Multi-tissue and multi-scale approach for nuclei segmentation in H&E stained images.

PubMed

Salvi, Massimo; Molinari, Filippo

2018-06-20

Accurate nuclei detection and segmentation in histological images is essential for many clinical purposes. While manual annotations are time-consuming and operator-dependent, full automated segmentation remains a challenging task due to the high variability of cells intensity, size and morphology. Most of the proposed algorithms for the automated segmentation of nuclei were designed for specific organ or tissues. The aim of this study was to develop and validate a fully multiscale method, named MANA (Multiscale Adaptive Nuclei Analysis), for nuclei segmentation in different tissues and magnifications. MANA was tested on a dataset of H&E stained tissue images with more than 59,000 annotated nuclei, taken from six organs (colon, liver, bone, prostate, adrenal gland and thyroid) and three magnifications (10×, 20×, 40×). Automatic results were compared with manual segmentations and three open-source software designed for nuclei detection. For each organ, MANA obtained always an F1-score higher than 0.91, with an average F1 of 0.9305 ± 0.0161. The average computational time was about 20 s independently of the number of nuclei to be detected (anyway, higher than 1000), indicating the efficiency of the proposed technique. To the best of our knowledge, MANA is the first fully automated multi-scale and multi-tissue algorithm for nuclei detection. Overall, the robustness and versatility of MANA allowed to achieve, on different organs and magnifications, performances in line or better than those of state-of-art algorithms optimized for single tissues.

Improved segmentation of abnormal cervical nuclei using a graph-search based approach

NASA Astrophysics Data System (ADS)

Zhang, Ling; Liu, Shaoxiong; Wang, Tianfu; Chen, Siping; Sonka, Milan

2015-03-01

Reliable segmentation of abnormal nuclei in cervical cytology is of paramount importance in automation-assisted screening techniques. This paper presents a general method for improving the segmentation of abnormal nuclei using a graph-search based approach. More specifically, the proposed method focuses on the improvement of coarse (initial) segmentation. The improvement relies on a transform that maps round-like border in the Cartesian coordinate system into lines in the polar coordinate system. The costs consisting of nucleus-specific edge and region information are assigned to the nodes. The globally optimal path in the constructed graph is then identified by dynamic programming. We have tested the proposed method on abnormal nuclei from two cervical cell image datasets, Herlev and H and E stained liquid-based cytology (HELBC), and the comparative experiments with recent state-of-the-art approaches demonstrate the superior performance of the proposed method.

Boundary-to-Marker Evidence-Controlled Segmentation and MDL-Based Contour Inference for Overlapping Nuclei.

PubMed

Song, Jie; Xiao, Liang; Lian, Zhichao

2017-03-01

This paper presents a novel method for automated morphology delineation and analysis of cell nuclei in histopathology images. Combining the initial segmentation information and concavity measurement, the proposed method first segments clusters of nuclei into individual pieces, avoiding segmentation errors introduced by the scale-constrained Laplacian-of-Gaussian filtering. After that a nuclear boundary-to-marker evidence computing is introduced to delineate individual objects after the refined segmentation process. The obtained evidence set is then modeled by the periodic B-splines with the minimum description length principle, which achieves a practical compromise between the complexity of the nuclear structure and its coverage of the fluorescence signal to avoid the underfitting and overfitting results. The algorithm is computationally efficient and has been tested on the synthetic database as well as 45 real histopathology images. By comparing the proposed method with several state-of-the-art methods, experimental results show the superior recognition performance of our method and indicate the potential applications of analyzing the intrinsic features of nuclei morphology.

Semi-automated segmentation of neuroblastoma nuclei using the gradient energy tensor: a user driven approach

NASA Astrophysics Data System (ADS)

Kromp, Florian; Taschner-Mandl, Sabine; Schwarz, Magdalena; Blaha, Johanna; Weiss, Tamara; Ambros, Peter F.; Reiter, Michael

2015-02-01

We propose a user-driven method for the segmentation of neuroblastoma nuclei in microscopic fluorescence images involving the gradient energy tensor. Multispectral fluorescence images contain intensity and spatial information about antigene expression, fluorescence in situ hybridization (FISH) signals and nucleus morphology. The latter serves as basis for the detection of single cells and the calculation of shape features, which are used to validate the segmentation and to reject false detections. Accurate segmentation is difficult due to varying staining intensities and aggregated cells. It requires several (meta-) parameters, which have a strong influence on the segmentation results and have to be selected carefully for each sample (or group of similar samples) by user interactions. Because our method is designed for clinicians and biologists, who may have only limited image processing background, an interactive parameter selection step allows the implicit tuning of parameter values. With this simple but intuitive method, segmentation results with high precision for a large number of cells can be achieved by minimal user interaction. The strategy was validated on handsegmented datasets of three neuroblastoma cell lines.

Automation-assisted cervical cancer screening in manual liquid-based cytology with hematoxylin and eosin staining.

PubMed

Zhang, Ling; Kong, Hui; Ting Chin, Chien; Liu, Shaoxiong; Fan, Xinmin; Wang, Tianfu; Chen, Siping

2014-03-01

Current automation-assisted technologies for screening cervical cancer mainly rely on automated liquid-based cytology slides with proprietary stain. This is not a cost-efficient approach to be utilized in developing countries. In this article, we propose the first automation-assisted system to screen cervical cancer in manual liquid-based cytology (MLBC) slides with hematoxylin and eosin (H&E) stain, which is inexpensive and more applicable in developing countries. This system consists of three main modules: image acquisition, cell segmentation, and cell classification. First, an autofocusing scheme is proposed to find the global maximum of the focus curve by iteratively comparing image qualities of specific locations. On the autofocused images, the multiway graph cut (GC) is performed globally on the a* channel enhanced image to obtain cytoplasm segmentation. The nuclei, especially abnormal nuclei, are robustly segmented by using GC adaptively and locally. Two concave-based approaches are integrated to split the touching nuclei. To classify the segmented cells, features are selected and preprocessed to improve the sensitivity, and contextual and cytoplasm information are introduced to improve the specificity. Experiments on 26 consecutive image stacks demonstrated that the dynamic autofocusing accuracy was 2.06 μm. On 21 cervical cell images with nonideal imaging condition and pathology, our segmentation method achieved a 93% accuracy for cytoplasm, and a 87.3% F-measure for nuclei, both outperformed state of the art works in terms of accuracy. Additional clinical trials showed that both the sensitivity (88.1%) and the specificity (100%) of our system are satisfyingly high. These results proved the feasibility of automation-assisted cervical cancer screening in MLBC slides with H&E stain, which is highly desirable in community health centers and small hospitals. © 2013 International Society for Advancement of Cytometry.

Serotonin projection patterns to the cochlear nucleus.

PubMed

Thompson, A M; Thompson, G C

2001-07-13

The cochlear nucleus is well known as an obligatory relay center for primary auditory nerve fibers. Perhaps not so well known is the neural input to the cochlear nucleus from cells containing serotonin that reside near the midline in the midbrain raphe region. Although the specific locations of the main, if not sole, sources of serotonin within the dorsal cochlear nucleus subdivision are known to be the dorsal and median raphe nuclei, sources of serotonin located within other cochlear nucleus subdivisions are not currently known. Anterograde tract tracing was used to label fibers originating from the dorsal and median raphe nuclei while fluorescence immunohistochemistry was used to simultaneously label specific serotonin fibers in cat. Biotinylated dextran amine was injected into the dorsal and median raphe nuclei and was visualized with Texas Red, while serotonin was visualized with fluorescein. Thus, double-labeled fibers were unequivocally identified as serotoninergic and originating from one of the labeled neurons within the dorsal and median raphe nuclei. Double-labeled fiber segments, typically of fine caliber with oval varicosities, were observed in many areas of the cochlear nucleus. They were found in the molecular layer of the dorsal cochlear nucleus, in the small cell cap region, and in the granule cell and external regions of the cochlear nuclei, bilaterally, of all cats. However, the density of these double-labeled fiber segments varied considerably depending upon the exact region in which they were found. Fiber segments were most dense in the dorsal cochlear nucleus (especially in the molecular layer) and the large spherical cell area of the anteroventral cochlear nucleus; they were moderately dense in the small cell cap region; and fiber segments were least dense in the octopus and multipolar cell regions of the posteroventral cochlear nucleus. Because of the presence of labeled fiber segments in subdivisions of the cochlear nucleus other than the dorsal cochlear nucleus, we concluded that the serotoninergic projection pattern to the cochlear nucleus is divergent and non-specific. Double-labeled fiber segments were also present, but sparse, in the superior olive, localized mainly in periolivary regions; this indicated that the divergence of dorsal and median raphe neurons that extends throughout regions of the cochlear nucleus also extended well beyond the cochlear nucleus to include at least the superior olivary complex as well.

Segmentation of White Blood Cells From Microscopic Images Using a Novel Combination of K-Means Clustering and Modified Watershed Algorithm.

PubMed

Ghane, Narjes; Vard, Alireza; Talebi, Ardeshir; Nematollahy, Pardis

2017-01-01

Recognition of white blood cells (WBCs) is the first step to diagnose some particular diseases such as acquired immune deficiency syndrome, leukemia, and other blood-related diseases that are usually done by pathologists using an optical microscope. This process is time-consuming, extremely tedious, and expensive and needs experienced experts in this field. Thus, a computer-aided diagnosis system that assists pathologists in the diagnostic process can be so effective. Segmentation of WBCs is usually a first step in developing a computer-aided diagnosis system. The main purpose of this paper is to segment WBCs from microscopic images. For this purpose, we present a novel combination of thresholding, k-means clustering, and modified watershed algorithms in three stages including (1) segmentation of WBCs from a microscopic image, (2) extraction of nuclei from cell's image, and (3) separation of overlapping cells and nuclei. The evaluation results of the proposed method show that similarity measures, precision, and sensitivity respectively were 92.07, 96.07, and 94.30% for nucleus segmentation and 92.93, 97.41, and 93.78% for cell segmentation. In addition, statistical analysis presents high similarity between manual segmentation and the results obtained by the proposed method.

Cell nuclei and cytoplasm joint segmentation using the sliding band filter.

PubMed

Quelhas, Pedro; Marcuzzo, Monica; Mendonça, Ana Maria; Campilho, Aurélio

2010-08-01

Microscopy cell image analysis is a fundamental tool for biological research. In particular, multivariate fluorescence microscopy is used to observe different aspects of cells in cultures. It is still common practice to perform analysis tasks by visual inspection of individual cells which is time consuming, exhausting and prone to induce subjective bias. This makes automatic cell image analysis essential for large scale, objective studies of cell cultures. Traditionally the task of automatic cell analysis is approached through the use of image segmentation methods for extraction of cells' locations and shapes. Image segmentation, although fundamental, is neither an easy task in computer vision nor is it robust to image quality changes. This makes image segmentation for cell detection semi-automated requiring frequent tuning of parameters. We introduce a new approach for cell detection and shape estimation in multivariate images based on the sliding band filter (SBF). This filter's design makes it adequate to detect overall convex shapes and as such it performs well for cell detection. Furthermore, the parameters involved are intuitive as they are directly related to the expected cell size. Using the SBF filter we detect cells' nucleus and cytoplasm location and shapes. Based on the assumption that each cell has the same approximate shape center in both nuclei and cytoplasm fluorescence channels, we guide cytoplasm shape estimation by the nuclear detections improving performance and reducing errors. Then we validate cell detection by gathering evidence from nuclei and cytoplasm channels. Additionally, we include overlap correction and shape regularization steps which further improve the estimated cell shapes. The approach is evaluated using two datasets with different types of data: a 20 images benchmark set of simulated cell culture images, containing 1000 simulated cells; a 16 images Drosophila melanogaster Kc167 dataset containing 1255 cells, stained for DNA and actin. Both image datasets present a difficult problem due to the high variability of cell shapes and frequent cluster overlap between cells. On the Drosophila dataset our approach achieved a precision/recall of 95%/69% and 82%/90% for nuclei and cytoplasm detection respectively and an overall accuracy of 76%.

Nucleus and cytoplasm segmentation in microscopic images using K-means clustering and region growing.

PubMed

Sarrafzadeh, Omid; Dehnavi, Alireza Mehri

2015-01-01

Segmentation of leukocytes acts as the foundation for all automated image-based hematological disease recognition systems. Most of the time, hematologists are interested in evaluation of white blood cells only. Digital image processing techniques can help them in their analysis and diagnosis. The main objective of this paper is to detect leukocytes from a blood smear microscopic image and segment them into their two dominant elements, nucleus and cytoplasm. The segmentation is conducted using two stages of applying K-means clustering. First, the nuclei are segmented using K-means clustering. Then, a proposed method based on region growing is applied to separate the connected nuclei. Next, the nuclei are subtracted from the original image. Finally, the cytoplasm is segmented using the second stage of K-means clustering. The results indicate that the proposed method is able to extract the nucleus and cytoplasm regions accurately and works well even though there is no significant contrast between the components in the image. In this paper, a method based on K-means clustering and region growing is proposed in order to detect leukocytes from a blood smear microscopic image and segment its components, the nucleus and the cytoplasm. As region growing step of the algorithm relies on the information of edges, it will not able to separate the connected nuclei more accurately in poor edges and it requires at least a weak edge to exist between the nuclei. The nucleus and cytoplasm segments of a leukocyte can be used for feature extraction and classification which leads to automated leukemia detection.

Nucleus and cytoplasm segmentation in microscopic images using K-means clustering and region growing

PubMed Central

Sarrafzadeh, Omid; Dehnavi, Alireza Mehri

2015-01-01

Background: Segmentation of leukocytes acts as the foundation for all automated image-based hematological disease recognition systems. Most of the time, hematologists are interested in evaluation of white blood cells only. Digital image processing techniques can help them in their analysis and diagnosis. Materials and Methods: The main objective of this paper is to detect leukocytes from a blood smear microscopic image and segment them into their two dominant elements, nucleus and cytoplasm. The segmentation is conducted using two stages of applying K-means clustering. First, the nuclei are segmented using K-means clustering. Then, a proposed method based on region growing is applied to separate the connected nuclei. Next, the nuclei are subtracted from the original image. Finally, the cytoplasm is segmented using the second stage of K-means clustering. Results: The results indicate that the proposed method is able to extract the nucleus and cytoplasm regions accurately and works well even though there is no significant contrast between the components in the image. Conclusions: In this paper, a method based on K-means clustering and region growing is proposed in order to detect leukocytes from a blood smear microscopic image and segment its components, the nucleus and the cytoplasm. As region growing step of the algorithm relies on the information of edges, it will not able to separate the connected nuclei more accurately in poor edges and it requires at least a weak edge to exist between the nuclei. The nucleus and cytoplasm segments of a leukocyte can be used for feature extraction and classification which leads to automated leukemia detection. PMID:26605213

High content image analysis for human H4 neuroglioma cells exposed to CuO nanoparticles.

PubMed

Li, Fuhai; Zhou, Xiaobo; Zhu, Jinmin; Ma, Jinwen; Huang, Xudong; Wong, Stephen T C

2007-10-09

High content screening (HCS)-based image analysis is becoming an important and widely used research tool. Capitalizing this technology, ample cellular information can be extracted from the high content cellular images. In this study, an automated, reliable and quantitative cellular image analysis system developed in house has been employed to quantify the toxic responses of human H4 neuroglioma cells exposed to metal oxide nanoparticles. This system has been proved to be an essential tool in our study. The cellular images of H4 neuroglioma cells exposed to different concentrations of CuO nanoparticles were sampled using IN Cell Analyzer 1000. A fully automated cellular image analysis system has been developed to perform the image analysis for cell viability. A multiple adaptive thresholding method was used to classify the pixels of the nuclei image into three classes: bright nuclei, dark nuclei, and background. During the development of our image analysis methodology, we have achieved the followings: (1) The Gaussian filtering with proper scale has been applied to the cellular images for generation of a local intensity maximum inside each nucleus; (2) a novel local intensity maxima detection method based on the gradient vector field has been established; and (3) a statistical model based splitting method was proposed to overcome the under segmentation problem. Computational results indicate that 95.9% nuclei can be detected and segmented correctly by the proposed image analysis system. The proposed automated image analysis system can effectively segment the images of human H4 neuroglioma cells exposed to CuO nanoparticles. The computational results confirmed our biological finding that human H4 neuroglioma cells had a dose-dependent toxic response to the insult of CuO nanoparticles.

