How to improve microscopic images obtained with consumer-type digital cameras

AIM: Digital imaging is useful in conventional photography because it immediately provides images, and the image quality can be improved afterwards by the use of computer programs. The major disadvantages of consumer-type digital cameras mounted on microscopes are (i) unequal illumination through the image, and (ii) a coloured background. A computer program was specifically adapted and refined to improve images obtained with consumer-type digital cameras mounted on microscopes. METHODS AND RESULTS: An approach using a division operation between the specimen image and a background image leads to homogeneous illumination throughout the image, with automatically corrected brightness and white background. The correct colour spectrum is preserved by correction of the histogram. This approach was obtained from the freeware computer program 'Image Arithmetic'. In a test, three different consumer-type digital cameras (Sony, Nikon, Olympus) on different microscopes were used to obtain images of different types of histological specimens (cervical smear, bone marrow biopsy, and colonic biopsy). The computer program dramatically improved the quality of images obtained with all tested cameras. CONCLUSION: Using this approach, even low-cost digital cameras mounted on microscopes produce brilliant images with homogeneous illumination and a white background, the image quality being comparable with expensive cameras especially designed for microscopes.     

From quantitative microscopy to automated image understanding

Quantitative microscopy has been extensively used in biomedical research and has provided significant insights into structure and dynamics at the cell and tissue level. The entire procedure of quantitative microscopy is comprised of specimen preparation, light absorption/reflection/emission from the specimen, microscope optical processing, optical/electrical conversion by a camera or detector, and computational processing of digitized images. Although many of the latest digital signal processing techniques have been successfully applied to compress, restore, and register digital microscope images, automated approaches for recognition and understanding of complex subcellular patterns in light microscope images have been far less widely used. We describe a systematic approach for interpreting protein subcellular distributions using various sets of subcellular location features (SLF), in combination with supervised classification and unsupervised clustering methods. These methods can handle complex patterns in digital microscope images, and the features can be applied for other purposes such as objectively choosing a representative image from a collection and performing statistical comparisons of image sets.     

Overlapped seed localization in seed implant brachytherapy

A procedure for the determination of the location of prostate implant seeds that are wholly overlapped in a projection view has been developed. The procedure mainly consists of a series of image processing and an in-house developed localization software based on a three-film technique. To verify the efficacy of the procedure, a simulation phantom was built and nine sets of simulation were performed. For the assessment of the location of the seeds in the phantom, three images, one in anterior-posterior direction and two others in oblique angles, were acquired and a series of image processing was applied to the images for the removal of unnecessary background and the improvement of imaging quality. In this study, three types were considered; first, when two seeds were overlapped in one of projection images, second, more than three seeds were overlapped in one of projection images, and the third, all images contained wholly overlapped seeds. The developed software separates wholly overlapped seeds by calculating the distance between seeds in each film. This software can provide valuable information for establishing effective quality assurance in permanent prostate brachytherapy.     

大鼠松质骨切片图像的三维重建与定量分析

本文研究动物松质骨连续切片图像数据集的获取、分割、配准、及三维重建的技术方法.利用病理切片和图像数码摄入技术,获取了大鼠腰椎松质骨连续切片图像数据集,用基于外置标记点和分割-计数法两种方法进行参数计算,依参数对图像进行刚体变换完成配准,将配准后的切片图像输入二维图像处理软件进行分割,提取感兴趣区域后输入三维重建软件进行三维重建与定量分析.重建后的松质骨三维立体图像呈均匀、致密的立体网状结构,骨小梁连接清晰可见.     

一种新计算机三维重建法用于猫肺连续切片

介绍了基于计算机工作站的连续组织学切片图像三维重建与显示技术。常规制作猫肺连续石蜡切片,切片图像经微机图像分析系统数字化后存在微机磁盘,将图像数据文件传输到Ｓｕｎ工作站硬盘上,作数字图像处理,采用“金字塔异或指数”方法完成图像对位,较好地解决了连续切片图像的精确对位问题,具体的重建算法采用光线追踪进行三维显示的体积显示算法,在计算机屏幕上从不同角度显示了肺内血管和细支气管等结构的立体图像。     

