Analysis of surface roughness parameters digital image identification

The article analyses some surface roughness parameters of metal parts determining the ability of the surface of digital image identification, covariance functions and Wavelet’s wave theory. Expressions of covariance functions are formed using random functions, made by spreading digital image pixel arrays by columns in the form of individual vectors. The digital images used for research may vary in scale, because the frequencies of colour waves with individual pixels remain constant in the images, therefore, the image change does not influence the scale in computing covariance functions. The colour spectrum of RGB format was applied to identify the surface images of the parts. There was analysed the influence of individual RGB colour tensor components on the estimates of digital image covariance functions. The identity of digital images was evaluated by the change of correlation coefficient values in the range of RGB colours. The software was applied to compute the above process.     

Digital stain separation for histological images

It is often desirable to perform digital image analyses on sections prepared for human interpretation, e.g. nuclear chromatin texture analysis or three-dimensional reconstructions using sections requiring human delineation of structures of interest. Unfortunately such analyses are often more effective using stains with less complex contrast. Here an automated selective 'de-staining' method for digital images is presented. The method separates an image into its red, green and blue and hue, saturation and intensity components. A mask of stained tissue is prepared by automatic percentile thresholding. A single weighted inverted colour channel is then added to each of the three primary colour channels separately by an iterative algorithm that adjusts the weights to give minimum variance within the mask. The modified red, green and blue channels are then recombined. This method is automatic requiring no pre-definition of stain colours or special hardware. The method is demonstrated to 'de-stain' nuclei in haematoxylin and eosin (H&E) sections (and a separate haematoxylin image can be derived from this). An image of isolated brown reaction product is produced with immunoperoxidase preparations counterstained with haematoxylin. Furthermore trichrome (haematoxylin van Gieson, picrosirius red) and other common stains may be separated into their components with modifications of the same algorithm. Although other methods for colour separation do exist (e.g. spectral pathology and colour deconvolution) these require special apparatus or precise calibration and foreknowledge of pure dye colour spectra. The present method of digital stain separation is fully automatic with no such prerequisites.     

Colour-coded stereo images from the tandem scanning reflected light microscope (TSRLM)

A method is described for the production of stereo-pair images in the tandem scanning reflected light microscope in which depths within the field imaged are coded in colour. The two images are recorded photographically whilst focusing vertically through the layer which is to be imaged. A horizontal component of motion is applied at the same time, but in opposing senses for the two images. Colour coding for depth is obtained by changing colour filters so that reflective features lying within different depth bands are imaged in corresponding colours.     

基因芯片荧光靶点显微成像自动调焦控制

在基因芯片荧光靶点阵列图像CCD扫描采集系统中,显微成像自动调焦控制是进行芯片杂交信号荧光靶点图像采集的先决条件.在定义图像清晰度评价函数的基础上,通过对实时采集的显微图像进行清晰度评价函数计算,采用基于粗精结合原则的自动调焦控制策略,实现了荧光靶点显微成像自动调焦控制.调焦控制实验表明,该方法显著提高了基因芯片荧光靶点图像聚焦和采集的效率.     

Chromatic shift in multicolour confocal microscopy

Multicolour confocal microscopy has proven to be a successful technique for the analysis of the spatial relationship between different biological structures in the same preparation. However, when the positions of objects are compared, e.g. co-localization and distance measurements, any positional shift that arises between the colour components is clearly unacceptable. This paper presents a simple technique for measuring with high accuracy the positional shifts that occur between the colour components of an image. Multi-labelled microbeads were scanned using two or three different detection channels. The position of each microbead was calculated separately for each detection channel. In general, the two calculated positions of the same microbead (one for each channel) are slightly different. This difference is a measure of the positional shift between the colours. This method enables the measurement of shift with a high accuracy (20 nm), and it has been applied to images from several experiments. The results of these experiments will give the reader an impression of typical contributions of different effects (such as chromatic aberration, misalignment of optical components and inaccuracy of the scanning unit) on the amount of positional shift.     