New breast cancer prognostic factors identified by computer-aided image analysis of HE stained histopathology images

PubMed Central

Chen, Jia-Mei; Qu, Ai-Ping; Wang, Lin-Wei; Yuan, Jing-Ping; Yang, Fang; Xiang, Qing-Ming; Maskey, Ninu; Yang, Gui-Fang; Liu, Juan; Li, Yan

2015-01-01

Computer-aided image analysis (CAI) can help objectively quantify morphologic features of hematoxylin-eosin (HE) histopathology images and provide potentially useful prognostic information on breast cancer. We performed a CAI workflow on 1,150 HE images from 230 patients with invasive ductal carcinoma (IDC) of the breast. We used a pixel-wise support vector machine classifier for tumor nests (TNs)-stroma segmentation, and a marker-controlled watershed algorithm for nuclei segmentation. 730 morphologic parameters were extracted after segmentation, and 12 parameters identified by Kaplan-Meier analysis were significantly associated with 8-year disease free survival (P < 0.05 for all). Moreover, four image features including TNs feature (HR 1.327, 95%CI [1.001 - 1.759], P = 0.049), TNs cell nuclei feature (HR 0.729, 95%CI [0.537 - 0.989], P = 0.042), TNs cell density (HR 1.625, 95%CI [1.177 - 2.244], P = 0.003), and stromal cell structure feature (HR 1.596, 95%CI [1.142 - 2.229], P = 0.006) were identified by multivariate Cox proportional hazards model to be new independent prognostic factors. The results indicated that CAI can assist the pathologist in extracting prognostic information from HE histopathology images for IDC. The TNs feature, TNs cell nuclei feature, TNs cell density, and stromal cell structure feature could be new prognostic factors. PMID:26022540

Fast globally optimal segmentation of cells in fluorescence microscopy images.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2011-01-01

Accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression in high-throughput screening applications. We propose a new approach for segmenting cell nuclei which is based on active contours and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images of different cell types. We have also performed a quantitative comparison with previous segmentation approaches.

Integrated profiling of three dimensional cell culture models and 3D microscopy

PubMed Central

Bilgin, Cemal Cagatay; Kim, Sun; Leung, Elle; Chang, Hang; Parvin, Bahram

2013-01-01

Motivation: Our goal is to develop a screening platform for quantitative profiling of colony organizations in 3D cell culture models. The 3D cell culture models, which are also imaged in 3D, are functional assays that mimic the in vivo characteristics of the tissue architecture more faithfully than the 2D cultures. However, they also introduce significant computational challenges, with the main barriers being the effects of growth conditions, fixations and inherent complexities in segmentation that need to be resolved in the 3D volume. Results: A segmentation strategy has been developed to delineate each nucleus in a colony that overcomes (i) the effects of growth conditions, (ii) variations in chromatin distribution and (iii) ambiguities formed by perceptual boundaries from adjacent nuclei. The strategy uses a cascade of geometric filters that are insensitive to spatial non-uniformity and partitions a clump of nuclei based on the grouping of points of maximum curvature at the interface of two neighboring nuclei. These points of maximum curvature are clustered together based on their coplanarity and proximity to define dissecting planes that separate the touching nuclei. The proposed curvature-based partitioning method is validated with both synthetic and real data, and is shown to have a superior performance against previous techniques. Validation and sensitivity analysis are coupled with the experimental design that includes a non-transformed cell line and three tumorigenic cell lines, which covers a wide range of phenotypic diversity in breast cancer. Colony profiling, derived from nuclear segmentation, reveals distinct indices for the morphogenesis of each cell line. Availability: All software are developed in ITK/VTK and are available at https://vision.lbl.gov/Software/3DMorphometry. Contact: b_parvin@lbl.gov or hchang@lbl.gov Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24045773

Algorithm for Automatic Segmentation of Nuclear Boundaries in Cancer Cells in Three-Channel Luminescent Images

NASA Astrophysics Data System (ADS)

Lisitsa, Y. V.; Yatskou, M. M.; Apanasovich, V. V.; Apanasovich, T. V.

2015-09-01

We have developed an algorithm for segmentation of cancer cell nuclei in three-channel luminescent images of microbiological specimens. The algorithm is based on using a correlation between fluorescence signals in the detection channels for object segmentation, which permits complete automation of the data analysis procedure. We have carried out a comparative analysis of the proposed method and conventional algorithms implemented in the CellProfiler and ImageJ software packages. Our algorithm has an object localization uncertainty which is 2-3 times smaller than for the conventional algorithms, with comparable segmentation accuracy.

Hit rates and radiation doses to nuclei of bone lining cells from alpha-particle-emitting radionuclides

NASA Technical Reports Server (NTRS)

Polig, E.; Jee, W. S.; Kruglikov, I. L.

1992-01-01

Factors relating the local concentration of a bone-seeking alpha-particle emitter to the mean hit rate have been determined for nuclei of bone lining cells using a Monte Carlo procedure. Cell nuclei were approximated by oblate spheroids with dimensions and location taken from a previous histomorphometric study. The Monte Carlo simulation is applicable for planar and diffuse labels at plane or cylindrical bone surfaces. Additionally, the mean nuclear dose per hit, the dose mean per hit, the mean track segment length and its second moment, the percentage of stoppers, and the frequency distribution of the dose have been determined. Some basic features of the hit statistics for bone lining cells have been outlined, and the consequences of existing standards of radiation protection with regard to the hit frequency to cell nuclei are discussed.

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

PubMed

Guo, Yanen; Xu, Xiaoyin; Wang, Yuanyuan; Wang, Yaming; Xia, Shunren; Yang, Zhong

2014-08-01

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. © 2014 Wiley Periodicals, Inc.

Automatic Cell Segmentation in Fluorescence Images of Confluent Cell Monolayers Using Multi-object Geometric Deformable Model.

PubMed

Yang, Zhen; Bogovic, John A; Carass, Aaron; Ye, Mao; Searson, Peter C; Prince, Jerry L

2013-03-13

With the rapid development of microscopy for cell imaging, there is a strong and growing demand for image analysis software to quantitatively study cell morphology. Automatic cell segmentation is an important step in image analysis. Despite substantial progress, there is still a need to improve the accuracy, efficiency, and adaptability to different cell morphologies. In this paper, we propose a fully automatic method for segmenting cells in fluorescence images of confluent cell monolayers. This method addresses several challenges through a combination of ideas. 1) It realizes a fully automatic segmentation process by first detecting the cell nuclei as initial seeds and then using a multi-object geometric deformable model (MGDM) for final segmentation. 2) To deal with different defects in the fluorescence images, the cell junctions are enhanced by applying an order-statistic filter and principal curvature based image operator. 3) The final segmentation using MGDM promotes robust and accurate segmentation results, and guarantees no overlaps and gaps between neighboring cells. The automatic segmentation results are compared with manually delineated cells, and the average Dice coefficient over all distinguishable cells is 0.88.

Coupled Segmentation of Nuclear and Membrane-bound Macromolecules through Voting and Multiphase Level Set

PubMed Central

Wen, Quan

2014-01-01

Membrane-bound macromolecules play an important role in tissue architecture and cell-cell communication, and is regulated by almost one-third of the genome. At the optical scale, one group of membrane proteins expresses themselves as linear structures along the cell surface boundaries, while others are sequestered; and this paper targets the former group. Segmentation of these membrane proteins on a cell-by-cell basis enables the quantitative assessment of localization for comparative analysis. However, such membrane proteins typically lack continuity, and their intensity distributions are often very heterogeneous; moreover, nuclei can form large clump, which further impedes the quantification of membrane signals on a cell-by-cell basis. To tackle these problems, we introduce a three-step process to (i) regularize the membrane signal through iterative tangential voting, (ii) constrain the location of surface proteins by nuclear features, where clumps of nuclei are segmented through a delaunay triangulation approach, and (iii) assign membrane-bound macromolecules to individual cells through an application of multi-phase geodesic level-set. We have validated our method using both synthetic data and a dataset of 200 images, and are able to demonstrate the efficacy of our approach with superior performance. PMID:25530633

3D Clumped Cell Segmentation Using Curvature Based Seeded Watershed.

PubMed

Atta-Fosu, Thomas; Guo, Weihong; Jeter, Dana; Mizutani, Claudia M; Stopczynski, Nathan; Sousa-Neves, Rui

2016-12-01

Image segmentation is an important process that separates objects from the background and also from each other. Applied to cells, the results can be used for cell counting which is very important in medical diagnosis and treatment, and biological research that is often used by scientists and medical practitioners. Segmenting 3D confocal microscopy images containing cells of different shapes and sizes is still challenging as the nuclei are closely packed. The watershed transform provides an efficient tool in segmenting such nuclei provided a reasonable set of markers can be found in the image. In the presence of low-contrast variation or excessive noise in the given image, the watershed transform leads to over-segmentation (a single object is overly split into multiple objects). The traditional watershed uses the local minima of the input image and will characteristically find multiple minima in one object unless they are specified (marker-controlled watershed). An alternative to using the local minima is by a supervised technique called seeded watershed, which supplies single seeds to replace the minima for the objects. Consequently, the accuracy of a seeded watershed algorithm relies on the accuracy of the predefined seeds. In this paper, we present a segmentation approach based on the geometric morphological properties of the 'landscape' using curvatures. The curvatures are computed as the eigenvalues of the Shape matrix, producing accurate seeds that also inherit the original shape of their respective cells. We compare with some popular approaches and show the advantage of the proposed method.

Efficient globally optimal segmentation of cells in fluorescence microscopy images using level sets and convex energy functionals.

PubMed

Bergeest, Jan-Philip; Rohr, Karl

2012-10-01

In high-throughput applications, accurate and efficient segmentation of cells in fluorescence microscopy images is of central importance for the quantification of protein expression and the understanding of cell function. We propose an approach for segmenting cell nuclei which is based on active contours using level sets and convex energy functionals. Compared to previous work, our approach determines the global solution. Thus, the approach does not suffer from local minima and the segmentation result does not depend on the initialization. We consider three different well-known energy functionals for active contour-based segmentation and introduce convex formulations of these functionals. We also suggest a numeric approach for efficiently computing the solution. The performance of our approach has been evaluated using fluorescence microscopy images from different experiments comprising different cell types. We have also performed a quantitative comparison with previous segmentation approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

Detection of immunocytological markers in photomicroscopic images

NASA Astrophysics Data System (ADS)

Friedrich, David; zur Jacobsmühlen, Joschka; Braunschweig, Till; Bell, André; Chaisaowong, Kraisorn; Knüchel-Clarke, Ruth; Aach, Til

2012-03-01

Early detection of cervical cancer can be achieved through visual analysis of cell anomalies. The established PAP smear achieves a sensitivity of 50-90%, most false negative results are caused by mistakes in the preparation of the specimen or reader variability in the subjective, visual investigation. Since cervical cancer is caused by human papillomavirus (HPV), the detection of HPV-infected cells opens new perspectives for screening of precancerous abnormalities. Immunocytochemical preparation marks HPV-positive cells in brush smears of the cervix with high sensitivity and specificity. The goal of this work is the automated detection of all marker-positive cells in microscopic images of a sample slide stained with an immunocytochemical marker. A color separation technique is used to estimate the concentrations of the immunocytochemical marker stain as well as of the counterstain used to color the nuclei. Segmentation methods based on Otsu's threshold selection method and Mean Shift are adapted to the task of segmenting marker-positive cells and their nuclei. The best detection performance of single marker-positive cells was achieved with the adapted thresholding method with a sensitivity of 95.9%. The contours differed by a modified Hausdorff Distance (MHD) of 2.8 μm. Nuclei of single marker positive cells were detected with a sensitivity of 95.9% and MHD = 1.02 μm.

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

PubMed

Chen, C; Li, H; Zhou, X; Wong, S T C

2008-05-01

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.

Adaptive segmentation of nuclei in H&S stained tendon microscopy

NASA Astrophysics Data System (ADS)

Chuang, Bo-I.; Wu, Po-Ting; Hsu, Jian-Han; Jou, I.-Ming; Su, Fong-Chin; Sun, Yung-Nien

2015-12-01

Tendiopathy is a popular clinical issue in recent years. In most cases like trigger finger or tennis elbow, the pathology change can be observed under H and E stained tendon microscopy. However, the qualitative analysis is too subjective and thus the results heavily depend on the observers. We develop an automatic segmentation procedure which segments and counts the nuclei in H and E stained tendon microscopy fast and precisely. This procedure first determines the complexity of images and then segments the nuclei from the image. For the complex images, the proposed method adopts sampling-based thresholding to segment the nuclei. While for the simple images, the Laplacian-based thresholding is employed to re-segment the nuclei more accurately. In the experiments, the proposed method is compared with the experts outlined results. The nuclei number of proposed method is closed to the experts counted, and the processing time of proposed method is much faster than the experts'.

Multifractal-based nuclei segmentation in fish images.

PubMed

Reljin, Nikola; Slavkovic-Ilic, Marijeta; Tapia, Coya; Cihoric, Nikola; Stankovic, Srdjan

2017-09-01

The method for nuclei segmentation in fluorescence in-situ hybridization (FISH) images, based on the inverse multifractal analysis (IMFA) is proposed. From the blue channel of the FISH image in RGB format, the matrix of Holder exponents, with one-by-one correspondence with the image pixels, is determined first. The following semi-automatic procedure is proposed: initial nuclei segmentation is performed automatically from the matrix of Holder exponents by applying predefined hard thresholding; then the user evaluates the result and is able to refine the segmentation by changing the threshold, if necessary. After successful nuclei segmentation, the HER2 (human epidermal growth factor receptor 2) scoring can be determined in usual way: by counting red and green dots within segmented nuclei, and finding their ratio. The IMFA segmentation method is tested over 100 clinical cases, evaluated by skilled pathologist. Testing results show that the new method has advantages compared to already reported methods.

Glioma grading using cell nuclei morphologic features in digital pathology images

NASA Astrophysics Data System (ADS)

Reza, Syed M. S.; Iftekharuddin, Khan M.

2016-03-01

This work proposes a computationally efficient cell nuclei morphologic feature analysis technique to characterize the brain gliomas in tissue slide images. In this work, our contributions are two-fold: 1) obtain an optimized cell nuclei segmentation method based on the pros and cons of the existing techniques in literature, 2) extract representative features by k-mean clustering of nuclei morphologic features to include area, perimeter, eccentricity, and major axis length. This clustering based representative feature extraction avoids shortcomings of extensive tile [1] [2] and nuclear score [3] based methods for brain glioma grading in pathology images. Multilayer perceptron (MLP) is used to classify extracted features into two tumor types: glioblastoma multiforme (GBM) and low grade glioma (LGG). Quantitative scores such as precision, recall, and accuracy are obtained using 66 clinical patients' images from The Cancer Genome Atlas (TCGA) [4] dataset. On an average ~94% accuracy from 10 fold crossvalidation confirms the efficacy of the proposed method.

An Automatic Segmentation Method Combining an Active Contour Model and a Classification Technique for Detecting Polycomb-group Proteinsin High-Throughput Microscopy Images.

PubMed

Gregoretti, Francesco; Cesarini, Elisa; Lanzuolo, Chiara; Oliva, Gennaro; Antonelli, Laura

2016-01-01

The large amount of data generated in biological experiments that rely on advanced microscopy can be handled only with automated image analysis. Most analyses require a reliable cell image segmentation eventually capable of detecting subcellular structures.We present an automatic segmentation method to detect Polycomb group (PcG) proteins areas isolated from nuclei regions in high-resolution fluorescent cell image stacks. It combines two segmentation algorithms that use an active contour model and a classification technique serving as a tool to better understand the subcellular three-dimensional distribution of PcG proteins in live cell image sequences. We obtained accurate results throughout several cell image datasets, coming from different cell types and corresponding to different fluorescent labels, without requiring elaborate adjustments to each dataset.

A Pulse Coupled Neural Network Segmentation Algorithm for Reflectance Confocal Images of Epithelial Tissue

PubMed Central

Malik, Bilal H.; Jabbour, Joey M.; Maitland, Kristen C.

