtained from a light microscope. When using these effects suitably, an NC-SEM image with the resolution equivalent to that of an SEM image can be acquired, though it is composed of an SEM image and a special video microscopy (VM) image with a resolution much lower than the SEM image of the identical view. The NC-SEM is more effective than the SEM in observation. interpretation, and analysis of various samples with important color information.     

Ion milling of materials science specimens for electron microscopy: A review

Ion bombardment to perforation is a common technique in the materials sciences by which thin specimens can be prepared for transmission electron microscopy. The process is not without complication and involves radiation damage to the specimen and tends not to preserve the initial specimen topology. Some of the more important facets of the ion-milling process, pertinent to such specimen preparations, are described.     

Polymethacrylate optodes: A potential for chemical digital color analysis

The paper describes results of implementing a new kind of optical analytical method, digital color analysis (DCA), in which colorimetric polymethacrylate sensors (optodes) are used. The optodes are made of optically transparent polymethacrylate matrix with physically immobilized analytical reagent which is responsible for the extraction of the analyte into the sensing material and changing its color. The developed optodes can be used in determination of various analytes using both solid-phase spectrophotometer and naked eye. In order to improve accuracy and sensitivity of the naked-eye determination, it is possible to measure visible color changes using digital imaging of the polymethacrylate optode. The digital images then can be represented as basic color (e.g. RGB) data. The DCA determinations of Cu (II), Ag (I) and Co (II) have indicated that the developed optodes are linear in concentration ranges 0.02–0.30; 0.02–0.20 and 0.05–0.25 mg L⁻¹ appropriately with corresponding practical detection limits of 0.01; 0.02 and 0.07 mg L⁻¹.