Dense nuclei segmentation based on graph cut and convexity–concavity analysis

With the rapid advancement of 3D confocal imaging technology, more and more 3D cellular images will be available. However, robust and automatic extraction of nuclei shape may be hindered by a highly cluttered environment, as for example, in fly eye tissues. In this paper, we present a novel and efficient nuclei segmentation algorithm based on the combination of graph cut and convex shape assumption. The main characteristic of the algorithm is that it segments nuclei foreground using a graph‐cut algorithm with our proposed new initialization method and splits overlapping or touching cell nuclei by simple convexity and concavity analysis. Experimental results show that the proposed algorithm can segment complicated nuclei clumps effectively in our fluorescent fruit fly eye images. Evaluation on a public hand‐labelled 2D benchmark demonstrates substantial quantitative improvement over other methods. For example, the proposed method achieves a 3.2 Hausdorff distance decrease and a 1.8 decrease in the merged nuclei error per slice.     

Detection of tubule boundaries based on circular shortest path and polar‐transformation of arbitrary shapes

In studies of germ cell transplantation, counting cells and measuring tubule diameters from different populations using labelled antibodies are important measurement processes. However, it is slow and sanity grinding to do these tasks manually. This paper proposes a way to accelerate these processes using a new image analysis framework based on several novel algorithms: centre points detection of tubules, tubule shape classification, skeleton‐based polar‐transformation, boundary weighting of polar‐transformed image, and circular shortest path smoothing. The framework has been tested on a dataset consisting of 27 images which contain a total of 989 tubules. Experiments show that the detection results of our algorithm are very close to the results obtained manually and the novel approach can achieve a better performance than two existing methods.     

An automatic segmentation algorithm for 3D cell cluster splitting using volumetric confocal images

With the rapid advance of three-dimensional (3D) confocal imaging technology, more and more 3D cellular images will be available. Segmentation of intact cells is a critical task in automated image analysis and quantification of cellular microscopic images. One of the major complications in the automatic segmentation of cellular images arises due to the fact that cells are often closely clustered. Several algorithms are proposed for segmenting cell clusters but most of them are 2D based. In other words, these algorithms are designed to segment 2D cell clusters from a single image. Given 2D segmentation methods developed, they can certainly be applied to each image slice with the 3D cellular volume to obtain the segmented cell clusters. Apparently, in such case, the 3D depth information with the volumetric images is not really used. Often, 3D reconstruction is conducted after the individualized segmentation to build the 3D cellular models from segmented 2D cellular contours. Such 2D native process is not appropriate as stacking of individually segmented 2D cells or nuclei do not necessarily form the correct and complete 3D cells or nuclei in 3D. This paper proposes a novel and efficient 3D cluster splitting algorithm based on concavity analysis and interslice spatial coherence. We have taken the advantage of using the 3D boundary points detected using higher order statistics as an input contour for performing the 3D cluster splitting algorithm. The idea is to separate the touching or overlapping cells or nuclei in a 3D native way. Experimental results show the efficiency of our algorithm for 3D microscopic cellular images.     

Path Searching Based Crease Detection for Large Scale Scanned Document Images

Since the large size documents are usually folded for preservation, creases will occur in the scanned images. In this paper, a crease detection method is proposed to locate the crease pixels for further processing. According to the imaging process of contactless scanners, the shading on both sides of the crease usually varies a lot. Based on this observation, a convex hull based algorithm is adopted to extract the shading information of the scanned image. Then, the possible crease path can be achieved by applying the vertical filter and morphological operations on the shading image. Finally, the accurate crease is detected via Dijkstra path searching. Experimental results on the dataset of real scanned newspapers demonstrate that the proposed method can obtain accurate locations of the creases in the large size document images.     