2015-01-01

Automatic segmentation of nuclei in reflectance confocal microscopy images is critical for visualization and rapid quantification of nuclear-to-cytoplasmic ratio, a useful indicator of epithelial precancer. Reflectance confocal microscopy can provide three-dimensional imaging of epithelial tissue in vivo with sub-cellular resolution. Changes in nuclear density or nuclear-to-cytoplasmic ratio as a function of depth obtained from confocal images can be used to determine the presence or stage of epithelial cancers. However, low nuclear to background contrast, low resolution at greater imaging depths, and significant variation in reflectance signal of nuclei complicate segmentation required for quantification of nuclear-to-cytoplasmic ratio. Here, we present an automated segmentation method to segment nuclei in reflectance confocal images using a pulse coupled neural network algorithm, specifically a spiking cortical model, and an artificial neural network classifier. The segmentation algorithm was applied to an image model of nuclei with varying nuclear to background contrast. Greater than 90% of simulated nuclei were detected for contrast of 2.0 or greater. Confocal images of porcine and human oral mucosa were used to evaluate application to epithelial tissue. Segmentation accuracy was assessed using manual segmentation of nuclei as the gold standard. PMID:25816131

Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

PubMed

Bunyak, Filiz; Palaniappan, Kannappan; Chagin, Vadim; Cardoso, M

2009-01-01

Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.

Automated cell analysis tool for a genome-wide RNAi screen with support vector machine based supervised learning

NASA Astrophysics Data System (ADS)

Remmele, Steffen; Ritzerfeld, Julia; Nickel, Walter; Hesser, Jürgen

2011-03-01

RNAi-based high-throughput microscopy screens have become an important tool in biological sciences in order to decrypt mostly unknown biological functions of human genes. However, manual analysis is impossible for such screens since the amount of image data sets can often be in the hundred thousands. Reliable automated tools are thus required to analyse the fluorescence microscopy image data sets usually containing two or more reaction channels. The herein presented image analysis tool is designed to analyse an RNAi screen investigating the intracellular trafficking and targeting of acylated Src kinases. In this specific screen, a data set consists of three reaction channels and the investigated cells can appear in different phenotypes. The main issue of the image processing task is an automatic cell segmentation which has to be robust and accurate for all different phenotypes and a successive phenotype classification. The cell segmentation is done in two steps by segmenting the cell nuclei first and then using a classifier-enhanced region growing on basis of the cell nuclei to segment the cells. The classification of the cells is realized by a support vector machine which has to be trained manually using supervised learning. Furthermore, the tool is brightness invariant allowing different staining quality and it provides a quality control that copes with typical defects during preparation and acquisition. A first version of the tool has already been successfully applied for an RNAi-screen containing three hundred thousand image data sets and the SVM extended version is designed for additional screens.

Proflavine sensitivity of RNA processing in isolated nuclei.

PubMed Central

Yannarell, A; Niemann, M; Schumm, D E; Webb, T E

1977-01-01

The intercalating agent proflavine inhibits the processing and subsequent release of preformed messenger RNA and ribosomal RNA from isolated liver nuclei to surrogate cytoplasm. The direct effect of proflavine on these processes, as monitored in a reconstituted cell-free system, supports the theory that base-paired segments (i.e. hairpin loops) in the precursor RNA's are involved as recognition sites in nuclear RNA processing. PMID:866181

Segmentation of fluorescence microscopy cell images using unsupervised mining.

PubMed

Du, Xian; Dua, Sumeet

2010-05-28

The accurate measurement of cell and nuclei contours are critical for the sensitive and specific detection of changes in normal cells in several medical informatics disciplines. Within microscopy, this task is facilitated using fluorescence cell stains, and segmentation is often the first step in such approaches. Due to the complex nature of cell issues and problems inherent to microscopy, unsupervised mining approaches of clustering can be incorporated in the segmentation of cells. In this study, we have developed and evaluated the performance of multiple unsupervised data mining techniques in cell image segmentation. We adapt four distinctive, yet complementary, methods for unsupervised learning, including those based on k-means clustering, EM, Otsu's threshold, and GMAC. Validation measures are defined, and the performance of the techniques is evaluated both quantitatively and qualitatively using synthetic and recently published real data. Experimental results demonstrate that k-means, Otsu's threshold, and GMAC perform similarly, and have more precise segmentation results than EM. We report that EM has higher recall values and lower precision results from under-segmentation due to its Gaussian model assumption. We also demonstrate that these methods need spatial information to segment complex real cell images with a high degree of efficacy, as expected in many medical informatics applications.

A generic nuclei detection method for histopathological breast images

NASA Astrophysics Data System (ADS)

Kost, Henning; Homeyer, André; Bult, Peter; Balkenhol, Maschenka C. A.; van der Laak, Jeroen A. W. M.; Hahn, Horst K.

2016-03-01

The detection of cell nuclei plays a key role in various histopathological image analysis problems. Considering the high variability of its applications, we propose a novel generic and trainable detection approach. Adaption to specific nuclei detection tasks is done by providing training samples. A trainable deconvolution and classification algorithm is used to generate a probability map indicating the presence of a nucleus. The map is processed by an extended watershed segmentation step to identify the nuclei positions. We have tested our method on data sets with different stains and target nuclear types. We obtained F1-measures between 0.83 and 0.93.

Breast Cancer Diagnostics Based on Spatial Genome Organization

DTIC Science & Technology

2012-07-01

using an already established imaging tool, called NMFA-FLO (Nuclei Manual and FISH automatic). In order to achieve accurate segmentation of nuclei...in tissue we used an artificial neuronal network (ANN)-based supervised pattern recognition approach to screen out well segmented nuclei, after image ... segmentation used to process images for automated nuclear segmentation . Part a) has been adapted from [15] and b) from [16]. Figure 4. Comparison of

Semi-automated discrimination of retinal pigmented epithelial cells in two-photon fluorescence images of mouse retinas.

PubMed

Alexander, Nathan S; Palczewska, Grazyna; Palczewski, Krzysztof

2015-08-01

Automated image segmentation is a critical step toward achieving a quantitative evaluation of disease states with imaging techniques. Two-photon fluorescence microscopy (TPM) has been employed to visualize the retinal pigmented epithelium (RPE) and provide images indicating the health of the retina. However, segmentation of RPE cells within TPM images is difficult due to small differences in fluorescence intensity between cell borders and cell bodies. Here we present a semi-automated method for segmenting RPE cells that relies upon multiple weak features that differentiate cell borders from the remaining image. These features were scored by a search optimization procedure that built up the cell border in segments around a nucleus of interest. With six images used as a test, our method correctly identified cell borders for 69% of nuclei on average. Performance was strongly dependent upon increasing retinosome content in the RPE. TPM image analysis has the potential of providing improved early quantitative assessments of diseases affecting the RPE.

Improved and Robust Detection of Cell Nuclei from Four Dimensional Fluorescence Images

PubMed Central

Bashar, Md. Khayrul; Yamagata, Kazuo; Kobayashi, Tetsuya J.

2014-01-01

Segmentation-free direct methods are quite efficient for automated nuclei extraction from high dimensional images. A few such methods do exist but most of them do not ensure algorithmic robustness to parameter and noise variations. In this research, we propose a method based on multiscale adaptive filtering for efficient and robust detection of nuclei centroids from four dimensional (4D) fluorescence images. A temporal feedback mechanism is employed between the enhancement and the initial detection steps of a typical direct method. We estimate the minimum and maximum nuclei diameters from the previous frame and feed back them as filter lengths for multiscale enhancement of the current frame. A radial intensity-gradient function is optimized at positions of initial centroids to estimate all nuclei diameters. This procedure continues for processing subsequent images in the sequence. Above mechanism thus ensures proper enhancement by automated estimation of major parameters. This brings robustness and safeguards the system against additive noises and effects from wrong parameters. Later, the method and its single-scale variant are simplified for further reduction of parameters. The proposed method is then extended for nuclei volume segmentation. The same optimization technique is applied to final centroid positions of the enhanced image and the estimated diameters are projected onto the binary candidate regions to segment nuclei volumes.Our method is finally integrated with a simple sequential tracking approach to establish nuclear trajectories in the 4D space. Experimental evaluations with five image-sequences (each having 271 3D sequential images) corresponding to five different mouse embryos show promising performances of our methods in terms of nuclear detection, segmentation, and tracking. A detail analysis with a sub-sequence of 101 3D images from an embryo reveals that the proposed method can improve the nuclei detection accuracy by 9 over the previous methods, which used inappropriate large valued parameters. Results also confirm that the proposed method and its variants achieve high detection accuracies ( 98 mean F-measure) irrespective of the large variations of filter parameters and noise levels. PMID:25020042

In situ mechanical characterization of the cell nucleus by atomic force microscopy.

PubMed

Liu, Haijiao; Wen, Jun; Xiao, Yun; Liu, Jun; Hopyan, Sevan; Radisic, Milica; Simmons, Craig A; Sun, Yu

2014-04-22

The study of nuclear mechanical properties can provide insights into nuclear dynamics and its role in cellular mechanotransduction. While several methods have been developed to characterize nuclear mechanical properties, direct intracellular probing of the nucleus in situ is challenging. Here, a modified AFM (atomic force microscopy) needle penetration technique is demonstrated to mechanically characterize cell nuclei in situ. Cytoplasmic and nuclear stiffness were determined based on two different segments on the AFM indentation curves and were correlated with simultaneous confocal Z-stack microscopy reconstructions. On the basis of direct intracellular measurement, we show that the isolated nuclei from fibroblast-like cells exhibited significantly lower Young's moduli than intact nuclei in situ. We also show that there is in situ nucleus softening in the highly metastatic bladder cancer cell line T24 when compared to its less metastatic counterpart RT4. This technique has potential to become a reliable quantitative measurement tool for intracellular mechanics studies.

Automatic Nuclei Segmentation in H&E Stained Breast Cancer Histopathology Images

PubMed Central

Veta, Mitko; van Diest, Paul J.; Kornegoor, Robert; Huisman, André; Viergever, Max A.; Pluim, Josien P. W.

2013-01-01

The introduction of fast digital slide scanners that provide whole slide images has led to a revival of interest in image analysis applications in pathology. Segmentation of cells and nuclei is an important first step towards automatic analysis of digitized microscopy images. We therefore developed an automated nuclei segmentation method that works with hematoxylin and eosin (H&E) stained breast cancer histopathology images, which represent regions of whole digital slides. The procedure can be divided into four main steps: 1) pre-processing with color unmixing and morphological operators, 2) marker-controlled watershed segmentation at multiple scales and with different markers, 3) post-processing for rejection of false regions and 4) merging of the results from multiple scales. The procedure was developed on a set of 21 breast cancer cases (subset A) and tested on a separate validation set of 18 cases (subset B). The evaluation was done in terms of both detection accuracy (sensitivity and positive predictive value) and segmentation accuracy (Dice coefficient). The mean estimated sensitivity for subset A was 0.875 (±0.092) and for subset B 0.853 (±0.077). The mean estimated positive predictive value was 0.904 (±0.075) and 0.886 (±0.069) for subsets A and B, respectively. For both subsets, the distribution of the Dice coefficients had a high peak around 0.9, with the vast majority of segmentations having values larger than 0.8. PMID:23922958

Automatic nuclei segmentation in H&E stained breast cancer histopathology images.

PubMed

Veta, Mitko; van Diest, Paul J; Kornegoor, Robert; Huisman, André; Viergever, Max A; Pluim, Josien P W

2013-01-01

The introduction of fast digital slide scanners that provide whole slide images has led to a revival of interest in image analysis applications in pathology. Segmentation of cells and nuclei is an important first step towards automatic analysis of digitized microscopy images. We therefore developed an automated nuclei segmentation method that works with hematoxylin and eosin (H&E) stained breast cancer histopathology images, which represent regions of whole digital slides. The procedure can be divided into four main steps: 1) pre-processing with color unmixing and morphological operators, 2) marker-controlled watershed segmentation at multiple scales and with different markers, 3) post-processing for rejection of false regions and 4) merging of the results from multiple scales. The procedure was developed on a set of 21 breast cancer cases (subset A) and tested on a separate validation set of 18 cases (subset B). The evaluation was done in terms of both detection accuracy (sensitivity and positive predictive value) and segmentation accuracy (Dice coefficient). The mean estimated sensitivity for subset A was 0.875 (±0.092) and for subset B 0.853 (±0.077). The mean estimated positive predictive value was 0.904 (±0.075) and 0.886 (±0.069) for subsets A and B, respectively. For both subsets, the distribution of the Dice coefficients had a high peak around 0.9, with the vast majority of segmentations having values larger than 0.8.

Computerized image analysis of cell-cell interactions in human renal tissue by using multi-channel immunoflourescent confocal microscopy

NASA Astrophysics Data System (ADS)

Peng, Yahui; Jiang, Yulei; Liarski, Vladimir M.; Kaverina, Natalya; Clark, Marcus R.; Giger, Maryellen L.

2012-03-01

Analysis of interactions between B and T cells in tubulointerstitial inflammation is important for understanding human lupus nephritis. We developed a computer technique to perform this analysis, and compared it with manual analysis. Multi-channel immunoflourescent-microscopy images were acquired from 207 regions of interest in 40 renal tissue sections of 19 patients diagnosed with lupus nephritis. Fresh-frozen renal tissue sections were stained with combinations of immunoflourescent antibodies to membrane proteins and counter-stained with a cell nuclear marker. Manual delineation of the antibodies was considered as the reference standard. We first segmented cell nuclei and cell membrane markers, and then determined corresponding cell types based on the distances between cell nuclei and specific cell-membrane marker combinations. Subsequently, the distribution of the shortest distance from T cell nuclei to B cell nuclei was obtained and used as a surrogate indicator of cell-cell interactions. The computer and manual analyses results were concordant. The average absolute difference was 1.1+/-1.2% between the computer and manual analysis results in the number of cell-cell distances of 3 μm or less as a percentage of the total number of cell-cell distances. Our computerized analysis of cell-cell distances could be used as a surrogate for quantifying cell-cell interactions as either an automated and quantitative analysis or for independent confirmation of manual analysis.

Combinational pixel-by-pixel and object-level classifying, segmenting, and agglomerating in performing quantitative image analysis that distinguishes between healthy non-cancerous and cancerous cell nuclei and delineates nuclear, cytoplasm, and stromal material objects from stained biological tissue materials

DOEpatents

Boucheron, Laura E

2013-07-16

Quantitative object and spatial arrangement-level analysis of tissue are detailed using expert (pathologist) input to guide the classification process. A two-step method is disclosed for imaging tissue, by classifying one or more biological materials, e.g. nuclei, cytoplasm, and stroma, in the tissue into one or more identified classes on a pixel-by-pixel basis, and segmenting the identified classes to agglomerate one or more sets of identified pixels into segmented regions. Typically, the one or more biological materials comprises nuclear material, cytoplasm material, and stromal material. The method further allows a user to markup the image subsequent to the classification to re-classify said materials. The markup is performed via a graphic user interface to edit designated regions in the image.

Early Diagnosis of Breast Cancer by Identifying Malignant Cells Within Neoplasias Histologically Classified as Benign

DTIC Science & Technology

2005-07-01

C Ortiz de Solorzano, R . Malladi , SA Leli&vre, and SJ Lockett. "Segmentation of nuclei and cells using membrane related protein markers." J...B•o derived, invasive carcinoma-type (malignant) T4-2 cells -- " expressing GFP-actin were plated in 3D at ratio 1/8 andi r cultured for 10 days

Automated Segmentation of Nuclei in Breast Cancer Histopathology Images.