Image analysis for discrimination of cervical neoplasia

Colposcopy involves visual imaging of the cervix for patients who have exhibited some prior indication of abnormality, and the major goals are to visually inspect for any malignancies and to guide biopsy sampling. Currently colposcopy equipment is being upgraded in many health care centers to incorporate digital image acquisition and archiving. These permanent images can be analyzed for characteristic features and color patterns which may enhance the specificity and objectivity of the routine exam. In this study a series of images from patients with biopsy confirmed cervical intraepithelia neoplasia stage 2/3 are compared with images from patients with biopsy confirmed immature squamous metaplasia, with the goal of determining optimal criteria for automated discrimination between them. All images were separated into their red, green, and blue channels, and comparisons were made between relative intensity, intensity variation, spatial frequencies, fractal dimension, and Euler number. This study indicates that computer-based processing of cervical images can provide some discrimination of the type of tissue features which are important for clinical evaluation, with the Euler number being the most clinically useful feature to discriminate metaplasia from neoplasia. Also there was a strong indication that morphology observed in the blue channel of the image provided more information about epithelial cell changes. Further research in this field can lead to advances in computer-aided diagnosis as well as the potential for online image enhancement in digital colposcopy.     

松质骨移植材料孔隙率的显微CT和组织切片测试方法比较

目的采用显微 CT法和组织切片法对松质骨移植材料的孔隙率进行对比研究,为骨组织工程支架材料的研制和开发提供理论依据.方法首先取新鲜尸体的股骨和新鲜猪股骨,进行去除多余组织、灭菌、低温冷冻保存等处理后,制备成松质骨移植材料,使用显微 CT分别扫描同种异体骨和动物源性猪骨移植材料获取显微 CT图像.然后对同种异体松质骨组织进行染色和切片等处理,制备成同种异体骨组织切片材料,显微镜下观察骨组织切片并获取其图像,各取10幅图像用于实验.设计matlab程序对所有图像进行灰度化、图像增强、二值化、移除干扰等处理,计算孔隙率的数值.结果显微CT和组织切片测试得到的同种异体松质骨孔隙率与动物源性猪松质骨孔隙率结果无显著性差异(P>0.05).结论显微 CT标本的制备较组织切片大为简化,可以无损地获取数据,并省去灰度化、图像增强、移除干扰等处理过程,比组织切片法测量松质骨孔隙率更加简便.     

Fracture characterization of human cortical bone under mode II loading using the end-notched flexure test

Fracture characterization of human cortical bone under mode II loading was analyzed using a miniaturized version of the end-notched flexure test. A data reduction scheme based on crack equivalent concept was employed to overcome uncertainties on crack length monitoring during the test. The crack tip shear displacement was experimentally measured using digital image correlation technique to determine the cohesive law that mimics bone fracture behavior under mode II loading. The developed procedure was validated by finite element analysis using cohesive zone modeling considering a trapezoidal with bilinear softening relationship. Experimental load-displacement curves, resistance curves and crack tip shear displacement versus applied displacement were used to validate the numerical procedure. The excellent agreement observed between the numerical and experimental results reveals the appropriateness of the proposed test and procedure to characterize human cortical bone fracture under mode II loading. The proposed methodology can be viewed as a novel valuable tool to be used in parametric and methodical clinical studies regarding features (e.g., age, diseases, drugs) influencing bone shear fracture under mode II loading.     

Melanomas non-invasive diagnosis application based on the ABCD rule and pattern recognition image processing algorithms

In this paper an automated dermatological tool for the parameterization of melanomas is presented. The system is based on the standard ABCD Rule and dermatological Pattern Recognition protocols. On the one hand, a complete stack of algorithms for the asymmetry, border, color, and diameter parameterization were developed. On the other hand, three automatic algorithms for digital image processing have been developed in order to detect the appropriate patterns. These allow one to calculate certain quantitative features based on the aspect and inner patterns of the melanoma using simple-operation algorithms, in order to minimize response time. The database used consists of 160 500×500-pixel RGB images (20 images per pattern) cataloged by dermatologists, and the results have turned out to be successful according to assessment by medical experts. While the ABCD algorithms are mathematically reliable, the proposed algorithms for pattern recognition produced a remarkable rate of globular, reticular, and blue veil Pattern recognition, with an average above 85% of accuracy. It thus proves to be a reliable system when performing a diagnosis.     