Automatic seamless mosaicing of microscopic images: enhancing appearance with colour degradation compensation and wavelet-based blending

In order to observe the fine details of biomedical specimens, various kinds of high-magnification microscopes are used. However, they suffer from a limited field of view when visualizing highly magnified specimens. Image mosaicing techniques are necessary to integrate two or more partially overlapping images into one and make the whole specimen visible. In this study, we propose a new system that automatically creates panoramic images by mosaicing all the microscopic images acquired from a specimen. Not only does it effectively compensate for the congenital narrowness in microscopic views, but it also results in the mosaiced image containing as little distortion with respect to the originals as possible. The system consists of four main steps: (1) feature point extraction using multiscale wavelet analysis, (2) image matching based on feature points or by projection profile alignment, (3) colour difference adjustment and optical degradation compensation with a Gaussian-like model and (4) wavelet-based image blending. In addition to providing a precise alignment, the proposed system also takes into account the colour deviations and degradation in image mosaicing. The visible seam lines are eliminated after image blending. The experimental results show that the system performs well on differently stained image sequences and is effective on acquired images with large colour variations and degradation. It is expected to be a practical tool for microscopic image mosaicing.     

Chromoxane cyanine R. II. Staining of animal tissues by the dye and its iron complexes

The staining properties of chromoxane cyanine R (Colour Index No. 43820, Mordant blue 3; also known as eriochrome cyanine R and solochrome cyanine R) have been studied. Used alone, the dye imparted its red colour to nuclei, cytoplasm and collagen. The dye was extracted by mild alkali but not by acids. Stainability required ionized amino groups in the tissue, and there was also evidence for non-ionic binding of the dye. The colours obtained by staining with mixtures of chromoxane cyanine R and ferric chloride varied with the molar iron: dye ratio and with the pH. Useful staining was seen only between pH 1 and 2. The tissues were coloured either all blue (when Fe: dye was high), or both red and blue (when Fe: dye was low). Lower pH favoured the deposition of red, higher pH the deposition of blue colour. The red was mainly in cytoplasm, blue in nuclei and myelin. Collagen fibres were red or purple, depending on pH and iron: dye ratio. Red colours were differentiated by acid and changed to blue, but not extracted, by mild alkali. The red substance in the stained sections was clearly not the free dye, so it was probably an iron-dye complex. From the effects of various differentiating agents, it was deduced that the red and blue dye-metal complex molecules were bound to the tissue by the dye moiety, not by interposition of iron atoms. Staining by the complexes of iron(III) with chromoxane cyanine R did not involve nucleic acids or other polyanions or the amino groups of proteins. There was evidence for only non-ionic binding of both red and blue complexes. It is suggested that the red colour in sections stained by solutions with low iron: dye ratio is due to a simple carboxylate complex, [Fe₂H(dye)]^?. The blue colour would then result from withdrawal of a proton from the red complex to give [Fe₂(dye)]^2-. The bases that remove the protons may be arginine-rich nucleoproteins of nuclei and phospholipid bases of myelin. Techniques are described for informative simultaneous staining in two colours, and for the selective staining of either nuclei or myelin.     

A low cost image and data acquisition system for use with the Vickers M85 scanning microdensitometer

A simple and inexpensive interface has been constructed between the Vickers M85 microdensitometer and a BBC model B microcomputer. The interface incorporates three sensitivity ranges and enables the production of pseudocolour images of the specimen using the two-dimensional scanning mode of the M85. The operator can select a 160times256 pixel image with eight colours or a 320times256 display using only four colours. Each colour represents a defined range of transmittance which is software controlled. The image histogram can be displayed and the interval between colours redefined so as to enable contrast stretching. Intervals between colours can be either linear or logarithmic and the images thus obtained can be stored on disc or videotape, or a hard copy can be obtained using a screen dump routine. Two-dimensional absorption images can thus be obtained at any single wavelength from 400 to 700 nm at normal magnifications of the light microscope. In addition, the system can be used to acquire, store and process data from one-dimensional scans to obtain quantitative information about variations in optical density within the specimen, so considerably increasing the usefulness of the instrument. Although obviously limited in its capabilities, the system produces images of very high quality and one-dimensional data of high sensitivity. The interface can be constructed for less than £40. A small modification to one of the M85 circuit boards is necessary to obtain maximum resolution.     

A protocol for registration and correction of multicolour STED superresolution images

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut‐shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co‐localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross‐correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co‐staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user‐friendly criteria, which – if fulfilled – support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co‐localisation analyses. Although the reference protocol is discussed exemplarily for two‐colour STED imaging, it can be readily expanded to three or more colours and STED channels.     