PubMed

Paramanandam, Maqlin; O'Byrne, Michael; Ghosh, Bidisha; Mammen, Joy John; Manipadam, Marie Therese; Thamburaj, Robinson; Pakrashi, Vikram

2016-01-01

The process of Nuclei detection in high-grade breast cancer images is quite challenging in the case of image processing techniques due to certain heterogeneous characteristics of cancer nuclei such as enlarged and irregularly shaped nuclei, highly coarse chromatin marginalized to the nuclei periphery and visible nucleoli. Recent reviews state that existing techniques show appreciable segmentation accuracy on breast histopathology images whose nuclei are dispersed and regular in texture and shape; however, typical cancer nuclei are often clustered and have irregular texture and shape properties. This paper proposes a novel segmentation algorithm for detecting individual nuclei from Hematoxylin and Eosin (H&E) stained breast histopathology images. This detection framework estimates a nuclei saliency map using tensor voting followed by boundary extraction of the nuclei on the saliency map using a Loopy Back Propagation (LBP) algorithm on a Markov Random Field (MRF). The method was tested on both whole-slide images and frames of breast cancer histopathology images. Experimental results demonstrate high segmentation performance with efficient precision, recall and dice-coefficient rates, upon testing high-grade breast cancer images containing several thousand nuclei. In addition to the optimal performance on the highly complex images presented in this paper, this method also gave appreciable results in comparison with two recently published methods-Wienert et al. (2012) and Veta et al. (2013), which were tested using their own datasets.

Automated Segmentation of Nuclei in Breast Cancer Histopathology Images

PubMed Central

Paramanandam, Maqlin; O’Byrne, Michael; Ghosh, Bidisha; Mammen, Joy John; Manipadam, Marie Therese; Thamburaj, Robinson; Pakrashi, Vikram

2016-01-01

The process of Nuclei detection in high-grade breast cancer images is quite challenging in the case of image processing techniques due to certain heterogeneous characteristics of cancer nuclei such as enlarged and irregularly shaped nuclei, highly coarse chromatin marginalized to the nuclei periphery and visible nucleoli. Recent reviews state that existing techniques show appreciable segmentation accuracy on breast histopathology images whose nuclei are dispersed and regular in texture and shape; however, typical cancer nuclei are often clustered and have irregular texture and shape properties. This paper proposes a novel segmentation algorithm for detecting individual nuclei from Hematoxylin and Eosin (H&E) stained breast histopathology images. This detection framework estimates a nuclei saliency map using tensor voting followed by boundary extraction of the nuclei on the saliency map using a Loopy Back Propagation (LBP) algorithm on a Markov Random Field (MRF). The method was tested on both whole-slide images and frames of breast cancer histopathology images. Experimental results demonstrate high segmentation performance with efficient precision, recall and dice-coefficient rates, upon testing high-grade breast cancer images containing several thousand nuclei. In addition to the optimal performance on the highly complex images presented in this paper, this method also gave appreciable results in comparison with two recently published methods—Wienert et al. (2012) and Veta et al. (2013), which were tested using their own datasets. PMID:27649496

Accurate Detection of Dysmorphic Nuclei Using Dynamic Programming and Supervised Classification.

PubMed

Verschuuren, Marlies; De Vylder, Jonas; Catrysse, Hannes; Robijns, Joke; Philips, Wilfried; De Vos, Winnok H

2017-01-01

A vast array of pathologies is typified by the presence of nuclei with an abnormal morphology. Dysmorphic nuclear phenotypes feature dramatic size changes or foldings, but also entail much subtler deviations such as nuclear protrusions called blebs. Due to their unpredictable size, shape and intensity, dysmorphic nuclei are often not accurately detected in standard image analysis routines. To enable accurate detection of dysmorphic nuclei in confocal and widefield fluorescence microscopy images, we have developed an automated segmentation algorithm, called Blebbed Nuclei Detector (BleND), which relies on two-pass thresholding for initial nuclear contour detection, and an optimal path finding algorithm, based on dynamic programming, for refining these contours. Using a robust error metric, we show that our method matches manual segmentation in terms of precision and outperforms state-of-the-art nuclear segmentation methods. Its high performance allowed for building and integrating a robust classifier that recognizes dysmorphic nuclei with an accuracy above 95%. The combined segmentation-classification routine is bound to facilitate nucleus-based diagnostics and enable real-time recognition of dysmorphic nuclei in intelligent microscopy workflows.

Accurate Detection of Dysmorphic Nuclei Using Dynamic Programming and Supervised Classification

PubMed Central

Verschuuren, Marlies; De Vylder, Jonas; Catrysse, Hannes; Robijns, Joke; Philips, Wilfried

2017-01-01

A vast array of pathologies is typified by the presence of nuclei with an abnormal morphology. Dysmorphic nuclear phenotypes feature dramatic size changes or foldings, but also entail much subtler deviations such as nuclear protrusions called blebs. Due to their unpredictable size, shape and intensity, dysmorphic nuclei are often not accurately detected in standard image analysis routines. To enable accurate detection of dysmorphic nuclei in confocal and widefield fluorescence microscopy images, we have developed an automated segmentation algorithm, called Blebbed Nuclei Detector (BleND), which relies on two-pass thresholding for initial nuclear contour detection, and an optimal path finding algorithm, based on dynamic programming, for refining these contours. Using a robust error metric, we show that our method matches manual segmentation in terms of precision and outperforms state-of-the-art nuclear segmentation methods. Its high performance allowed for building and integrating a robust classifier that recognizes dysmorphic nuclei with an accuracy above 95%. The combined segmentation-classification routine is bound to facilitate nucleus-based diagnostics and enable real-time recognition of dysmorphic nuclei in intelligent microscopy workflows. PMID:28125723

Using multimodal information for the segmentation of fluorescent micrographs with application to virology and microbiology.

PubMed

Held, Christian; Wenzel, Jens; Webel, Rike; Marschall, Manfred; Lang, Roland; Palmisano, Ralf; Wittenberg, Thomas

2011-01-01

In order to improve reproducibility and objectivity of fluorescence microscopy based experiments and to enable the evaluation of large datasets, flexible segmentation methods are required which are able to adapt to different stainings and cell types. This adaption is usually achieved by the manual adjustment of the segmentation methods parameters, which is time consuming and challenging for biologists with no knowledge on image processing. To avoid this, parameters of the presented methods automatically adapt to user generated ground truth to determine the best method and the optimal parameter setup. These settings can then be used for segmentation of the remaining images. As robust segmentation methods form the core of such a system, the currently used watershed transform based segmentation routine is replaced by a fast marching level set based segmentation routine which incorporates knowledge on the cell nuclei. Our evaluations reveal that incorporation of multimodal information improves segmentation quality for the presented fluorescent datasets.

Current automated 3D cell detection methods are not a suitable replacement for manual stereologic cell counting

PubMed Central

Schmitz, Christoph; Eastwood, Brian S.; Tappan, Susan J.; Glaser, Jack R.; Peterson, Daniel A.; Hof, Patrick R.

2014-01-01

Stereologic cell counting has had a major impact on the field of neuroscience. A major bottleneck in stereologic cell counting is that the user must manually decide whether or not each cell is counted according to three-dimensional (3D) stereologic counting rules by visual inspection within hundreds of microscopic fields-of-view per investigated brain or brain region. Reliance on visual inspection forces stereologic cell counting to be very labor-intensive and time-consuming, and is the main reason why biased, non-stereologic two-dimensional (2D) “cell counting” approaches have remained in widespread use. We present an evaluation of the performance of modern automated cell detection and segmentation algorithms as a potential alternative to the manual approach in stereologic cell counting. The image data used in this study were 3D microscopic images of thick brain tissue sections prepared with a variety of commonly used nuclear and cytoplasmic stains. The evaluation compared the numbers and locations of cells identified unambiguously and counted exhaustively by an expert observer with those found by three automated 3D cell detection algorithms: nuclei segmentation from the FARSIGHT toolkit, nuclei segmentation by 3D multiple level set methods, and the 3D object counter plug-in for ImageJ. Of these methods, FARSIGHT performed best, with true-positive detection rates between 38 and 99% and false-positive rates from 3.6 to 82%. The results demonstrate that the current automated methods suffer from lower detection rates and higher false-positive rates than are acceptable for obtaining valid estimates of cell numbers. Thus, at present, stereologic cell counting with manual decision for object inclusion according to unbiased stereologic counting rules remains the only adequate method for unbiased cell quantification in histologic tissue sections. PMID:24847213

Automatic Segmentation of High-Throughput RNAi Fluorescent Cellular Images

PubMed Central

Yan, Pingkum; Zhou, Xiaobo; Shah, Mubarak; Wong, Stephen T. C.

2010-01-01

High-throughput genome-wide RNA interference (RNAi) screening is emerging as an essential tool to assist biologists in understanding complex cellular processes. The large number of images produced in each study make manual analysis intractable; hence, automatic cellular image analysis becomes an urgent need, where segmentation is the first and one of the most important steps. In this paper, a fully automatic method for segmentation of cells from genome-wide RNAi screening images is proposed. Nuclei are first extracted from the DNA channel by using a modified watershed algorithm. Cells are then extracted by modeling the interaction between them as well as combining both gradient and region information in the Actin and Rac channels. A new energy functional is formulated based on a novel interaction model for segmenting tightly clustered cells with significant intensity variance and specific phenotypes. The energy functional is minimized by using a multiphase level set method, which leads to a highly effective cell segmentation method. Promising experimental results demonstrate that automatic segmentation of high-throughput genome-wide multichannel screening can be achieved by using the proposed method, which may also be extended to other multichannel image segmentation problems. PMID:18270043

Iterative h-minima-based marker-controlled watershed for cell nucleus segmentation.

PubMed

Koyuncu, Can Fahrettin; Akhan, Ece; Ersahin, Tulin; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem

2016-04-01

Automated microscopy imaging systems facilitate high-throughput screening in molecular cellular biology research. The first step of these systems is cell nucleus segmentation, which has a great impact on the success of the overall system. The marker-controlled watershed is a technique commonly used by the previous studies for nucleus segmentation. These studies define their markers finding regional minima on the intensity/gradient and/or distance transform maps. They typically use the h-minima transform beforehand to suppress noise on these maps. The selection of the h value is critical; unnecessarily small values do not sufficiently suppress the noise, resulting in false and oversegmented markers, and unnecessarily large ones suppress too many pixels, causing missing and undersegmented markers. Because cell nuclei show different characteristics within an image, the same h value may not work to define correct markers for all the nuclei. To address this issue, in this work, we propose a new watershed algorithm that iteratively identifies its markers, considering a set of different h values. In each iteration, the proposed algorithm defines a set of candidates using a particular h value and selects the markers from those candidates provided that they fulfill the size requirement. Working with widefield fluorescence microscopy images, our experiments reveal that the use of multiple h values in our iterative algorithm leads to better segmentation results, compared to its counterparts. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

[Structure of the retina of Pacific salmon fry in twilight illumination during the geomagnetic field changes].

PubMed

Maksimovich, A A; Gniubkina, V P

2010-01-01

The retinomotor response of the masu salmon Oncorhynchus masou fry retina was studied under the conditions of mesopic (twilight) illumination after experimental geomagnetic field (GMF) compensation which was reached using the Helmholtz coils. In the control group, the retinomotor response of masu salmon fry to twilight illumination was usual: the nuclei of the neurosensory rod cells were located immediately above the external limiting layer, while the nuclei of the neurosensory cone cells were displaced closer to the pigment epithelium. After experimental GMF compensation, the masu salmon fry retina reaction was unusual: the neurosensory cone cell nuclei adhered to the external limiting membrane, while the nuclei of the neurosensory rod cells were displaced closer to the pigment epithelium layer. Double and central neurosensory cone cells occupied the position that was inadequate to normal reaction to twilight: the bodies of these cells were considerably elongated, and the external segments reached the pigment epithelium layer. Thus, in the experiment with GMF compensation, we have found the unusual structure of the retina, which only vaguely corresponded to a reaction to mesopic adaptation. The results suggest, that the visible light is not a unique variety of the electromagnetic field, that could be perceived by the fish retina.

Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy.

PubMed

Schmid, Volker J; Cremer, Marion; Cremer, Thomas

2017-07-01

Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction. In addition, these tools allow 3D mapping of specific DNA/RNA sequences and nuclear proteins relative to the 3D chromatin compaction maps and comparisons of multiple cell nuclei. The tools are available in the free and open source R packages nucim and bioimagetools. We discuss the use of masks for the segmentation of nuclei and the use of DNA stains, such as DAPI, as a proxy for local differences in chromatin compaction. We further discuss the limitations of 3D maps of the nuclear landscape as well as problems of the biological interpretation of such data. Copyright © 2017 Elsevier Inc. All rights reserved.

New decision support tool for acute lymphoblastic leukemia classification

NASA Astrophysics Data System (ADS)

Madhukar, Monica; Agaian, Sos; Chronopoulos, Anthony T.

2012-03-01

In this paper, we build up a new decision support tool to improve treatment intensity choice in childhood ALL. The developed system includes different methods to accurately measure furthermore cell properties in microscope blood film images. The blood images are exposed to series of pre-processing steps which include color correlation, and contrast enhancement. By performing K-means clustering on the resultant images, the nuclei of the cells under consideration are obtained. Shape features and texture features are then extracted for classification. The system is further tested on the classification of spectra measured from the cell nuclei in blood samples in order to distinguish normal cells from those affected by Acute Lymphoblastic Leukemia. The results show that the proposed system robustly segments and classifies acute lymphoblastic leukemia based on complete microscopic blood images.

Multiscale image analysis reveals structural heterogeneity of the cell microenvironment in homotypic spheroids.

PubMed

Schmitz, Alexander; Fischer, Sabine C; Mattheyer, Christian; Pampaloni, Francesco; Stelzer, Ernst H K

2017-03-03

Three-dimensional multicellular aggregates such as spheroids provide reliable in vitro substitutes for tissues. Quantitative characterization of spheroids at the cellular level is fundamental. We present the first pipeline that provides three-dimensional, high-quality images of intact spheroids at cellular resolution and a comprehensive image analysis that completes traditional image segmentation by algorithms from other fields. The pipeline combines light sheet-based fluorescence microscopy of optically cleared spheroids with automated nuclei segmentation (F score: 0.88) and concepts from graph analysis and computational topology. Incorporating cell graphs and alpha shapes provided more than 30 features of individual nuclei, the cellular neighborhood and the spheroid morphology. The application of our pipeline to a set of breast carcinoma spheroids revealed two concentric layers of different cell density for more than 30,000 cells. The thickness of the outer cell layer depends on a spheroid's size and varies between 50% and 75% of its radius. In differently-sized spheroids, we detected patches of different cell densities ranging from 5 × 10 5 to 1 × 10 6  cells/mm 3 . Since cell density affects cell behavior in tissues, structural heterogeneities need to be incorporated into existing models. Our image analysis pipeline provides a multiscale approach to obtain the relevant data for a system-level understanding of tissue architecture.

Deep Learning Nuclei Detection in Digitized Histology Images by Superpixels.

PubMed

Sornapudi, Sudhir; Stanley, Ronald Joe; Stoecker, William V; Almubarak, Haidar; Long, Rodney; Antani, Sameer; Thoma, George; Zuna, Rosemary; Frazier, Shelliane R

2018-01-01

Advances in image analysis and computational techniques have facilitated automatic detection of critical features in histopathology images. Detection of nuclei is critical for squamous epithelium cervical intraepithelial neoplasia (CIN) classification into normal, CIN1, CIN2, and CIN3 grades. In this study, a deep learning (DL)-based nuclei segmentation approach is investigated based on gathering localized information through the generation of superpixels using a simple linear iterative clustering algorithm and training with a convolutional neural network. The proposed approach was evaluated on a dataset of 133 digitized histology images and achieved an overall nuclei detection (object-based) accuracy of 95.97%, with demonstrated improvement over imaging-based and clustering-based benchmark techniques. The proposed DL-based nuclei segmentation Method with superpixel analysis has shown improved segmentation results in comparison to state-of-the-art methods.

Object Segmentation and Ground Truth in 3D Embryonic Imaging.

PubMed

Rajasekaran, Bhavna; Uriu, Koichiro; Valentin, Guillaume; Tinevez, Jean-Yves; Oates, Andrew C

2016-01-01

Many questions in developmental biology depend on measuring the position and movement of individual cells within developing embryos. Yet, tools that provide this data are often challenged by high cell density and their accuracy is difficult to measure. Here, we present a three-step procedure to address this problem. Step one is a novel segmentation algorithm based on image derivatives that, in combination with selective post-processing, reliably and automatically segments cell nuclei from images of densely packed tissue. Step two is a quantitative validation using synthetic images to ascertain the efficiency of the algorithm with respect to signal-to-noise ratio and object density. Finally, we propose an original method to generate reliable and experimentally faithful ground truth datasets: Sparse-dense dual-labeled embryo chimeras are used to unambiguously measure segmentation errors within experimental data. Together, the three steps outlined here establish a robust, iterative procedure to fine-tune image analysis algorithms and microscopy settings associated with embryonic 3D image data sets.

