Linear phase imaging using differential interference contrast microscopy

We propose an extension to Nomarski differential interference contrast microscopy that enables isotropic linear phase imaging. The method combines phase shifting, two directions of shear and Fourier‐space integration using a modified spiral phase transform. We simulated the method using a phantom object with spatially varying amplitude and phase. Simulated results show good agreement between the final phase image and the object phase, and demonstrate resistance to imaging noise.     

Differential phase contrast in scanning optical microscopy

High-quality high-resolution transmission and reflection images produced using a scanning optical microscope and the split-detector technique are presented. These images exhibit differential phase contrast, the method avoiding some drawbacks of the usual Nomarski DIC arrangement. Imaging is treated theoretically and compared with the Nomarski method.     

Collection deficiencies of scanning electron microscopy signal contrasts measured and corrected by differential hysteresis image processing

Limitations of scanning electron microscopy (SEM) image resolution and quality were measured in digital image data and their effect on image contrasts was analyzed and corrected by differential hysteresis (DH) processing. DH processing is a mathematical procedure that utilizes hysteresis properties of intensity variations in the image for a segmentation of differential contrast patterns. These patterns display contrast properties of the data as coherent full-frame images. The contrast segmentation is revertible so that the original image can be restored from the sum of the sequentially extracted DH contrast patterns. DH imaging enhances weak contrast components so that they are more easily recognizable and displays SEM image data free of signal collection efficiency contrasts. Example image data include environmental SEM (ESEM) and SEM images of low and mediumhigh magnifications where collection deficiencies included charging of the specimen surface, obstructions from specimen topography, and uneven signal collection properties of the detector. ESEM low-vacuum image data, which appear to be of high quality, contained local areas of reduced contrasts due to residual surface charging. In such areas, signal contrasts were reduced up to 80%, which suppressed most of the weak short-range contrasts. In low-magnification SEM images, up to 93% of the local high precision contrast was lost from the various adverse effects which diminished the pixel-related contrast resolution of the microscope and resulted in images with low detail. Also, at medium magnification, surface charging effects dramatically reduced the image quality because contrasts resulting from local electron beam/specimen interactions were reduced by as much as 71%. DH imaging restored the local contrast losses by elimination of the collected distorted fraction of signal contrasts and reconstitution of the collected maintained fraction. Restored DH images are of superior quality and enhance the imaging capability of the conventional SEM. DH contrast segmentation provides an improved basis for the measurement of various signal contrast components and detector performances. The DH analysis will ultimately facilitate a precise deduction of specimen properties from extracted contrast patterns.     

Elimination of scanning electron microscopy image periodic distortions with digital signal-processing methods

Electromagnetic interference is one of the main distortion sources in scanning electron microscopy. Electromagnetic interference‐generated scanning electron microscopy image distortions are usually visible as edge blur (at low scan rates) or vibration (at high scan rates). Hardware solutions to this problem, e.g. electrostatic and magnetic shielding, are expensive and, in some cases, difficult to implement. The current investigations led to a significant decrease in the periodic distortions by a novel adaptation of software‐based digital signal processing to scanning electron microscopy problems, without any hardware modification.     

Ribosome substructure investigated by scanning force microscopy and image processing

Scanning force microscopy was used to study the ultrastructure of eukaryotic ribosomes from Chironomus pallidivittatus in the polysomal complex. Positively stained polysomes were imaged, and the resulting three-dimensional ribosomal structures were further processed by statistical analyses of virtual cross-sections parallel to the substrate plane. Structural investigations were based on parameters which are minimally influenced by the tip geometry, like section plane centre or axis ratio. In the lower part of the structure a shift of the section centres was observed, suggesting an attached structure. The geometry of the sections revealed an elliptical shape in the upper part (axis ratio 1.52 ± 0.22), with a less elongated shape in the lower region (axis ratio 1.41 ± 0.18). A model for the surface topography of a positively stained ribosome exhibiting a small subunit attached along the long side of an elliptical large structure is proposed.     