采用联合变换相关的透镜中心偏差测量

基于双相位编码光学联合变换相关技术提出了一种测量透镜中心偏差的方法以确定偏差方向并提高测量精度。在经典联合变换相关原理基础之上,使用两个相位函数分别对参考图像和联合功率谱进行编码,并选用合适的滤波器消除旁瓣干扰,得到单个互相关峰的输出。利用该双相位编码后的联合变换相关技术探测不同目标图像相对于参考图像的位移矢量,并拟合圆。此拟合圆圆心到圆上点的矢量即为经自准直光学系统放大后的偏心矢量,从而同时确定了中心偏差的大小和方向。实验结果表明,通过双相位编码后的相关输出仅保留一个尖锐的相关峰,实现了位移矢量的亚像元探测;使用联合变换相关技术准确地测量了透镜的中心偏差,其测量结果的实验标准差为0.1μm,误差绝对值最大为0.3μm,满足一般透镜中心偏差测量的要求。     

色彩设计的控制研究

在满足生活需要的大前提下,当代设计的主流仍然是讲究功能主义设计,其色彩将依其不同的功能根据不同阶层人的不同品味和审美而定.色彩设计不是单一的实现色彩美学价值.而是一个复合操作过程.因此,要充分控制色彩的滥用,重视色彩设计在信息传递的诸多应用环节的作用.     

Color‐based tumor tissue segmentation for the automated estimation of oral cancer parameters

This article presents an automatic color‐based feature extraction system for parameter estimation of oral cancer from optical microscopic images. The system first reduces image‐to‐image variations by means of color normalization. We then construct a database which consists of typical cancer images. The color parameters extracted from this database are then used in automated online sampling from oral cancer images. Principal component analysis is subsequently used to divide the color features into four tissue types. Each pixel in the cancer image is then classified into the corresponding tissue types based on the Mahalanobis distance. The aforementioned procedures are all fully automated; in particular, the automated sampling step greatly reduces the need for intensive labor in manual sampling and training. Experiments reveal high levels of consistency among the results achieved using the manual, semiautomatic, and fully automatic methods. Parameter comparisons between the four cancer stages are conducted, and only the mean parameters between early and late cancer stages are statistically different. In summary, the proposed system provides a useful and convenient tool for automatic segmentation and evaluation for stained biopsy samples of oral cancer. This tool can also be modified and applied to other tissue images with similar staining conditions. Microsc. Res. Tech. 2009. © 2009 Wiley‐Liss, Inc.     

Digitally adjusting chromogenic dye proportions in brightfield microscopy images

We present an algorithm to adjust the contrast of individual dyes from colour (red-green-blue) images of dye mixtures. Our technique is based on first decomposing the colour image into individual dye components, then adjusting each of the dye components and finally mixing the individual dyes to generate colour images. Specifically in this paper, we digitally adjust the staining proportions of hematoxylin and eosin (H&E) chromogenic dyes in tissue images. We formulate the physical dye absorption process as a non-negative mixing equation, and solve the individual components using non-negative matrix factorisation (NMF). Our NMF formulation includes camera dark current in addition to the mixing proportions and the individual H and E components. The novelty of our approach is to adjust the dye proportions while preserving the color of nonlinear dye interactions, such as pigments and red blood cells. In this paper we present results for only H&E images, our technique can easily be extended to other staining techniques.     

Accurate representation of interference colours (Michel–Lévy chart): from rendering to image colour correction

Here a work flow towards an accurate representation of interference colours (Michel‐Lévy chart) digitally captured on a polarised light microscope using dry and oil immersion objectives is presented. The work flow includes accurate rendering of interference colours considering the colour temperature of the light source of the microscope and chromatic adaptation to white points of RGB colour spaces as well as the colour correction of the camera using readily available colour targets. The quality of different colour correction profiles was tested independently on an IT8.7/1 target. The best performing profile was using the XYZ cLUT algorithm and it revealed a ΔE₀₀ of 1.9 (6.4 no profile) at 5× and 1.1 (8.4 no profile) at 100× magnification, respectively. The overall performance of the workflow was tested by comparing rendered interference colours with colour‐corrected images of a quartz wedge captured over a retardation range from 80–2500 nm at 5× magnification. Uncorrected images of the quartz wedge in sRGB colour space revealed a mean ΔE₀₀ of 12.3, which could be reduced to a mean of 4.9 by applying a camera correction profile based on an IT8.7/1 target and the Matrix only algorithm (ΔE₀₀ < 1.0 signifies colour differences imperceptible by the human eye). ΔE₀₀ varied significantly over the retardation range of 80–2500 nm of the quartz wedge, but the reasons for this variation is not well understood and the quality of colour correction might be further improved in future by using custom made colour targets specifically designed for the analysis of high‐order interference colours.     