Object Segmentation and Ground Truth in 3D Embryonic Imaging

PubMed Central

Rajasekaran, Bhavna; Uriu, Koichiro; Valentin, Guillaume; Tinevez, Jean-Yves; Oates, Andrew C.

2016-01-01

Many questions in developmental biology depend on measuring the position and movement of individual cells within developing embryos. Yet, tools that provide this data are often challenged by high cell density and their accuracy is difficult to measure. Here, we present a three-step procedure to address this problem. Step one is a novel segmentation algorithm based on image derivatives that, in combination with selective post-processing, reliably and automatically segments cell nuclei from images of densely packed tissue. Step two is a quantitative validation using synthetic images to ascertain the efficiency of the algorithm with respect to signal-to-noise ratio and object density. Finally, we propose an original method to generate reliable and experimentally faithful ground truth datasets: Sparse-dense dual-labeled embryo chimeras are used to unambiguously measure segmentation errors within experimental data. Together, the three steps outlined here establish a robust, iterative procedure to fine-tune image analysis algorithms and microscopy settings associated with embryonic 3D image data sets. PMID:27332860

Physical insight into light scattering by photoreceptor cell nuclei.

PubMed

Kreysing, Moritz; Boyde, Lars; Guck, Jochen; Chalut, Kevin J

2010-08-01

A recent study showed that the rod photoreceptor cell nuclei in the retina of nocturnal and diurnal mammals differ considerably in architecture: the location of euchromatin and heterochromatin in the nucleus is interchanged. This inversion has significant implications for the refractive index distribution and the light scattering properties of the nucleus. Here, we extend previous two-dimensional analysis to three dimensions (3D) by using both a numerical finite-difference time-domain and an analytic Mie theory approach. We find that the specific arrangement of the chromatin phases in the nuclear core-shell models employed have little impact on the far-field scattering cross section. However, scattering in the near field, which is the relevant regime inside the retina, shows a significant difference between the two architectures. The "inverted" photoreceptor cell nuclei of nocturnal mammals act as collection lenses, with the lensing effect being much more pronounced in 3D than in two dimensions. This lensing helps to deliver light efficiently to the light-sensing outer segments of the rod photoreceptor cells and thereby improve night vision.

Deep Learning Nuclei Detection in Digitized Histology Images by Superpixels

PubMed Central

Sornapudi, Sudhir; Stanley, Ronald Joe; Stoecker, William V.; Almubarak, Haidar; Long, Rodney; Antani, Sameer; Thoma, George; Zuna, Rosemary; Frazier, Shelliane R.

2018-01-01

Background: Advances in image analysis and computational techniques have facilitated automatic detection of critical features in histopathology images. Detection of nuclei is critical for squamous epithelium cervical intraepithelial neoplasia (CIN) classification into normal, CIN1, CIN2, and CIN3 grades. Methods: In this study, a deep learning (DL)-based nuclei segmentation approach is investigated based on gathering localized information through the generation of superpixels using a simple linear iterative clustering algorithm and training with a convolutional neural network. Results: The proposed approach was evaluated on a dataset of 133 digitized histology images and achieved an overall nuclei detection (object-based) accuracy of 95.97%, with demonstrated improvement over imaging-based and clustering-based benchmark techniques. Conclusions: The proposed DL-based nuclei segmentation Method with superpixel analysis has shown improved segmentation results in comparison to state-of-the-art methods. PMID:29619277

Computer assisted detection and analysis of tall cell variant papillary thyroid carcinoma in histological images

NASA Astrophysics Data System (ADS)

Kim, Edward; Baloch, Zubair; Kim, Caroline

2015-03-01

The number of new cases of thyroid cancer are dramatically increasing as incidences of this cancer have more than doubled since the early 1970s. Tall cell variant (TCV-PTC) papillary thyroid carcinoma is one type of thyroid cancer that is more aggressive and usually associated with higher local recurrence and distant metastasis. This variant can be identified through visual characteristics of cells in histological images. Thus, we created a fully automatic algorithm that is able to segment cells using a multi-stage approach. Our method learns the statistical characteristics of nuclei and cells during the segmentation process and utilizes this information for a more accurate result. Furthermore, we are able to analyze the detected regions and extract characteristic cell data that can be used to assist in clinical diagnosis.

Multi-scale Gaussian representation and outline-learning based cell image segmentation.

PubMed

Farhan, Muhammad; Ruusuvuori, Pekka; Emmenlauer, Mario; Rämö, Pauli; Dehio, Christoph; Yli-Harja, Olli

2013-01-01

High-throughput genome-wide screening to study gene-specific functions, e.g. for drug discovery, demands fast automated image analysis methods to assist in unraveling the full potential of such studies. Image segmentation is typically at the forefront of such analysis as the performance of the subsequent steps, for example, cell classification, cell tracking etc., often relies on the results of segmentation. We present a cell cytoplasm segmentation framework which first separates cell cytoplasm from image background using novel approach of image enhancement and coefficient of variation of multi-scale Gaussian scale-space representation. A novel outline-learning based classification method is developed using regularized logistic regression with embedded feature selection which classifies image pixels as outline/non-outline to give cytoplasm outlines. Refinement of the detected outlines to separate cells from each other is performed in a post-processing step where the nuclei segmentation is used as contextual information. We evaluate the proposed segmentation methodology using two challenging test cases, presenting images with completely different characteristics, with cells of varying size, shape, texture and degrees of overlap. The feature selection and classification framework for outline detection produces very simple sparse models which use only a small subset of the large, generic feature set, that is, only 7 and 5 features for the two cases. Quantitative comparison of the results for the two test cases against state-of-the-art methods show that our methodology outperforms them with an increase of 4-9% in segmentation accuracy with maximum accuracy of 93%. Finally, the results obtained for diverse datasets demonstrate that our framework not only produces accurate segmentation but also generalizes well to different segmentation tasks.

Multi-scale Gaussian representation and outline-learning based cell image segmentation

PubMed Central

2013-01-01

Background High-throughput genome-wide screening to study gene-specific functions, e.g. for drug discovery, demands fast automated image analysis methods to assist in unraveling the full potential of such studies. Image segmentation is typically at the forefront of such analysis as the performance of the subsequent steps, for example, cell classification, cell tracking etc., often relies on the results of segmentation. Methods We present a cell cytoplasm segmentation framework which first separates cell cytoplasm from image background using novel approach of image enhancement and coefficient of variation of multi-scale Gaussian scale-space representation. A novel outline-learning based classification method is developed using regularized logistic regression with embedded feature selection which classifies image pixels as outline/non-outline to give cytoplasm outlines. Refinement of the detected outlines to separate cells from each other is performed in a post-processing step where the nuclei segmentation is used as contextual information. Results and conclusions We evaluate the proposed segmentation methodology using two challenging test cases, presenting images with completely different characteristics, with cells of varying size, shape, texture and degrees of overlap. The feature selection and classification framework for outline detection produces very simple sparse models which use only a small subset of the large, generic feature set, that is, only 7 and 5 features for the two cases. Quantitative comparison of the results for the two test cases against state-of-the-art methods show that our methodology outperforms them with an increase of 4-9% in segmentation accuracy with maximum accuracy of 93%. Finally, the results obtained for diverse datasets demonstrate that our framework not only produces accurate segmentation but also generalizes well to different segmentation tasks. PMID:24267488

Quantitative diagnosis of bladder cancer by morphometric analysis of HE images

NASA Astrophysics Data System (ADS)

Wu, Binlin; Nebylitsa, Samantha V.; Mukherjee, Sushmita; Jain, Manu

2015-02-01

In clinical practice, histopathological analysis of biopsied tissue is the main method for bladder cancer diagnosis and prognosis. The diagnosis is performed by a pathologist based on the morphological features in the image of a hematoxylin and eosin (HE) stained tissue sample. This manuscript proposes algorithms to perform morphometric analysis on the HE images, quantify the features in the images, and discriminate bladder cancers with different grades, i.e. high grade and low grade. The nuclei are separated from the background and other types of cells such as red blood cells (RBCs) and immune cells using manual outlining, color deconvolution and image segmentation. A mask of nuclei is generated for each image for quantitative morphometric analysis. The features of the nuclei in the mask image including size, shape, orientation, and their spatial distributions are measured. To quantify local clustering and alignment of nuclei, we propose a 1-nearest-neighbor (1-NN) algorithm which measures nearest neighbor distance and nearest neighbor parallelism. The global distributions of the features are measured using statistics of the proposed parameters. A linear support vector machine (SVM) algorithm is used to classify the high grade and low grade bladder cancers. The results show using a particular group of nuclei such as large ones, and combining multiple parameters can achieve better discrimination. This study shows the proposed approach can potentially help expedite pathological diagnosis by triaging potentially suspicious biopsies.

Cell nuclei attributed relational graphs for efficient representation and classification of gastric cancer in digital histopathology

NASA Astrophysics Data System (ADS)

Sharma, Harshita; Zerbe, Norman; Heim, Daniel; Wienert, Stephan; Lohmann, Sebastian; Hellwich, Olaf; Hufnagl, Peter

2016-03-01

This paper describes a novel graph-based method for efficient representation and subsequent classification in histological whole slide images of gastric cancer. Her2/neu immunohistochemically stained and haematoxylin and eosin stained histological sections of gastric carcinoma are digitized. Immunohistochemical staining is used in practice by pathologists to determine extent of malignancy, however, it is laborious to visually discriminate the corresponding malignancy levels in the more commonly used haematoxylin and eosin stain, and this study attempts to solve this problem using a computer-based method. Cell nuclei are first isolated at high magnification using an automatic cell nuclei segmentation strategy, followed by construction of cell nuclei attributed relational graphs of the tissue regions. These graphs represent tissue architecture comprehensively, as they contain information about cell nuclei morphology as vertex attributes, along with knowledge of neighborhood in the form of edge linking and edge attributes. Global graph characteristics are derived and ensemble learning is used to discriminate between three types of malignancy levels, namely, non-tumor, Her2/neu positive tumor and Her2/neu negative tumor. Performance is compared with state of the art methods including four texture feature groups (Haralick, Gabor, Local Binary Patterns and Varma Zisserman features), color and intensity features, and Voronoi diagram and Delaunay triangulation. Texture, color and intensity information is also combined with graph-based knowledge, followed by correlation analysis. Quantitative assessment is performed using two cross validation strategies. On investigating the experimental results, it can be concluded that the proposed method provides a promising way for computer-based analysis of histopathological images of gastric cancer.

Her2Net: A Deep Framework for Semantic Segmentation and Classification of Cell Membranes and Nuclei in Breast Cancer Evaluation.

PubMed

Saha, Monjoy; Chakraborty, Chandan

2018-05-01

We present an efficient deep learning framework for identifying, segmenting, and classifying cell membranes and nuclei from human epidermal growth factor receptor-2 (HER2)-stained breast cancer images with minimal user intervention. This is a long-standing issue for pathologists because the manual quantification of HER2 is error-prone, costly, and time-consuming. Hence, we propose a deep learning-based HER2 deep neural network (Her2Net) to solve this issue. The convolutional and deconvolutional parts of the proposed Her2Net framework consisted mainly of multiple convolution layers, max-pooling layers, spatial pyramid pooling layers, deconvolution layers, up-sampling layers, and trapezoidal long short-term memory (TLSTM). A fully connected layer and a softmax layer were also used for classification and error estimation. Finally, HER2 scores were calculated based on the classification results. The main contribution of our proposed Her2Net framework includes the implementation of TLSTM and a deep learning framework for cell membrane and nucleus detection, segmentation, and classification and HER2 scoring. Our proposed Her2Net achieved 96.64% precision, 96.79% recall, 96.71% F-score, 93.08% negative predictive value, 98.33% accuracy, and a 6.84% false-positive rate. Our results demonstrate the high accuracy and wide applicability of the proposed Her2Net in the context of HER2 scoring for breast cancer evaluation.

A note on the stability and discriminability of graph-based features for classification problems in digital pathology

NASA Astrophysics Data System (ADS)

Cruz-Roa, Angel; Xu, Jun; Madabhushi, Anant

2015-01-01

Nuclear architecture or the spatial arrangement of individual cancer nuclei on histopathology images has been shown to be associated with different grades and differential risk for a number of solid tumors such as breast, prostate, and oropharyngeal. Graph-based representations of individual nuclei (nuclei representing the graph nodes) allows for mining of quantitative metrics to describe tumor morphology. These graph features can be broadly categorized into global and local depending on the type of graph construction method. While a number of local graph (e.g. Cell Cluster Graphs) and global graph (e.g. Voronoi, Delaunay Triangulation, Minimum Spanning Tree) features have been shown to associated with cancer grade, risk, and outcome for different cancer types, the sensitivity of the preceding segmentation algorithms in identifying individual nuclei can have a significant bearing on the discriminability of the resultant features. This therefore begs the question as to which features while being discriminative of cancer grade and aggressiveness are also the most resilient to the segmentation errors. These properties are particularly desirable in the context of digital pathology images, where the method of slide preparation, staining, and type of nuclear segmentation algorithm employed can all dramatically affect the quality of the nuclear graphs and corresponding features. In this paper we evaluated the trade off between discriminability and stability of both global and local graph-based features in conjunction with a few different segmentation algorithms and in the context of two different histopathology image datasets of breast cancer from whole-slide images (WSI) and tissue microarrays (TMA). Specifically in this paper we investigate a few different performance measures including stability, discriminability and stability vs discriminability trade off, all of which are based on p-values from the Kruskal-Wallis one-way analysis of variance for local and global graph features. Apart from identifying the set of local and global features that satisfied the trade off between stability and discriminability, our most interesting finding was that a simple segmentation method was sufficient to identify the most discriminant features for invasive tumour detection in TMAs, whereas for tumour grading in WSI, the graph based features were more sensitive to the accuracy of the segmentation algorithm employed.

Cancer diagnostics using neural network sorting of processed images

NASA Astrophysics Data System (ADS)

Wyman, Charles L.; Schreeder, Marshall; Grundy, Walt; Kinser, Jason M.

1996-03-01

A combination of image processing with neural network sorting was conducted to demonstrate feasibility of automated cervical smear screening. Nuclei were isolated to generate a series of data points relating to the density and size of individual nuclei. This was followed by segmentation to isolate entire cells for subsequent generation of data points to bound the size of the cytoplasm. Data points were taken on as many as ten cells per image frame and included correlation against a series of filters providing size and density readings on nuclei. Additional point data was taken on nuclei images to refine size information and on whole cells to bound the size of the cytoplasm, twenty data points per assessed cell were generated. These data point sets, designated as neural tensors, comprise the inputs for training and use of a unique neural network to sort the images and identify those indicating evidence of disease. The neural network, named the Fast Analog Associative Memory, accumulates data and establishes lookup tables for comparison against images to be assessed. Six networks were trained to differentiate normal cells from those evidencing various levels abnormality that may lead to cancer. A blind test was conducted on 77 images to evaluate system performance. The image set included 31 positives (diseased) and 46 negatives (normal). Our system correctly identified all 31 positives and 41 of the negatives with 5 false positives. We believe this technology can lead to more efficient automated screening of cervical smears.