An automated feedback system with the hybrid model of scoring and classification for solving over-segmentation problems in RNAi high content screening

Background: High content screening (HCS) via automated fluorescence microscopy is a powerful technology for generating cellular images that are rich in phenotypic information. RNA interference is a revolutionary approach for silencing gene expression and has become an important method for studying genes through RNA interference‐induced cellular phenotype analysis. The convergence of the two technologies has led to large‐scale, image‐based studies of cellular phenotypes under systematic perturbations of RNA interference. However, existing high content screening image analysis tools are inadequate to extract content regarding cell morphology from the complex images, thus they limit the potential of genome‐wide RNA interference high content screening screening for simple marker readouts. In particular, over‐segmentation is one of the persistent problems of cell segmentation; this paper describes a new method to alleviate this problem. Methods: To solve the issue of over‐segmentation, we propose a novel feedback system with a hybrid model for automated cell segmentation of images from high content screening. A Hybrid learning model is developed based on three scoring models to capture specific characteristics of over‐segmented cells. Dead nuclei are also removed through a statistical model. Results: Experimental validation showed that the proposed method had 93.7% sensitivity and 94.23% specificity. When applied to a set of images of F‐actin‐stained Drosophila cells, 91.3% of over‐segmented cells were detected and only 2.8% were under‐segmented. Conclusions: The proposed feedback system significantly reduces over‐segmentation of cell bodies caused by over‐segmented nuclei, dead nuclei, and dividing cells. This system can be used in the automated analysis system of high content screening images.     

偏振探测系统的图像配准

分振幅式偏振探测成像系统的各分光路图像之间存在位置误差,率先完成各分光路图像之间的图像配准是进行偏振探测的前提条件。针对探测过程中,目标特征不明显、图像特征难以提取、各分光路图像间灰度变化较大的问题,提出适用于分振幅式偏振探测成像系统各分光路图像的相似性度量函数,并在此基础上,完成各分光路图像间的配准工作。首先,根据图像间的位置误差会造成偏振信息图像中出现信息异常区域的原理,研究了相似性度量函数的提取算法;接着,根据探测系统的各分光路的成像特点,确定图像间的几何变换参数;以遗传算法作为参数优化搜索算法,搜索得到最优的几何变换参数,完成整个图像配准算法的设计;最后,分别利用构造图像和实际采集图像,对配准算法进行了验证,并以图像间互信息值(MI)衡量图像配准的精度。实验结果表明:配准后的构造图像的MI为2.692 5,高于特征配准方法的实现精度;实际采集图像配准后的MI达1.849 3,同样高于特征配准方法的实现精度。基本满足偏振探测系统的图像配准需求。     

Fusion of GFP and phase contrast images with complex shearlet transform and Haar wavelet‐based energy rule

Image fusion techniques can integrate the information from different imaging modalities to get a composite image which is more suitable for human visual perception and further image processing tasks. Fusing green fluorescent protein (GFP) and phase contrast images is very important for subcellular localization, functional analysis of protein and genome expression. The fusion method of GFP and phase contrast images based on complex shearlet transform (CST) is proposed in this paper. Firstly the GFP image is converted to IHS model and its intensity component is obtained. Secondly the CST is performed on the intensity component and the phase contrast image to acquire the low‐frequency subbands and the high‐frequency subbands. Then the high‐frequency subbands are merged by the absolute‐maximum rule while the low‐frequency subbands are merged by the proposed Haar wavelet‐based energy (HWE) rule. Finally the fused image is obtained by performing the inverse CST on the merged subbands and conducting IHS‐to‐RGB conversion. The proposed fusion method is tested on a number of GFP and phase contrast images and compared with several popular image fusion methods. The experimental results demonstrate that the proposed fusion method can provide better fusion results in terms of subjective quality and objective evaluation.     

改进Demons算法的非刚性医学图像配准

非刚性配准是医学图像处理的一个重要的研究方向。基于光流场模型的Demons算法由于仅依赖图像灰度梯度使图像变形,当缺乏梯度信息时图像的变形方向不能确定,因而容易造成误配准,且该算法只适合于单模态图像配准。本文针对最大互信息配准方法在多模态刚性配准中的成功应用,提出了一种可用于多模态图像配准的改进Demons算法。该方法在原有驱动图像变形力的基础上,增加两幅图像间互信息对当前变换的梯度作为附加力作用,使浮动图像向两图像间互信息增大的方向变形,正确地配准图像。为避免陷入局部极值并提高算法的运行速度,该方法在多分辨率策略下实现。使用单模态、多模态图像分别进行实验来验证此算法,并与原始Demons算法进行比较,实验表明,该方法能够快速地产生准确的配准变换。     

齿面干涉测量系统的成像畸变分析与光路优化

针对由物体光路误差导致的齿面像形状畸变现象,利用精确光线追迹算法分析了可能导致齿面像形状畸变的三种因素,明确了齿面像形状畸变的主要误差来源,建立了齿面像形状畸变的判断模型,确立了齿面像形状畸变和齿轮轴线倾斜之间的映射关系,提出了基于齿面像形状畸变特征的物体光路优化方法。最后进行了实验验证,结果表明误差控制在2%左右,证明了所提方法的合理性与正确性。     