Palm print image recovery and de-noising via multi-channel wavelet filter bank

In this paper, a 3-channel wavelet transform method is introduced to recover palm print images following serious deformation. The deformation processing is actually a kind of digital re-sampling. A filter bank consisting of three filters is implemented for wavelet decomposition of the palm print image, and then a procedure of binary interpolation is performed after the image is reconstructed by another filter bank consisting of another three filters. The design of multi-channel wavelet filter bank is based on the Quadrature Mirror Filter (QMF) method. Because the noise is caused by the Morie stripe, the images are further de-noised after the geometry deformation is addressed. Acceptable results have been obtained.     

Palm print image recovery and de-noising via multi-channel wavelet filter bank

In this paper, a 3-channel wavelet transform method is introduced to recover palm print images following serious deformation. The deformation processing is actually a kind of digital re-sampling. A filter bank consisting of three filters is implemented for wavelet decomposition of the palm print image, and then a procedure of binary interpolation is performed after the image is reconstructed by another filter bank consisting of another three filters. The design of multi-channel wavelet filter bank is based on the Quadrature Mirror Filter (QMF) method. Because the noise is caused by the Morie stripe, the images are further de-noised after the geometry deformation is addressed. Acceptable results have been obtained.     

基于螺旋CT扫描图像的人牙齿的计算机三维重建

目的:研究基于螺旋CT扫描图像的人牙齿的计算机三维重建。方法:采用美国PQ6000型螺旋CT扫描机,自下颌颏部下缘开始至鼻骨底做连续横断超薄扫描,得到了人牙齿的二维CT图像。应用图像处理与识别合成软件,读取二维CT图像数据文件,通过灰度分割方法对二维图像进行边缘提取,再利用三维表面重建算法对所截取的二维CT图像进行三维重建,最终获得人牙齿的三维重建影像。结果:获得的人牙齿三维重建影像,细致逼真、可被任意旋转、剖开、透视、截取,从不同角度观察重建影像形态还原性良好、重建效果满意。结论:基于螺旋CT扫描图像的人牙齿的计算机三维重建,可为有限元实体建模提供快捷、方便的方法。     

Digital imaging of bone and tooth modification

Digital cameras are capable of producing images of cut marks and other three dimensional subjects comparable to those obtained from the scanning electron microscope (SEM). Until now, the SEM has offered unparalleled depth of field and surface rendition. SEM units are, however, expensive to acquire and maintain, and SEM image production is time-intensive. Furthermore, SEM images lack color and are often incapable of imaging bone modifications because of magnification and chamber size limitations. A digital imaging method for producing extremely high depth of field enlargements of three dimensional, sub-millimeter scale objects circumvents these problems. In our presentation we employ four case studies of hard tissue modification related to: (1) the origins of meat-eating in the human lineage; (2) evidence for cannibalistic behavior among Neanderthals; (3) pre-Columbian evidence of North American dentistry; and (4) the earliest evidence of cranial surgery in North America. Procedures for replica production, image-capture, and image enhancement are provided. The digital images produced are compared to those obtained with the SEM. Image processing software, a crucial component of this method, allows complete control of graphic data, making graphic fraud a larger threat than ever before. Issues in ethics stemming from the application of this technology to scientific analysis are considered.     

瓣后血流湍流剪应力在体无创测量技术

本文提出并实现了基于彩色超声多普勒速度图像分析的瓣后血流湍流剪应力的在体无创测量的数值方法及软件。首先通过图像处理和图像分析得到待测位点至少5个心动周期的速度曲线，然后在每个周期获取该位点N(N≥5)个时刻的瞬时速度信息，由此计算该位点的速度扰动并进一步计算法向雷诺应力和湍流剪应力。软件包括图像捕获、图像预处理、交互式速度获取以及湍流剪应力计算等功能，并使用了多媒体数据库对数据和图像进行管理。实际应用表明，该方法和软件是一种方便可行的在体无创测量瓣后血流湍流剪应力的手段，可为一些瓣膜疾病的诊断和人工心瓣的性能评估提供有效的血液动力学依据。     

基于模糊空间的骨膜显微图像识别

医学图像的目标描述往往是带有模糊性的。随着计算技术的发展,模糊逻辑理论渐被引入医学图像处理的理论研究和实际应用中。而采色医学图像和灰度图像具有不同的空间表达和模糊描述方式,其分析处理方法也有区别。我们阐述了一种基于模糊空间的彩色色骨膜显微图像识别,提出用模糊算法进行彩色图像增强、特征撮和自动分割,完成了对骨膜显微图像中的骨细胞的分割过程。通过对多幅实际采集的彩色图像进行处理,实验结果结果表明是一种     