Numerical analysis of dysplasia-associated changes in depth-dependent light scattering profile of cervical epithelium

NASA Astrophysics Data System (ADS)

Arifler, Dizem; MacAulay, Calum; Follen, Michele; Guillaud, Martial

2013-06-01

Dysplastic progression is known to be associated with changes in morphology and internal structure of cells. A detailed assessment of the influence of these changes on cellular scattering response is needed to develop and optimize optical diagnostic techniques. In this study, we first analyzed a set of quantitative histopathologic images from cervical biopsies and we obtained detailed information on morphometric and photometric features of segmented epithelial cell nuclei. Morphometric parameters included average size and eccentricity of the best-fit ellipse. Photometric parameters included optical density measures that can be related to dielectric properties and texture characteristics of the nuclei. These features enabled us to construct realistic three-dimensional computational models of basal, parabasal, intermediate, and superficial cell nuclei that were representative of four diagnostic categories, namely normal (or negative for dysplasia), mild dysplasia, moderate dysplasia, and severe dysplasia or carcinoma in situ. We then employed the finite-difference time-domain method, a popular numerical tool in electromagnetics, to compute the angle-resolved light scattering properties of these representative models. Results indicated that a high degree of variability can characterize a given diagnostic category, but scattering from moderately and severely dysplastic or cancerous nuclei was generally observed to be stronger compared to scattering from normal and mildly dysplastic nuclei. Simulation results also pointed to significant intensity level variations among different epithelial depths. This suggests that intensity changes associated with dysplastic progression need to be analyzed in a depth-dependent manner.

Joint multiple fully connected convolutional neural network with extreme learning machine for hepatocellular carcinoma nuclei grading.

PubMed

Li, Siqi; Jiang, Huiyan; Pang, Wenbo

2017-05-01

Accurate cell grading of cancerous tissue pathological image is of great importance in medical diagnosis and treatment. This paper proposes a joint multiple fully connected convolutional neural network with extreme learning machine (MFC-CNN-ELM) architecture for hepatocellular carcinoma (HCC) nuclei grading. First, in preprocessing stage, each grayscale image patch with the fixed size is obtained using center-proliferation segmentation (CPS) method and the corresponding labels are marked under the guidance of three pathologists. Next, a multiple fully connected convolutional neural network (MFC-CNN) is designed to extract the multi-form feature vectors of each input image automatically, which considers multi-scale contextual information of deep layer maps sufficiently. After that, a convolutional neural network extreme learning machine (CNN-ELM) model is proposed to grade HCC nuclei. Finally, a back propagation (BP) algorithm, which contains a new up-sample method, is utilized to train MFC-CNN-ELM architecture. The experiment comparison results demonstrate that our proposed MFC-CNN-ELM has superior performance compared with related works for HCC nuclei grading. Meanwhile, external validation using ICPR 2014 HEp-2 cell dataset shows the good generalization of our MFC-CNN-ELM architecture. Copyright © 2017 Elsevier Ltd. All rights reserved.

High-resolution magnetic resonance imaging reveals nuclei of the human amygdala: manual segmentation to automatic atlas.

PubMed

Saygin, Z M; Kliemann, D; Iglesias, J E; van der Kouwe, A J W; Boyd, E; Reuter, M; Stevens, A; Van Leemput, K; McKee, A; Frosch, M P; Fischl, B; Augustinack, J C

2017-07-15

The amygdala is composed of multiple nuclei with unique functions and connections in the limbic system and to the rest of the brain. However, standard in vivo neuroimaging tools to automatically delineate the amygdala into its multiple nuclei are still rare. By scanning postmortem specimens at high resolution (100-150µm) at 7T field strength (n = 10), we were able to visualize and label nine amygdala nuclei (anterior amygdaloid, cortico-amygdaloid transition area; basal, lateral, accessory basal, central, cortical medial, paralaminar nuclei). We created an atlas from these labels using a recently developed atlas building algorithm based on Bayesian inference. This atlas, which will be released as part of FreeSurfer, can be used to automatically segment nine amygdala nuclei from a standard resolution structural MR image. We applied this atlas to two publicly available datasets (ADNI and ABIDE) with standard resolution T1 data, used individual volumetric data of the amygdala nuclei as the measure and found that our atlas i) discriminates between Alzheimer's disease participants and age-matched control participants with 84% accuracy (AUC=0.915), and ii) discriminates between individuals with autism and age-, sex- and IQ-matched neurotypically developed control participants with 59.5% accuracy (AUC=0.59). For both datasets, the new ex vivo atlas significantly outperformed (all p < .05) estimations of the whole amygdala derived from the segmentation in FreeSurfer 5.1 (ADNI: 75%, ABIDE: 54% accuracy), as well as classification based on whole amygdala volume (using the sum of all amygdala nuclei volumes; ADNI: 81%, ABIDE: 55% accuracy). This new atlas and the segmentation tools that utilize it will provide neuroimaging researchers with the ability to explore the function and connectivity of the human amygdala nuclei with unprecedented detail in healthy adults as well as those with neurodevelopmental and neurodegenerative disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

Interpreting CARS images of tissue within the C-H-stretching region

NASA Astrophysics Data System (ADS)

Dietzek, Benjamin; Meyer, Tobias; Medyukhina, Anna; Bergner, Norbert; Krafft, Christoph; Romeike, Bernd F. M.; Reichart, Rupert; Kalff, Rolf; Schmitt, Michael; Popp, Jürgen

2014-03-01

Single band coherent anti-Stokes Raman scattering (CARS) microscopy within the CH-stretching region is applied to detect individual cells and nuclei of human brain tissue and brain tumors - an information which allows for histopathologic grading of the tissue. The CARS image contrast within the C-H-stretching region correlated to the tissue composition. Based on the specific application example of identifying nuclei within (coherent) Raman images of neurotissue sections, we shall derive general design parameters for lasers optimally suited to serve in a clinical environment and discuss the potential of recently developed methods to analyze spectrally resolved CARS images and image segmentation algorithms.

Optimization of a Widefield Structured Illumination Microscope for Non-Destructive Assessment and Quantification of Nuclear Features in Tumor Margins of a Primary Mouse Model of Sarcoma

PubMed Central

Fu, Henry L.; Mueller, Jenna L.; Javid, Melodi P.; Mito, Jeffrey K.; Kirsch, David G.; Ramanujam, Nimmi; Brown, J. Quincy

2013-01-01

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1×1.6 mm (3.4 mm2) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm−1 used in conjunction with a 4×0.1 NA objective ( = 0.165). This yielded an optical section thickness of 128 µm and an 8.9×contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy. PMID:23894357

Automated recognition of cell phenotypes in histology images based on membrane- and nuclei-targeting biomarkers

PubMed Central

Karaçalı, Bilge; Vamvakidou, Alexandra P; Tözeren, Aydın

2007-01-01

Background Three-dimensional in vitro culture of cancer cells are used to predict the effects of prospective anti-cancer drugs in vivo. In this study, we present an automated image analysis protocol for detailed morphological protein marker profiling of tumoroid cross section images. Methods Histologic cross sections of breast tumoroids developed in co-culture suspensions of breast cancer cell lines, stained for E-cadherin and progesterone receptor, were digitized and pixels in these images were classified into five categories using k-means clustering. Automated segmentation was used to identify image regions composed of cells expressing a given biomarker. Synthesized images were created to check the accuracy of the image processing system. Results Accuracy of automated segmentation was over 95% in identifying regions of interest in synthesized images. Image analysis of adjacent histology slides stained, respectively, for Ecad and PR, accurately predicted regions of different cell phenotypes. Image analysis of tumoroid cross sections from different tumoroids obtained under the same co-culture conditions indicated the variation of cellular composition from one tumoroid to another. Variations in the compositions of cross sections obtained from the same tumoroid were established by parallel analysis of Ecad and PR-stained cross section images. Conclusion Proposed image analysis methods offer standardized high throughput profiling of molecular anatomy of tumoroids based on both membrane and nuclei markers that is suitable to rapid large scale investigations of anti-cancer compounds for drug development. PMID:17822559

Automatic Segmentation of Granular Objects in Images: Combining Local Density Clustering and Gradient-Barrier Watershed

DTIC Science & Technology

2014-01-01

in applications, such as the recognition of biological cells [12,17], cell nuclei [8–10], colonies, and pollen [34,35], as well as nanoparticles [6... pollen image shown in Fig. 8, we see that two pollen grains on the upper-right are merged in the gradient-weighted distance trans- form watershed...10.1016/j.patcog.2013.11.004i images, pollen images, as well as physical nanoparticle images. This is only a small selection of this approach’s

A three-dimensional image processing program for accurate, rapid, and semi-automated segmentation of neuronal somata with dense neurite outgrowth

PubMed Central

Ross, James D.; Cullen, D. Kacy; Harris, James P.; LaPlaca, Michelle C.; DeWeerth, Stephen P.

2015-01-01

Three-dimensional (3-D) image analysis techniques provide a powerful means to rapidly and accurately assess complex morphological and functional interactions between neural cells. Current software-based identification methods of neural cells generally fall into two applications: (1) segmentation of cell nuclei in high-density constructs or (2) tracing of cell neurites in single cell investigations. We have developed novel methodologies to permit the systematic identification of populations of neuronal somata possessing rich morphological detail and dense neurite arborization throughout thick tissue or 3-D in vitro constructs. The image analysis incorporates several novel automated features for the discrimination of neurites and somata by initially classifying features in 2-D and merging these classifications into 3-D objects; the 3-D reconstructions automatically identify and adjust for over and under segmentation errors. Additionally, the platform provides for software-assisted error corrections to further minimize error. These features attain very accurate cell boundary identifications to handle a wide range of morphological complexities. We validated these tools using confocal z-stacks from thick 3-D neural constructs where neuronal somata had varying degrees of neurite arborization and complexity, achieving an accuracy of ≥95%. We demonstrated the robustness of these algorithms in a more complex arena through the automated segmentation of neural cells in ex vivo brain slices. These novel methods surpass previous techniques by improving the robustness and accuracy by: (1) the ability to process neurites and somata, (2) bidirectional segmentation correction, and (3) validation via software-assisted user input. This 3-D image analysis platform provides valuable tools for the unbiased analysis of neural tissue or tissue surrogates within a 3-D context, appropriate for the study of multi-dimensional cell-cell and cell-extracellular matrix interactions. PMID:26257609

Two-phase deep convolutional neural network for reducing class skewness in histopathological images based breast cancer detection.

PubMed

Wahab, Noorul; Khan, Asifullah; Lee, Yeon Soo

2017-06-01

Different types of breast cancer are affecting lives of women across the world. Common types include Ductal carcinoma in situ (DCIS), Invasive ductal carcinoma (IDC), Tubular carcinoma, Medullary carcinoma, and Invasive lobular carcinoma (ILC). While detecting cancer, one important factor is mitotic count - showing how rapidly the cells are dividing. But the class imbalance problem, due to the small number of mitotic nuclei in comparison to the overwhelming number of non-mitotic nuclei, affects the performance of classification models. This work presents a two-phase model to mitigate the class biasness issue while classifying mitotic and non-mitotic nuclei in breast cancer histopathology images through a deep convolutional neural network (CNN). First, nuclei are segmented out using blue ratio and global binary thresholding. In Phase-1 a CNN is then trained on the segmented out 80×80 pixel patches based on a standard dataset. Hard non-mitotic examples are identified and augmented; mitotic examples are oversampled by rotation and flipping; whereas non-mitotic examples are undersampled by blue ratio histogram based k-means clustering. Based on this information from Phase-1, the dataset is modified for Phase-2 in order to reduce the effects of class imbalance. The proposed CNN architecture and data balancing technique yielded an F-measure of 0.79, and outperformed all the methods relying on specific handcrafted features, as well as those using a combination of handcrafted and CNN-generated features. Copyright © 2017 Elsevier Ltd. All rights reserved.

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments.

PubMed

Van Valen, David A; Kudo, Takamasa; Lane, Keara M; Macklin, Derek N; Quach, Nicolas T; DeFelice, Mialy M; Maayan, Inbal; Tanouchi, Yu; Ashley, Euan A; Covert, Markus W

2016-11-01

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments

DOE PAGES

Van Valen, David A.; Kudo, Takamasa; Lane, Keara M.; ...

2016-11-04

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domainsmore » of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.« less

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Van Valen, David A.; Kudo, Takamasa; Lane, Keara M.

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domainsmore » of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems.« less

Deep Learning Automates the Quantitative Analysis of Individual Cells in Live-Cell Imaging Experiments

PubMed Central

Van Valen, David A.; Lane, Keara M.; Quach, Nicolas T.; Maayan, Inbal

2016-01-01

Live-cell imaging has opened an exciting window into the role cellular heterogeneity plays in dynamic, living systems. A major critical challenge for this class of experiments is the problem of image segmentation, or determining which parts of a microscope image correspond to which individual cells. Current approaches require many hours of manual curation and depend on approaches that are difficult to share between labs. They are also unable to robustly segment the cytoplasms of mammalian cells. Here, we show that deep convolutional neural networks, a supervised machine learning method, can solve this challenge for multiple cell types across the domains of life. We demonstrate that this approach can robustly segment fluorescent images of cell nuclei as well as phase images of the cytoplasms of individual bacterial and mammalian cells from phase contrast images without the need for a fluorescent cytoplasmic marker. These networks also enable the simultaneous segmentation and identification of different mammalian cell types grown in co-culture. A quantitative comparison with prior methods demonstrates that convolutional neural networks have improved accuracy and lead to a significant reduction in curation time. We relay our experience in designing and optimizing deep convolutional neural networks for this task and outline several design rules that we found led to robust performance. We conclude that deep convolutional neural networks are an accurate method that require less curation time, are generalizable to a multiplicity of cell types, from bacteria to mammalian cells, and expand live-cell imaging capabilities to include multi-cell type systems. PMID:27814364

Automated analysis of siRNA screens of cells infected by hepatitis C and dengue viruses based on immunofluorescence microscopy images

NASA Astrophysics Data System (ADS)

Matula, Petr; Kumar, Anil; Wörz, Ilka; Harder, Nathalie; Erfle, Holger; Bartenschlager, Ralf; Eils, Roland; Rohr, Karl

2008-03-01

We present an image analysis approach as part of a high-throughput microscopy siRNA-based screening system using cell arrays for the identification of cellular genes involved in hepatitis C and dengue virus replication. Our approach comprises: cell nucleus segmentation, quantification of virus replication level in the neighborhood of segmented cell nuclei, localization of regions with transfected cells, cell classification by infection status, and quality assessment of an experiment and single images. In particular, we propose a novel approach for the localization of regions of transfected cells within cell array images, which combines model-based circle fitting and grid fitting. By this scheme we integrate information from single cell array images and knowledge from the complete cell arrays. The approach is fully automatic and has been successfully applied to a large number of cell array images from screening experiments. The experimental results show a good agreement with the expected behaviour of positive as well as negative controls and encourage the application to screens from further high-throughput experiments.

[Colocalization of nucleoli in cell nuclei of HeLa line].

PubMed

Petrov, Iu P; Neguliaev, Iu A; Tsupkina, N V

2014-01-01

The pattern of localization of nucleoli relative to each other and to cell nucleus was studied in M-HeLa cell line. For this puspose, the following morphometric parameters were introduced. For the two-nucleolar cells: 1) the ratio of the nucleus long axis to the length of a segment between the centers of the nucleoli, and 2) the angle between the segment connecting the centers of the nucleoli and a longitudinal axis of cell nucleus. For the three-nucleolar cells: the ratio perimeter of the nucleus to perimeter of a triangle with vertexes in the centre of nucleoli. We have shown that the values of these parameters are individual for each cell but their values remain constant for the cell in spite of the changes in cell shape. These results allow us to conclude that, on the one hand, the nucleoli colocalization is individual for each cell, and, on the other hand, location of nucleoli in relation to nucleus is not changed during interphase. Thereby, the distance between nucleoli increases proportionally with nucleus growth.

Nucleus segmentation in histology images with hierarchical multilevel thresholding

NASA Astrophysics Data System (ADS)

Ahmady Phoulady, Hady; Goldgof, Dmitry B.; Hall, Lawrence O.; Mouton, Peter R.

2016-03-01

Automatic segmentation of histological images is an important step for increasing throughput while maintaining high accuracy, avoiding variation from subjective bias, and reducing the costs for diagnosing human illnesses such as cancer and Alzheimer's disease. In this paper, we present a novel method for unsupervised segmentation of cell nuclei in stained histology tissue. Following an initial preprocessing step involving color deconvolution and image reconstruction, the segmentation step consists of multilevel thresholding and a series of morphological operations. The only parameter required for the method is the minimum region size, which is set according to the resolution of the image. Hence, the proposed method requires no training sets or parameter learning. Because the algorithm requires no assumptions or a priori information with regard to cell morphology, the automatic approach is generalizable across a wide range of tissues. Evaluation across a dataset consisting of diverse tissues, including breast, liver, gastric mucosa and bone marrow, shows superior performance over four other recent methods on the same dataset in terms of F-measure with precision and recall of 0.929 and 0.886, respectively.