Semi-automated imaging system to quantitate estrogen and progesterone receptor immunoreactivity in human breast cancer

A semi‐automated imaging system is described to quantitate estrogen and progesterone receptor immunoreactivity in human breast cancer. The system works for any conventional method of image acquisition using microscopic slides that have been processed for immunohistochemical analysis of the estrogen receptor and progesterone receptor. Estrogen receptor and progesterone receptor immunohistochemical staining produce colorimetric differences in nuclear staining that conventionally have been interpreted manually by pathologists and expressed as percentage of positive tumoral nuclei. The estrogen receptor and progesterone receptor status of human breast cancer represent important prognostic and predictive markers of human breast cancer that dictate therapeutic decisions but their subjective interpretation result in interobserver, intraobserver and fatigue variability. Subjective measurements are traditionally limited to a determination of percentage of tumoral nuclei that show positive immunoreactivity. To address these limitations, imaging algorithms utilizing both colorimetric (RGB) as well as intensity (gray scale) determinations were used to analyze pixels of the acquired image. Image acquisition utilized either scanner or microscope with attached digital or analogue camera capable of producing images with a resolution of 20 pixels /10 μ. Areas of each image were screened and the area of interest richest in tumour cells manually selected for image processing. Images were processed initially by JPG conversion of SVS scanned virtual slides or direct JPG photomicrograph capture. Following image acquisition, images were screened for quality, enhanced and processed. The algorithm‐based values for estrogen receptor and progesterone receptor percentage nuclear positivity both strongly correlated with the subjective measurements (intraclass correlation: 0.77; 95% confidence interval: 0.59, 0.95) yet exhibited no interobserver, intraobserver or fatigue variability. In addition the algorithms provided measurements of nuclear estrogen receptor and progesterone receptor staining intensity (mean, mode and median staining intensity of positive staining nuclei), parameters that subjective review could not assess. Other semi‐automated image analysis systems have been used to measure estrogen receptor and progesterone receptor immunoreactivity but these either have required proprietary hardware or have been based on luminosity differences alone. By contrast our algorithms were independent of proprietary hardware and were based on not just luminosity and colour but also many other imaging features including epithelial pattern recognition and nuclear morphology. These features provide a more accurate, versatile and robust imaging analysis platform that can be fully automated in the near future. Because of all these properties, our semi‐automated imaging system ‘adds value’ as a means of measuring these important nuclear biomarkers of human breast cancer.     

Characterization of ovarian nuclei with the parameter of power ratio

The nucleoli and chromatin clumps of ovarian cells contain important features in discriminating malignant cells from normal ones. In geometric properties, the ovarian nucleoli and chromatin clumps appear as irregularly shaped dark spots in the nuclear images from specimens immunohistochemically stained with antibody to Mib-1. Malignant cells often have more active and larger nucleoli and chromatin clumps. However, estimating the size of the nucleoli or chromatin clumps is a difficult task since it is not easy to recognize and accurately separate the regions of nucleoli and chromatin clumps from the rest of the nuclei that are highly irregular and variant in contents and intensities. In this paper, we develop a method to derive a parameter called power ratio that is proportionally related to the size of nucleoli and chromatin clumps based on an ideal nuclear model without the region segmentation of nucleoli or chromatin clumps. Results of characterization of the parameter and comparison between malignant and normal cells are provided.     

一种适用于特征稀少零件的图像精确拼接方法

针对特征稀少零件的图像精确拼接难题,提出了一种基于相位相关法和闭环运动控制的图像精确拼接方法,以充分发挥软硬件的综合优势。该方法以具有足够特征信息的零件为对象,获取成像系统分别沿X轴和Y轴运动时的零件等距序列图像并预处理,再利用相位相关法求解图像配准参数。在闭环运动控制系统的良好重复定位精度支持下,将上述图像配准参数视为系统配准参数,以进行特征稀少零件的图像拼接。典型零件的图像拼接实验表明,该拼接方法用于特征稀少零件的图像拼接具有无像素级拼接错位和拼接速度快等优点。     