Orientation-independent differential interference contrast microscopy and its combination with an orientation-independent polarization system

We describe a combined orientation-independent differential interference contrast OI-DIC and polarization microscope and its biological applications. Several conventional DIC images were recorded with the specimen oriented in different directions followed by digital alignment and processing of the images. Then the obtained images are used for computation of the phase gradient magnitude and azimuth distribution and, further, the phase image. The OI-DIC images were obtained using optics having numerical aperture (NA) 1.4, thus achieving a level of resolution not previously achieved with phase contrast or interference microscope. The combined system yields two complementary phase images of thin optical sections of the specimen: distribution of refractive index and distribution of birefringence due to anisotropy of the cell structure. For instance, in a live dividing cell, the OI-DIC image clearly shows the detailed shape of the chromosomes, while the polarization image quantitatively depicts the distribution of birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell. We present pseudo-color combined images of a crane fly spermatocyte at diakinesis and metaphase of meiosis I. Those images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live cells without perturbation associated with staining or fluorescent labeling.     

Single Particle Tracking Across Sequences of Microscopical Images: Application to Platelet Adhesion Under Flow

A versatile and automated image processing technique and data extraction procedure from videomicroscopic data is presented. The motivation is a detailed quantification of blood platelet adhesion from laminar flow onto a surface. The characteristics of the system under observation (type of cells, their speed of movement, and the quality of the optical image to analyze) provided the criteria for developing a new procedure enabling tracking for long image sequences. Specific features of the novel method include: automatic segmentation methodology which removes operator bias; platelet recognition across the series of images based on a probability density function (two-dimensional, Gaussian-like) tailored to the physics of platelet motion on the surface; options to automatically tune the procedure parameters to explore different applications; integrated analysis of the results (platelet trajectories) to obtain relevant information, such as deposition and removal rates, displacement distributions, pause times and rolling velocities. Synthetic images, providing known reference conditions, are used to test the method. The algorithm operation is illustrated by application to images obtained by fluorescence microscopy of the interaction between platelets and von Willebrand factor-coated surfaces in parallel-plate flow chambers. Potentials and limits are discussed, together with evaluation of errors resulting from an inaccurate tracking.     

Recommendations for the biopsy procedure and assessment of skeletal muscle biopsies

 Muscle biopsy has a valuable diagnostic role in many neuromuscular diseases, but it is an invasive investigation that should not be undertaken lightly. Furthermore, the biopsy procedure and subsequent laboratory processing of the specimen may significantly influence the results. The following guide is designed to optimise the diagnostic information that can be obtained from muscle biopsy. It covers the whole procedure, from selection of the biopsy site and technique of biopsy, progressing through freezing of the specimen for histochemistry, and the role of immunocytochemistry and electron microscopy. Finally, the contents of a model biopsy report are considered. Received: 7 April 1997 / Accepted: 7 April 1997     

Measures of complexity for cancellous bone.

The problem of quantifying the structure of cancellous bone has been addressed in the past by histomorphometry and more recently by imaging techniques using X-ray attenuation. The current approaches compute and describe parts of the construction of the trabecular net. We developed a new technique which quantifies cancellous bone of human lumbar vertebrae as a whole. The interactions, transactions, and interrelationships of all parts of the structural composition of the trabeculae are accounted for and quantified. The method is based on the concept of structural complexity within the framework of nonlinear dynamics. The methodology was developed by using axial high resolution computed tomography images. The technique was transferred to quantitative computed tomography images and is based on the non-invasive assessment of 50 human L3 specimens. The value of Houndsfield units per pixel representing trabecular bone of the vertebrae was transformed into color-encoded and alphabet-encoded symbols. The procedure of transformation of the X-ray attenuation pixels into symbols was necessary as a basis on which measures of complexity were introduced to assess the composition of symbols within the images. The development of a generalization of symbolic dynamics, a mathematical method, to work with two-dimensional images was a prerequisite. The results of this study demonstrate that the structural composition of cancellous bone declines more rapidly than bone mineral density during the loss of bone. This outcome strongly suggests an exponential relationship between bone mineral density and the architectural composition of cancellous bone. Normal trabecular bone has a complex ordered structure. The structural composition during the osteopenic phase of bone loss is characterized by lower structural complexity and a significantly higher level of architectural disorder. A high grade of osteoporosis leads again to an ordered structure, although its structural complexity is minimal.