The iRoCS Toolbox--3D analysis of the plant root apical meristem at cellular resolution.

PubMed

Schmidt, Thorsten; Pasternak, Taras; Liu, Kun; Blein, Thomas; Aubry-Hivet, Dorothée; Dovzhenko, Alexander; Duerr, Jasmin; Teale, William; Ditengou, Franck A; Burkhardt, Hans; Ronneberger, Olaf; Palme, Klaus

2014-03-01

To achieve a detailed understanding of processes in biological systems, cellular features must be quantified in the three-dimensional (3D) context of cells and organs. We described use of the intrinsic root coordinate system (iRoCS) as a reference model for the root apical meristem of plants. iRoCS enables direct and quantitative comparison between the root tips of plant populations at single-cell resolution. The iRoCS Toolbox automatically fits standardized coordinates to raw 3D image data. It detects nuclei or segments cells, automatically fits the coordinate system, and groups the nuclei/cells into the root's tissue layers. The division status of each nucleus may also be determined. The only manual step required is to mark the quiescent centre. All intermediate outputs may be refined if necessary. The ability to learn the visual appearance of nuclei by example allows the iRoCS Toolbox to be easily adapted to various phenotypes. The iRoCS Toolbox is provided as an open-source software package, licensed under the GNU General Public License, to make it accessible to a broad community. To demonstrate the power of the technique, we measured subtle changes in cell division patterns caused by modified auxin flux within the Arabidopsis thaliana root apical meristem. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Effects of ionizing radiation on the light sensing elements of the retina. [Structural and physiological effects of carbon, helium, and neon ions on rods and cones of salamanders and mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malachowski, M.J.

1978-07-01

This investigation was undertaken to quantitate possible morphological and physiological effects of particles of high linear energy transfer on the retina, in comparison with x-ray effects. The particles used were accelerated atomic nuclei of helium, carbon, and neon at kinetic energies of several hundred MeV/nucleon. For morphological studies, scanning and transmission electron microscopy and light microscopy were used. Physiological studies consisted of autoradiographic data of the rate of incorporation of labeled protein in the structures (opsin) of the outer segment of visual cells. Structural changes were found in the nuclei, as well as the inner and outer segments of visualmore » cells, rods and cones. At a low dose of 10 rad, x rays and helium had no statistically significant morphological effects, but carbon and neon beams did cause significant degeneration of individual cells, pointing to the existence of a linear dose--effect relationship. At high doses of several hundred rads, a Pathologic Index determined the relative biological effectiveness of neon against alpha particles to have a value of greater than 6. The severity of effects per particle increased with atomic number. Labeling studies demonstrated a decreased rate of incorporation of labeled proteins in the structural organization of the outer segments of visual rods. The rate of self-renewal of visual rod discs was punctuated by irradiation and the structures themselves were depleted of amino acids. A model of rod discs (metabolic and catabolic) was postulated for correlated early and late effects to high and low doses.« less

Automatic layer segmentation of H&E microscopic images of mice skin

NASA Astrophysics Data System (ADS)

Hussein, Saif; Selway, Joanne; Jassim, Sabah; Al-Assam, Hisham

2016-05-01

Mammalian skin is a complex organ composed of a variety of cells and tissue types. The automatic detection and quantification of changes in skin structures has a wide range of applications for biological research. To accurately segment and quantify nuclei, sebaceous gland, hair follicles, and other skin structures, there is a need for a reliable segmentation of different skin layers. This paper presents an efficient segmentation algorithm to segment the three main layers of mice skin, namely epidermis, dermis, and subcutaneous layers. It also segments the epidermis layer into two sub layers, basal and cornified layers. The proposed algorithm uses adaptive colour deconvolution technique on H&E stain images to separate different tissue structures, inter-modes and Otsu thresholding techniques were effectively combined to segment the layers. It then uses a set of morphological and logical operations on each layer to removing unwanted objects. A dataset of 7000 H&E microscopic images of mutant and wild type mice were used to evaluate the effectiveness of the algorithm. Experimental results examined by domain experts have confirmed the viability of the proposed algorithms.

Influence of nuclei segmentation on breast cancer malignancy classification

NASA Astrophysics Data System (ADS)

Jelen, Lukasz; Fevens, Thomas; Krzyzak, Adam

2009-02-01

Breast Cancer is one of the most deadly cancers affecting middle-aged women. Accurate diagnosis and prognosis are crucial to reduce the high death rate. Nowadays there are numerous diagnostic tools for breast cancer diagnosis. In this paper we discuss a role of nuclear segmentation from fine needle aspiration biopsy (FNA) slides and its influence on malignancy classification. Classification of malignancy plays a very important role during the diagnosis process of breast cancer. Out of all cancer diagnostic tools, FNA slides provide the most valuable information about the cancer malignancy grade which helps to choose an appropriate treatment. This process involves assessing numerous nuclear features and therefore precise segmentation of nuclei is very important. In this work we compare three powerful segmentation approaches and test their impact on the classification of breast cancer malignancy. The studied approaches involve level set segmentation, fuzzy c-means segmentation and textural segmentation based on co-occurrence matrix. Segmented nuclei were used to extract nuclear features for malignancy classification. For classification purposes four different classifiers were trained and tested with previously extracted features. The compared classifiers are Multilayer Perceptron (MLP), Self-Organizing Maps (SOM), Principal Component-based Neural Network (PCA) and Support Vector Machines (SVM). The presented results show that level set segmentation yields the best results over the three compared approaches and leads to a good feature extraction with a lowest average error rate of 6.51% over four different classifiers. The best performance was recorded for multilayer perceptron with an error rate of 3.07% using fuzzy c-means segmentation.

The development of a virtual 3D model of the renal corpuscle from serial histological sections for E-learning environments.

PubMed

Roth, Jeremy A; Wilson, Timothy D; Sandig, Martin

2015-01-01

Histology is a core subject in the anatomical sciences where learners are challenged to interpret two-dimensional (2D) information (gained from histological sections) to extrapolate and understand the three-dimensional (3D) morphology of cells, tissues, and organs. In gross anatomical education 3D models and learning tools have been associated with improved learning outcomes, but similar tools have not been created for histology education to visualize complex cellular structure-function relationships. This study outlines steps in creating a virtual 3D model of the renal corpuscle from serial, semi-thin, histological sections obtained from epoxy resin-embedded kidney tissue. The virtual renal corpuscle model was generated by digital segmentation to identify: Bowman's capsule, nuclei of epithelial cells in the parietal capsule, afferent arteriole, efferent arteriole, proximal convoluted tubule, distal convoluted tubule, glomerular capillaries, podocyte nuclei, nuclei of extraglomerular mesangial cells, nuclei of epithelial cells of the macula densa in the distal convoluted tubule. In addition to the imported images of the original sections the software generates, and allows for visualization of, images of virtual sections generated in any desired orientation, thus serving as a "virtual microtome". These sections can be viewed separately or with the 3D model in transparency. This approach allows for the development of interactive e-learning tools designed to enhance histology education of microscopic structures with complex cellular interrelationships. Future studies will focus on testing the efficacy of interactive virtual 3D models for histology education. © 2015 American Association of Anatomists.

Scanning and transmission electron microscopic analysis of ampullary segment of oviduct during estrous cycle in caprines.

PubMed

Sharma, R K; Singh, R; Bhardwaj, J K

2015-01-01

The ampullary segment of the mammalian oviduct provides suitable milieu for fertilization and development of zygote before implantation into uterus. It is, therefore, in the present study, the cyclic changes in the morphology of ampullary segment of goat oviduct were studied during follicular and luteal phases using scanning and transmission electron microscopy techniques. Topographical analysis revealed the presence of uniformly ciliated ampullary epithelia, concealing apical processes of non-ciliated cells along with bulbous secretory cells during follicular phase. The luteal phase was marked with decline in number of ciliated cells with increased occurrence of secretory cells. The ultrastructure analysis has demonstrated the presence of indented nuclear membrane, supranuclear cytoplasm, secretory granules, rough endoplasmic reticulum, large lipid droplets, apically located glycogen masses, oval shaped mitochondria in the secretory cells. The ciliated cells were characterized by the presence of elongated nuclei, abundant smooth endoplasmic reticulum, oval or spherical shaped mitochondria with crecentric cristae during follicular phase. However, in the luteal phase, secretory cells were possessing highly indented nucleus with diffused electron dense chromatin, hyaline nucleosol, increased number of lipid droplets. The ciliated cells had numerous fibrous granules and basal bodies. The parallel use of scanning and transmission electron microscopy techniques has enabled us to examine the cyclic and hormone dependent changes occurring in the topography and fine structure of epithelium of ampullary segment and its cells during different reproductive phases that will be great help in understanding major bottle neck that limits success rate in vitro fertilization and embryo transfer technology. © Wiley Periodicals, Inc.

Gebiss: an ImageJ plugin for the specification of ground truth and the performance evaluation of 3D segmentation algorithms

PubMed Central

2011-01-01

Background Image segmentation is a crucial step in quantitative microscopy that helps to define regions of tissues, cells or subcellular compartments. Depending on the degree of user interactions, segmentation methods can be divided into manual, automated or semi-automated approaches. 3D image stacks usually require automated methods due to their large number of optical sections. However, certain applications benefit from manual or semi-automated approaches. Scenarios include the quantification of 3D images with poor signal-to-noise ratios or the generation of so-called ground truth segmentations that are used to evaluate the accuracy of automated segmentation methods. Results We have developed Gebiss; an ImageJ plugin for the interactive segmentation, visualisation and quantification of 3D microscopic image stacks. We integrated a variety of existing plugins for threshold-based segmentation and volume visualisation. Conclusions We demonstrate the application of Gebiss to the segmentation of nuclei in live Drosophila embryos and the quantification of neurodegeneration in Drosophila larval brains. Gebiss was developed as a cross-platform ImageJ plugin and is freely available on the web at http://imaging.bii.a-star.edu.sg/projects/gebiss/. PMID:21668958

A Multiscale Parallel Computing Architecture for Automated Segmentation of the Brain Connectome

PubMed Central

Knobe, Kathleen; Newton, Ryan R.; Schlimbach, Frank; Blower, Melanie; Reid, R. Clay

2015-01-01

Several groups in neurobiology have embarked into deciphering the brain circuitry using large-scale imaging of a mouse brain and manual tracing of the connections between neurons. Creating a graph of the brain circuitry, also called a connectome, could have a huge impact on the understanding of neurodegenerative diseases such as Alzheimer’s disease. Although considerably smaller than a human brain, a mouse brain already exhibits one billion connections and manually tracing the connectome of a mouse brain can only be achieved partially. This paper proposes to scale up the tracing by using automated image segmentation and a parallel computing approach designed for domain experts. We explain the design decisions behind our parallel approach and we present our results for the segmentation of the vasculature and the cell nuclei, which have been obtained without any manual intervention. PMID:21926011

Phenotypic feature quantification of patient derived 3D cancer spheroids in fluorescence microscopy image

NASA Astrophysics Data System (ADS)

Kang, Mi-Sun; Rhee, Seon-Min; Seo, Ji-Hyun; Kim, Myoung-Hee

2017-03-01

Patients' responses to a drug differ at the cellular level. Here, we present an image-based cell phenotypic feature quantification method for predicting the responses of patient-derived glioblastoma cells to a particular drug. We used high-content imaging to understand the features of patient-derived cancer cells. A 3D spheroid culture formation resembles the in vivo environment more closely than 2D adherent cultures do, and it allows for the observation of cellular aggregate characteristics. However, cell analysis at the individual level is more challenging. In this paper, we demonstrate image-based phenotypic screening of the nuclei of patient-derived cancer cells. We first stitched the images of each well of the 384-well plate with the same state. We then used intensity information to detect the colonies. The nuclear intensity and morphological characteristics were used for the segmentation of individual nuclei. Next, we calculated the position of each nucleus that is appeal of the spatial pattern of cells in the well environment. Finally, we compared the results obtained using 3D spheroid culture cells with those obtained using 2D adherent culture cells from the same patient being treated with the same drugs. This technique could be applied for image-based phenotypic screening of cells to determine the patient's response to the drug.

Reproduction in the male honey possum (Tarsipes rostratus: Marsupialia): the epididymis.

PubMed

Cummins, J M; Temple-Smith, P D; Renfree, M B

1986-11-01

The epididymis of the adult honey possum, Tarsipes rostratus, is enclosed by a heavily pigmented tunica vaginalis and lies with the testis in a prominent prepenile scrotum. It is connected to the testis by a single ductus efferentis and is lined by approximately equal numbers of cuboidal ciliated and principal cells. It is unusual for marsupials in having no well-defined compartments or fibrous septae and in having extensive convolutions of the duct only at the caudal flexure. Three principal functional zones (initial, middle, and terminal segments) were identified in the epididymis, based on epithelial type and ultrastructural evidence of sperm maturation. Luminal diameter increases progressively throughout the tract, and epithelial height variations (from about 2 to 20 microns) are greatest in the terminal segment. The epithelium itself is remarkably low (maximum of 21.6 microns) compared with that seen in the epididymis of other mammals. The thickness of the peritubular smooth muscle coat increases close to the junction of the epididymis and ductus deferens. Sperm concentrations were estimated from counts of sperm nuclei and thus can be no more than approximations. The figures are consistent, however, with a rapid increase in concentration in the initial segment, indicating extensive fluid resorption. Sperm concentrations appear to peak in the distal zone of the terminal segment, although sampling problems and wide variations in count make such a conclusion only tentative. Principal and basal cells are the predominant cell types in the epididymal epithelium. Basal cells are most abundant in the initial and distal middle segment. Principal cells show structural evidence of active exchange with the luminal contents and have abundant apical stereocilia, the structure of which depends on the epididymal zone. Other cell types occur less commonly in the epithelium. Lipid-rich and phagocytic principal cells are restricted to the middle and distal zones of the middle segment, respectively. Clear cells, restricted to the terminal segment, and halo cells were found in very low numbers. As in some other marsupials, principal cells (possibly specialized for this function) selectively remove cytoplasmic droplets and probably other cellular debris from the luminal contents. In Tarsipes, however, this process is not very efficient, and many discarded droplets pass through to the terminal segment where they form large masses of debris associated with aggregates of degenerating spermatozoa.

Essential roles for lines in mediating leg and antennal proximodistal patterning and generating a stable Notch signaling interface at segment borders.

PubMed

Greenberg, Lina; Hatini, Victor

2009-06-01

The Drosophila leg imaginal disc provides a paradigm with which to understand the fundamental developmental mechanisms that generate an intricate appendage structure. Leg formation depends on the subdivision of the leg proximodistal (PD) axis into broad domains by the leg gap genes. The leg gap genes act combinatorially to initiate the expression of the Notch ligands Delta (Dl) and Serrate (Ser) in a segmental pattern. Dl and Ser induce the expression of a set of transcriptional regulators along the segment border, which mediate leg segment growth and joint morphogenesis. Here we show that Lines accumulates in nuclei in the presumptive tarsus and the inter-joints of proximal leg segments and governs the formation of these structures by destabilizing the nuclear protein Bowl. Across the presumptive tarsus, lines modulates the opposing expression landscapes of the leg gap gene dachshund (dac) and the tarsal PD genes, bric-a-brac 2 (bab), apterous (ap) and BarH1 (Bar). In this manner, lines inhibits proximal tarsal fates and promotes medial and distal tarsal fates. Across proximal leg segments, lines antagonizes bowl to promote Dl expression by relief-of-repression. In turn, Dl signals asymmetrically to stabilize Bowl in adjacent distal cells. Bowl, then, acts cell-autonomously, together with one or more redundant factors, to repress Dl expression. Together, lines and bowl act as a binary switch to generate a stable Notch signaling interface between Dl-expressing cells and adjacent distal cell. lines plays analogous roles in developing antennae, which are serially homologous to legs, suggesting evolutionarily conserved roles for lines in ventral appendage formation.