Landmarks in biological light microscopy

The pioneering seventeenth-century work of Hooke (using the compound microscope) and Leeuwenhoek (using the simple microscope) was followed in the eighteenth century by a period of comparative stagnation until the early nineteenth-century work of Lister (who evolved the principle of aplanatic focal points in lens design). The founding of the Microscopical Society of London in 1839 reflected the increasing importance of the microscope as a scientific instrument and its involvement in specimen preparation techniques, cell theory and bacteriology is described. The pursuit of increased resolving power—in particular the development of apochromatic lenses by Abbe and Zeiss and enhanced contrast techniques—polarized fight, u.v. microscopy, u.v. absorption and fluorescence, phase contrast and the differential interference contrast techniques, etc., are discussed. The story of microscopy in the twentieth century is one of interlocking discoveries and advances and the techniques of epi-illumination in scanning fluorescence microscopy, video acquisition and analyses of images in digital form and confocal scanning microscopy are surveyed. It is concluded that these techniques have opened up whole new areas for the future development of light microscopy.     

Confocal microscopy and image processing in the study of plant nuclear structure

We describe measurements of the point spread function (PSF) for a confocal microscope and compare them with the PSF for a conventional (wide-field) fluorescence microscope. In situ hybridization with probes to telomere and ribosomal rDNA sequences, combined with three-dimensional (3-D) microscopy, has been used to study interphase nuclei in root tissue of Pisum sativum and Vicia faba. Nearly all the telomeres in both species are located at the nuclear envelope, and are highly clustered in the Vicia tissues, suggesting specific binding interactions. rDNA labelling in P. sativum shows four brightly staining knobs, corresponding to condensed regions of the rDNA genes from the two pairs of nucleolar organizer genes in this species, arranged approximately tetrahedrally around each nucleolus. Deconvolution using the measured PSFs can be used to improve these images, revealing a fibrous substructure in the perinucleolar knobs, and a large amount of interconnecting internal structure, which we suggest represents rDNA both in the fibrillar centres and also more diffuse, widely dispersed rDNA. Finally we show that accurate conventional data coupled with deconvolution can produce 3-D reconstructions comparable to those obtainable with confocal microscopy, but that the clearest images are obtained by applying deconvolution to the confocal data.     

由形态学边缘模式匹配实现数字稳像

针对在一些实际应用场合摄像机设备的震动造成的视频序列失稳,提出了一种基于形态学边缘模式匹配的数字稳像方法.该方法首先利用形态学方法提取视频图像边缘,然后利用当前帧子块与参考帧子块的边缘进行特征匹配,以确定当前帧子块相对于参考帧的局部运动矢量.对得到的局部运动矢量进行分析判决,以确定该局部运动矢量是否为真正的抖动偏移量,消除视频帧本身因存在局部运动目标、低对比度、纹理区域等因素对全局运动矢量估计的影响;最后,通过判别确认的局部运动矢量确定全局运动矢量并进行运动补偿,实现数字稳像.仿真实验表明,由于该方法的块局部运动矢量采用1 bit边缘进行最大相关值估计,因而复杂度低,而采用异常情况判决准则消除了其对真正运动矢量估计的影响,稳像精度高,可应用于因摄像头平移震动等因素造成的视频序列失稳.     

基于微分干涉相衬的相位分析法研究

通过对微分干涉相衬显微定量测量方法进行研究,提出了一种更有效的相位分析法。即在不对双光束干涉光路进行改造或处理的前提下,通过对光学成像进行处理而得到理想的结果。即把图像中的光强信号转变成相位信号,并通过维纳滤波对噪声进行了消除,最后获得表面微观形貌定量参数。     

Measurement of the geometric features of a cutting tool edge with the aid of a digital image processing technique

For the further development of new machining systems such as flexible manufacturing systems (FMS), this paper reports on a new measuring system developed for the multi-purpose understanding of the geometric features of cutting tool edges having serious influences on the machining outputs. In this system, to detect three-dimensional (3D) features of the toolface and tool flank only from the two-dimensional (2D) microscopic observation of the toolface and to describe an overall 3D geometric feature of the tooledge as a digital 2D image as well as to enable inexpensive system development, two convenient measuring principles are used. These are the scanning optical section method for the toolface contour mapping and the cutting edge profile comparison method for the flank wear landwidth estimation. Based on these, experimental hardware was arranged and various processing softwares were developed. They were applied to an observation of the cutting edge wear development in some cylindrical turning experiments, to show that various effective cutting edge parameters could be easily extracted from the integrated toolface image recorded, and that sufficient accuracy and resolution for practical use could be obtained.     