A cross-correlation technique as a system evaluation tool; application to blood flow measurement in extra-corporeal circuits

Auto-correlation and cross-correlation techniques have been favored by engineers and scientists as powerful analysis tools, especially when noise is present in the system. Since cross-correlation and power spectral density functions are tightly coupled, cross-correlation is a well suited instrument for the analysis of time invariant systems. This paper discusses the conditions under which cross-correlation techniques can be used to identify a system model. Random signals, either arbitrarily generated or inherent in the systems, can be used as test signals for the purpose of identification. A real time cross-correlator is designed and used to evaluate blood carrying vessels for the purpose of flow measurement in extra-corporeal circuits. Naturally occurring random signals due to ‘impurities’ in the blood such as red and white blood cells are correlated by means of two pairs of ultrasonic sensors. The correlation functions obtained under different flow rates manifest themselves as unit impulse responses of the fluid system between upstream and downstream sensors. The results obtained were evaluated for accurate modeling of the fluid path and transit time flow measurement.     

Video microscopic image processing facilitates the evaluation of light microscopic autoradiography at high magnification

Light microscopic autoradiographs of H-thymidine labelled unstained semithin sections of Xenopus laevis embryonic nuclei were examined with conventional Nomarski differential interference contrast, phase-contrast and video microscopy. Whereas at low magnification it was possible to obtain a photograph of the nuclear structure and the silver grains in one focal plain, at high magnification, with small depths of focus, a satisfactory image was not attainable. Therefore, we stored the images of the two different focus levels with a digital image processing system and combined both images by an arithmetic operation. This video microscopic technique allows the use of high magnification light microscopy with oil immersion objectives and the application of additional electronic contrast enhancing methods for an adequate and rapid analysis of light microscopic autoradiographs.     

The application of a normalized correlation function in image analysis

This paper describes the use of normalized correlation functions, such as auto- and cross-correlation functions for the analysis of images. The normalization is introduced to avoid problems of interpretation of the resulting correlation functions caused by the finite dimension of the image used for calculation. Due to a reduction of noise in the correlation function it is possible to detect periodic structures, which are hidden by noise in the original image. Besides the normalization of the correlation functions, a modification of the correlation function by periodic continuation of the image is proposed. This leads in all cases where the structure allows this to a further drastic reduction of noise. Besides the two-dimensional correlation functions one-dimensional correlation functions are proposed. It is shown that there are cases where it is difficult to find an adequate interpretation of these one-dimensional functions.     

温场对CaF2色心晶体形成的影响

我们采用月Bridgman-Stockbarger法生长出具有黄, 紫红, 蓝, 无色四种颜色的晶体, 这些不同颜色的晶体与生长过程中的温场有关.通过辐照、吸收退火实验.我们分析三种颜色色心晶体的形成及热稳定性.     

HisTOOLogy: an open‐source tool for quantitative analysis of histological sections

HisTOOLogy is an open‐source software for the quantification of digital colour images of histological sections. The simple graphical user interface enables both expert and non‐expert users to rapidly extract useful information from stained tissue sections. The software's main feature is a generalizable colour separation algorithm based on k‐means clustering which accurately and reproducibly returns the amount of colour per unit area for any stain, thus allowing the quantification of tissue components. Here we describe HisTOOLogy's algorithms and graphical user interface structure, showing how it can be used to separate different dye colours in several classical stains. In addition, to demonstrate how the tool can be employed to obtain quantitative information on biological tissues, the effect of different hepatic tissue decellularization protocols on cell removal and matrix preservation was assessed through image analysis using HisTOOLogy and compared with conventional DNA and total protein content assays. HisTOOLogy's performance was also compared with ImageJ's colour deconvolution plug‐in, demonstrating its advantages in terms of ease of use and speed of colour separation.     

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.