Segmentation of nuclei and cells using membrane related protein markers

Segmenting individual cell nuclei from microscope images normally involves volume labelling of the nuclei with a DNA stain. However, this method often fails when the nuclei are tightly clustered in the tissue, because there is little evidence from the images on where the borders of the nuclei are. In this paper we present a method which solves this limitation and furthermore enables segmentation of whole cells. Instead of using volume stains, we used stains that specifically label the surface of nuclei or cells: lamins for the nuclear envelope and alpha-6 or beta-1 integrins for the cellular surface. The segmentation is performed by identifying unique seeds for each nucleus/cell and expanding the boundaries of the seeds until they reach the limits of the nucleus/cell, as delimited by the lamin or integrin staining, using gradient-curvature flow techniques. We tested the algorithm using computer-generated objects to evaluate its robustness against noise and applied it to cells in culture and to tissue specimens. In all the cases that we present the algorithm gave accurate results.     

Overlapping nuclei segmentation based on Bayesian networks and stepwise merging strategy

This paper presents a new approach to the segmentation of fluorescence in situ hybridization images. First, to segment the cell nuclei from the background, a threshold is estimated using a Gaussian mixture model and maximizing the likelihood function of the grey values for the cell images. After the nuclei segmentation, the overlapping and isolated nuclei are classified to facilitate a more accurate nuclei analysis. To do this, the morphological features of the nuclei, such their compactness, smoothness and moments, are extracted from training data to generate three probability distribution functions that are then applied to a Bayesian network as evidence. Following the nuclei classification, the overlapping nuclei are segmented into isolated nuclei using an intensity gradient transform and watershed algorithm. A new stepwise merging strategy is also proposed to merge fragments into a major nucleus. Experimental results using fluorescence in situ hybridization images confirm that the proposed system produced better segmentation results when compared to previous methods, because of the nuclei classification before separating the overlapping nuclei.     

An image processing pipeline to detect and segment nuclei in muscle fiber microscopic images

Muscle fiber images play an important role in the medical diagnosis and treatment of many muscular diseases. The number of nuclei in skeletal muscle fiber images is a key bio‐marker of the diagnosis of muscular dystrophy. In nuclei segmentation one primary challenge is to correctly separate the clustered nuclei. In this article, we developed an image processing pipeline to automatically detect, segment, and analyze nuclei in microscopic image of muscle fibers. The pipeline consists of image pre‐processing, identification of isolated nuclei, identification and segmentation of clustered nuclei, and quantitative analysis. Nuclei are initially extracted from background by using local Otsu's threshold. Based on analysis of morphological features of the isolated nuclei, including their areas, compactness, and major axis lengths, a Bayesian network is trained and applied to identify isolated nuclei from clustered nuclei and artifacts in all the images. Then a two‐step refined watershed algorithm is applied to segment clustered nuclei. After segmentation, the nuclei can be quantified for statistical analysis. Comparing the segmented results with those of manual analysis and an existing technique, we find that our proposed image processing pipeline achieves good performance with high accuracy and precision. The presented image processing pipeline can therefore help biologists increase their throughput and objectivity in analyzing large numbers of nuclei in muscle fiber images. Microsc. Res. Tech. 77:547–559, 2014. © 2014 Wiley Periodicals, Inc.     

A statistical approach for intensity loss compensation of confocal microscopy images

In this paper, a probabilistic technique for compensation of intensity loss in confocal microscopy images is presented. For single-colour-labelled specimen, confocal microscopy images are modelled as a mixture of two Gaussian probability distribution functions, one representing the background and another corresponding to the foreground. Images are segmented into foreground and background by applying Expectation Maximization algorithm to the mixture. Final intensity compensation is carried out by scaling and shifting the original intensities with the help of parameters estimated for the foreground. Since foreground is separated to calculate the compensation parameters, the method is effective even when image structure changes from frame to frame. As intensity decay function is not used, complexity associated with estimation of the intensity decay function parameters is eliminated. In addition, images can be compensated out of order, as only information from the reference image is required for the compensation of any image. These properties make our method an ideal tool for intensity compensation of confocal microscopy images that suffer intensity loss due to absorption/scattering of light as well as photobleaching and the image can change structure from optical/temporal section-to-section due to changes in the depth of specimen or due to a live specimen. The proposed method was tested with a number of confocal microscopy image stacks and results are presented to demonstrate the effectiveness of the method.     