Segmentation of ascidian notochord cells in DIC timelapse images

We have developed a method to automatically segment notochord cell boundaries from differential interference contrast (DIC) timelapse images of the elongating ascidian tail. The method is based on a specialized parametric active contour, the network snake, which can be initialized as a network of arbitrary but fixed topology and provides an effective framework for simultaneously segmenting multiple touching cells. Several modifications to the original network snake were necessary for high-quality segmentation, including linear Gaussian derivative filtering to reconstruct edge maps from DIC images and a new energy function to improve the segmentation of critical cell-cell vertices. We find that post-intercalation ascidian notochord cells exhibit two distinct cell behaviors: lateral cell edges expand along the AP axis while showing a rapid pulsatile behavior, whereas anterior and posterior cell edges contract smoothly.     

Porosity determination of ceramic materials by digital image analysis--a critical evaluation

Measuring the porosity of materials by digital image analysis of micrographs is a well-established and convenient method for the testing of metallic samples. However, when applied to ceramic materials, this method has been shown to be much less reliable and poorly reproducible. The purpose of this present work is to clarify the reason for this deficiency, involving many porosity measurements, performed on plasma-sprayed zirconia, under systematically varied microscopic imaging conditions, and the porosities being calculated using various evaluation methods. Comparison between of the results has shown that the present state of the image analysis method is not satisfactory for absolute porosity measurements on ceramic materials. It can be useful as a convenient tool for comparative measurements, however, if the imaging conditions maintained in the microscope and the evaluation method are held to be exactly identical.     

Identification of fat,protein matrix,and water/starch on microscopy images of sausages by a principal component analysis-based segmentation scheme

A color-based segmentation scheme applied to microscopy images of cryosectioned sausages is proposed. The segmentation scheme is capable of segmenting three different levels on the microscopy images: the fat particles, the protein matrix, and water/starch. The method is based on principal component analysis. A user-friendly program was developed for the manual segmentation of a selection of image pixels by microscopists. Principal component models based on the manually classified pixels are then used to segment fat, protein matrix, and starch/water on microscopy images. The program can also be used as a training tool for microscopists.     

Quantitative fractography by digital image processing: NIH Image macro tools for stereo pair analysis and 3-D reconstruction

A set of NIH Image macro programs was developed to make qualitative and quantitative analyses from digital stereo pictures produced by scanning electron microscopes. These tools were designed for image alignment, anaglyph representation, animation, reconstruction of true elevation surfaces, reconstruction of elevation profiles, true-scale elevation mapping and, for the quantitative approach, surface area and roughness calculations. Limitations on time processing, scanning techniques and programming concepts are also discussed.     

Reconstruction of optical pathlength distributions from images obtained by a wide-field differential interference contrast microscope

An image processing algorithm is presented to reconstruct optical pathlength distributions from images of nonabsorbing weak phase objects, obtained by a differential interference contrast (DIC) microscope, equipped with a charge-coupled device camera. The method is demonstrated on DIC images of transparent latex spheres and unstained bovine spermatozoa. The images were obtained with a wide-field DIC microscope, using monochromatic light. After image acquisition, the measured intensities were converted to pathlength differences. Filtering in the Fourier domain was applied to correct for the typical shadow-cast effect of DIC images. The filter was constructed using the lateral shift introduced in the microscope, and parameters describing the spectral distribution of the signal-to-noise ratio. By varying these parameters and looking at the resulting images, an appropriate setting for the filter parameters was found. In the reconstructed image each grey value represents the optical pathlength at that particular location, enabling quantitative analysis of object parameters using standard image processing techniques. The advantage of using interferometric techniques is that measurements can be done on transparent objects, without staining, enabling observations on living cells. Quantitative use of images obtained by a wide-field DIC microscope becomes possible with this technique, using relatively simple means.