Automated nuclei segmentation of malignant using level sets

Segmentation of objects from a noisy and complex image is still a challenging task that needs to be addressed. This article proposed a new method to detect and segment nuclei to determine whether they are malignant or not (determination of the region of interest, noise removal, enhance the image, candidate detection is employed on the centroid transform to evaluate the centroid of each object, the level set [LS] is applied to segment the nuclei). The proposed method consists of three main stages: preprocessing, seed detection, and segmentation. Preprocessing stage involves the preparation of the image conditions to ensure that they meet the segmentation requirements. Seed detection detects the seed point to be used in the segmentation stage, which refers to the process of segmenting the nuclei using the LS method. In this research work, 58 H&E breast cancer images from the UCSB Bio‐Segmentation Benchmark dataset are evaluated. The proposed method reveals the high performance and accuracy in comparison to the techniques reported in literature. The experimental results are also harmonized with the ground truth images.     

Fusion of mis‐registered GFP and phase contrast images with convolutional sparse representation and adaptive region energy rule

Biomedical image fusion is the process of combining the information from different imaging modalities to get a synthetic image. Fusion of phase contrast and green fluorescent protein (GFP) images is significant to predict the role of unknown proteins, analyze the function of proteins, locate the subcellular structure, and so forth. Generally, the fusion performance largely depends on the registration of GFP and phase contrast images. However, accurate registration of multi‐modal images is a very challenging task. Hence, we propose a novel fusion method based on convolutional sparse representation (CSR) to fuse the mis‐registered GFP and phase contrast images. At first, the GFP and phase contrast images are decomposed by CSR to get the coefficients of base layers and detail layers. Secondly, the coefficients of detail layers are fused by the sum modified Laplacian (SML) rule while the coefficients of base layers are fused by the proposed adaptive region energy (ARE) rule. ARE rule is calculated by discussion mechanism based brain storm optimization (DMBSO) algorithm. Finally, the fused image is achieved by carrying out the inverse CSR. The proposed fusion method is tested on 100 pairs of mis‐registered GFP and phase contrast images. The experimental results reveal that our proposed fusion method exhibits better fusion results and superior robustness than several existing fusion methods.     

A novel segmentation approach for noisy medical images using Intuitionistic fuzzy divergence with neighbourhood‐based membership function

Medical image segmentation demands higher segmentation accuracy especially when the images are affected by noise. This paper proposes a novel technique to segment medical images efficiently using an intuitionistic fuzzy divergence–based thresholding. A neighbourhood‐based membership function is defined here. The intuitionistic fuzzy divergence–based image thresholding technique using the neighbourhood‐based membership functions yield lesser degradation of segmentation performance in noisy environment. Its ability in handling noisy images has been validated. The algorithm is independent of any parameter selection. Moreover, it provides robustness to both additive and multiplicative noise. The proposed scheme has been applied on three types of medical image datasets in order to establish its novelty and generality. The performance of the proposed algorithm has been compared with other standard algorithms viz. Otsu's method, fuzzy C‐means clustering, and fuzzy divergence–based thresholding with respect to (1) noise‐free images and (2) ground truth images labelled by experts/clinicians. Experiments show that the proposed methodology is effective, more accurate and efficient for segmenting noisy images.     

Constraint factor graph cut–based active contour method for automated cellular image segmentation in RNAi screening

Image‐based, high throughput genome‐wide RNA interference (RNAi) experiments are increasingly carried out to facilitate the understanding of gene functions in intricate biological processes. Automated screening of such experiments generates a large number of images with great variations in image quality, which makes manual analysis unreasonably time‐consuming. Therefore, effective techniques for automatic image analysis are urgently needed, in which segmentation is one of the most important steps. This paper proposes a fully automatic method for cells segmentation in genome‐wide RNAi screening images. The method consists of two steps: nuclei and cytoplasm segmentation. Nuclei are extracted and labelled to initialize cytoplasm segmentation. Since the quality of RNAi image is rather poor, a novel scale‐adaptive steerable filter is designed to enhance the image in order to extract long and thin protrusions on the spiky cells. Then, constraint factor GCBAC method and morphological algorithms are combined to be an integrated method to segment tight clustered cells. Compared with the results obtained by using seeded watershed and the ground truth, that is, manual labelling results by experts in RNAi screening data, our method achieves higher accuracy. Compared with active contour methods, our method consumes much less time. The positive results indicate that the proposed method can be applied in automatic image analysis of multi‐channel image screening data.