Small intestine histomorphometry of beef cattle with divergent feed efficiency

PubMed Central

2013-01-01

Background The provision of feed is a major cost in beef production. Therefore, the improvement of feed efficiency is warranted. The direct assessment of feed efficiency has limitations and alternatives are needed. Small intestine micro-architecture is associated with function and may be related to feed efficiency. The objective was to verify the potential histomorphological differences in the small intestine of animals with divergent feed efficiency. Methods From a population of 45 feedlot steers, 12 were selected with low-RFI (superior feed efficiency) and 12 with high-RFI (inferior feed efficiency) at the end of the finishing period. The animals were processed at 13.79 ± 1.21 months of age. Within 1.5 h of slaughter the gastrointestinal tract was collected and segments from duodenum and ileum were harvested. Tissue fragments were processed, sectioned and stained with hematoxylin and eosin. Photomicroscopy images were taken under 1000x magnification. For each animal 100 intestinal crypts were imaged, in a cross section view, from each of the two intestinal segments. Images were analyzed using the software ImageJ®. The measurements taken were: crypt area, crypt perimeter, crypt lumen area, nuclei number and the cell size was indirectly calculated. Data were analyzed using general linear model and correlation procedures of SAS®. Results Efficient beef steers (low-RFI) have a greater cellularity (indicated by nuclei number) in the small intestinal crypts, both in duodenum and ileum, than less efficient beef steers (high-RFI) (P < 0.05). The mean values for the nuclei number of the low-RFI and high-RFI groups were 33.16 and 30.30 in the duodenum and 37.21 and 33.65 in the ileum, respectively. The average size of the cells did not differ between feed efficiency groups in both segments (P ≥ 0.10). A trend was observed (P ≤ 0.10) for greater crypt area and crypt perimeter in the ileum for cattle with improved feed efficiency. Conclusion Improved feed efficiency is associated with greater cellularity and no differences on average cell size in the crypts of the small intestine in the bovine. These observations are likely to lead to an increase in the energy demand by the small intestine regardless of the more desirable feed efficiency. PMID:23379622

Age of heart disease presentation and dysmorphic nuclei in patients with LMNA mutations

PubMed Central

Core, Jason Q.; Mehrabi, Mehrsa; Robinson, Zachery R.; Ochs, Alexander R.; McCarthy, Linda A.; Zaragoza, Michael V.

2017-01-01

Nuclear shape defects are a distinguishing characteristic in laminopathies, cancers, and other pathologies. Correlating these defects to the symptoms, mechanisms, and progression of disease requires unbiased, quantitative, and high-throughput means of quantifying nuclear morphology. To accomplish this, we developed a method of automatically segmenting fluorescently stained nuclei in 2D microscopy images and then classifying them as normal or dysmorphic based on three geometric features of the nucleus using a package of Matlab codes. As a test case, cultured skin-fibroblast nuclei of individuals possessing LMNA splice-site mutation (c.357-2A>G), LMNA nonsense mutation (c.736 C>T, pQ246X) in exon 4, LMNA missense mutation (c.1003C>T, pR335W) in exon 6, Hutchinson-Gilford Progeria Syndrome, and no LMNA mutations were analyzed. For each cell type, the percentage of dysmorphic nuclei, and other morphological features such as average nuclear area and average eccentricity were obtained. Compared to blind observers, our procedure implemented in Matlab codes possessed similar accuracy to manual counting of dysmorphic nuclei while being significantly more consistent. The automatic quantification of nuclear defects revealed a correlation between in vitro results and age of patients for initial symptom onset. Our results demonstrate the method’s utility in experimental studies of diseases affecting nuclear shape through automated, unbiased, and accurate identification of dysmorphic nuclei. PMID:29149195

Age of heart disease presentation and dysmorphic nuclei in patients with LMNA mutations.

PubMed

Core, Jason Q; Mehrabi, Mehrsa; Robinson, Zachery R; Ochs, Alexander R; McCarthy, Linda A; Zaragoza, Michael V; Grosberg, Anna

2017-01-01

Nuclear shape defects are a distinguishing characteristic in laminopathies, cancers, and other pathologies. Correlating these defects to the symptoms, mechanisms, and progression of disease requires unbiased, quantitative, and high-throughput means of quantifying nuclear morphology. To accomplish this, we developed a method of automatically segmenting fluorescently stained nuclei in 2D microscopy images and then classifying them as normal or dysmorphic based on three geometric features of the nucleus using a package of Matlab codes. As a test case, cultured skin-fibroblast nuclei of individuals possessing LMNA splice-site mutation (c.357-2A>G), LMNA nonsense mutation (c.736 C>T, pQ246X) in exon 4, LMNA missense mutation (c.1003C>T, pR335W) in exon 6, Hutchinson-Gilford Progeria Syndrome, and no LMNA mutations were analyzed. For each cell type, the percentage of dysmorphic nuclei, and other morphological features such as average nuclear area and average eccentricity were obtained. Compared to blind observers, our procedure implemented in Matlab codes possessed similar accuracy to manual counting of dysmorphic nuclei while being significantly more consistent. The automatic quantification of nuclear defects revealed a correlation between in vitro results and age of patients for initial symptom onset. Our results demonstrate the method's utility in experimental studies of diseases affecting nuclear shape through automated, unbiased, and accurate identification of dysmorphic nuclei.

Morphology and innervation pattern of the feline urogenital junction.

PubMed

Wrobel, K H; Gürtler, A

2004-12-01

The feline urogenital junction is situated between the extratesticular rete and the spacious initial segments of the efferent ductules. The rete epithelium is cuboidal to low columnar. The rete cells forming the junction rest on a wavy basal lamina, display deep mutual invaginations, possess central nuclei with several infoldings and form a distinct border with the columnar epithelial cells of the initial segments of the ductuli efferentes. The epithelium of the initial segments is composed of ciliated cells and non-ciliated principal cells. The latter are the dominating type and characterized by an apical brush-border and a supranuclear endocytotic apparatus. The stroma of the extratesticular rete contains an abundance of collagen whereas contractile cells are here generally absent. In contrast, the initial segments of the efferent ductules are surrounded by elastic fibres and a layer of contractile cells. All nerves for the feline urogenital junction come from the nervus spermaticus superior. In the epididymal head, small nerve bundles deviate into the septa between the ductules. Single fibres establish a dense network within the muscular coat of the ductuli. At the transition to the extratesticular rete, this network ends abruptly. Nerve fibres in the confines of the rete are associated with blood vessels or proceed to the testicular interior, but establish no relationships with the rete epithelium or the myofibroblasts of the mediastinum. The nervous network in the walls of the efferent ductules and their initial segments is not only composed of sympathetic but also parasympathetic, non-myelinated fibres. Particularly noteworthy is the abundance of calcitonin gene-related peptide (CGRP)- and substance P (SP)-containing axons around the initial segments. Both neuroproteins are consistent markers for sensory neurones. Taken together, it can be assumed that the entry of seminal fluid and spermatozoa into the efferent ductules is controlled by a regulatory nervous chain provided with afferent and efferent components.

Blue intensity matters for cell cycle profiling in fluorescence DAPI-stained images.

PubMed

Ferro, Anabela; Mestre, Tânia; Carneiro, Patrícia; Sahumbaiev, Ivan; Seruca, Raquel; Sanches, João M

2017-05-01

In the past decades, there has been an amazing progress in the understanding of the molecular mechanisms of the cell cycle. This has been possible largely due to a better conceptualization of the cycle itself, but also as a consequence of technological advances. Herein, we propose a new fluorescence image-based framework targeted at the identification and segmentation of stained nuclei with the purpose to determine DNA content in distinct cell cycle stages. The method is based on discriminative features, such as total intensity and area, retrieved from in situ stained nuclei by fluorescence microscopy, allowing the determination of the cell cycle phase of both single and sub-population of cells. The analysis framework was built on a modified k-means clustering strategy and refined with a Gaussian mixture model classifier, which enabled the definition of highly accurate classification clusters corresponding to G1, S and G2 phases. Using the information retrieved from area and fluorescence total intensity, the modified k-means (k=3) cluster imaging framework classified 64.7% of the imaged nuclei, as being at G1 phase, 12.0% at G2 phase and 23.2% at S phase. Performance of the imaging framework was ascertained with normal murine mammary gland cells constitutively expressing the Fucci2 technology, exhibiting an overall sensitivity of 94.0%. Further, the results indicate that the imaging framework has a robust capacity to both identify a given DAPI-stained nucleus to its correct cell cycle phase, as well as to determine, with very high probability, true negatives. Importantly, this novel imaging approach is a non-disruptive method that allows an integrative and simultaneous quantitative analysis of molecular and morphological parameters, thus awarding the possibility of cell cycle profiling in cytological and histological samples.

NucliTrack: an integrated nuclei tracking application.

PubMed

Cooper, Sam; Barr, Alexis R; Glen, Robert; Bakal, Chris

2017-10-15

Live imaging studies give unparalleled insight into dynamic single cell behaviours and fate decisions. However, the challenge of reliably tracking single cells over long periods of time limits both the throughput and ease with which such studies can be performed. Here, we present NucliTrack, a cross platform solution for automatically segmenting, tracking and extracting features from fluorescently labelled nuclei. NucliTrack performs similarly to other state-of-the-art cell tracking algorithms, but NucliTrack's interactive, graphical interface makes it significantly more user friendly. NucliTrack is available as a free, cross platform application and open source Python package. Installation details and documentation are at: http://nuclitrack.readthedocs.io/en/latest/ A video guide can be viewed online: https://www.youtube.com/watch?v=J6e0D9F-qSU Source code is available through Github: https://github.com/samocooper/nuclitrack. A Matlab toolbox is also available at: https://uk.mathworks.com/matlabcentral/fileexchange/61479-samocooper-nuclitrack-matlab. sam@socooper.com. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

NucliTrack: an integrated nuclei tracking application

PubMed Central

Cooper, Sam; Barr, Alexis R.; Glen, Robert; Bakal, Chris

2017-01-01

Abstract Summary Live imaging studies give unparalleled insight into dynamic single cell behaviours and fate decisions. However, the challenge of reliably tracking single cells over long periods of time limits both the throughput and ease with which such studies can be performed. Here, we present NucliTrack, a cross platform solution for automatically segmenting, tracking and extracting features from fluorescently labelled nuclei. NucliTrack performs similarly to other state-of-the-art cell tracking algorithms, but NucliTrack’s interactive, graphical interface makes it significantly more user friendly. Availability and implementation NucliTrack is available as a free, cross platform application and open source Python package. Installation details and documentation are at: http://nuclitrack.readthedocs.io/en/latest/ A video guide can be viewed online: https://www.youtube.com/watch?v=J6e0D9F-qSU Source code is available through Github: https://github.com/samocooper/nuclitrack. A Matlab toolbox is also available at: https://uk.mathworks.com/matlabcentral/fileexchange/61479-samocooper-nuclitrack-matlab. Contact sam@socooper.com Supplementary information Supplementary data are available at Bioinformatics online. PMID:28637183

Automated seeding-based nuclei segmentation in nonlinear optical microscopy.

PubMed

Medyukhina, Anna; Meyer, Tobias; Heuke, Sandro; Vogler, Nadine; Dietzek, Benjamin; Popp, Jürgen

2013-10-01

Nonlinear optical (NLO) microscopy based, e.g., on coherent anti-Stokes Raman scattering (CARS) or two-photon-excited fluorescence (TPEF) is a fast label-free imaging technique, with a great potential for biomedical applications. However, NLO microscopy as a diagnostic tool is still in its infancy; there is a lack of robust and durable nuclei segmentation methods capable of accurate image processing in cases of variable image contrast, nuclear density, and type of investigated tissue. Nonetheless, such algorithms specifically adapted to NLO microscopy present one prerequisite for the technology to be routinely used, e.g., in pathology or intraoperatively for surgical guidance. In this paper, we compare the applicability of different seeding and boundary detection methods to NLO microscopic images in order to develop an optimal seeding-based approach capable of accurate segmentation of both TPEF and CARS images. Among different methods, the Laplacian of Gaussian filter showed the best accuracy for the seeding of the image, while a modified seeded watershed segmentation was the most accurate in the task of boundary detection. The resulting combination of these methods followed by the verification of the detected nuclei performs high average sensitivity and specificity when applied to various types of NLO microscopy images.

A fourth order PDE based fuzzy c- means approach for segmentation of microscopic biopsy images in presence of Poisson noise for cancer detection.

PubMed

Kumar, Rajesh; Srivastava, Subodh; Srivastava, Rajeev

2017-07-01

For cancer detection from microscopic biopsy images, image segmentation step used for segmentation of cells and nuclei play an important role. Accuracy of segmentation approach dominate the final results. Also the microscopic biopsy images have intrinsic Poisson noise and if it is present in the image the segmentation results may not be accurate. The objective is to propose an efficient fuzzy c-means based segmentation approach which can also handle the noise present in the image during the segmentation process itself i.e. noise removal and segmentation is combined in one step. To address the above issues, in this paper a fourth order partial differential equation (FPDE) based nonlinear filter adapted to Poisson noise with fuzzy c-means segmentation method is proposed. This approach is capable of effectively handling the segmentation problem of blocky artifacts while achieving good tradeoff between Poisson noise removals and edge preservation of the microscopic biopsy images during segmentation process for cancer detection from cells. The proposed approach is tested on breast cancer microscopic biopsy data set with region of interest (ROI) segmented ground truth images. The microscopic biopsy data set contains 31 benign and 27 malignant images of size 896 × 768. The region of interest selected ground truth of all 58 images are also available for this data set. Finally, the result obtained from proposed approach is compared with the results of popular segmentation algorithms; fuzzy c-means, color k-means, texture based segmentation, and total variation fuzzy c-means approaches. The experimental results shows that proposed approach is providing better results in terms of various performance measures such as Jaccard coefficient, dice index, Tanimoto coefficient, area under curve, accuracy, true positive rate, true negative rate, false positive rate, false negative rate, random index, global consistency error, and variance of information as compared to other segmentation approaches used for cancer detection. Copyright © 2017 Elsevier B.V. All rights reserved.

Phagocytosis of photoreceptor outer segments by transplanted human neural stem cells as a neuroprotective mechanism in retinal degeneration.

PubMed

Cuenca, Nicolás; Fernández-Sánchez, Laura; McGill, Trevor J; Lu, Bin; Wang, Shaomei; Lund, Raymond; Huhn, Stephen; Capela, Alexandra

2013-10-15

Transplantation of human central nervous system stem cells (HuCNS-SC) into the subretinal space of Royal College of Surgeons (RCS) rats preserves photoreceptors and visual function. To explore possible mechanism(s) of action underlying this neuroprotective effect, we performed a detailed morphologic and ultrastructure analysis of HuCNS-SC transplanted retinas. The HuCNS-SC were transplanted into the subretinal space of RCS rats. Histologic examination of the transplanted retinas was performed by light and electron microscopy. Areas of the retina adjacent to HuCNS-SC graft (treated regions) were analyzed and compared to control sections obtained from the same retina, but distant from the transplant site (untreated regions). The HuCNS-SC were detected as a layer of STEM 121 immunopositive cells in the subretinal space. In treated regions, preserved photoreceptor nuclei, as well as inner and outer segments were identified readily. In contrast, classic signs of degeneration were observed in the untreated regions. Interestingly, detailed ultrastructure analysis revealed a striking preservation of the photoreceptor-bipolar-horizontal cell synaptic contacts in the outer plexiform layer (OPL) of treated areas, in stark contrast with untreated areas. Finally, the presence of phagosomes and vesicles exhibiting the lamellar structure of outer segments also was detected within the cytosol of HuCNS-SC, indicating that these cells have phagocytic capacity in vivo. This study reveals the novel finding that preservation of specialized synaptic contacts between photoreceptors and second order neurons, as well as phagocytosis of photoreceptor outer segments, are potential mechanism(s) of HuCNS-SC transplantation, mediating functional rescue in retinal degeneration.

Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies

PubMed Central

Riera, Marina; Fontrodona, Laura; Albert, Silvia; Ramirez, Diana Mora; Seriola, Anna; Salas, Anna; Muñoz, Yolanda; Ramos, David; Villegas-Perez, Maria Paz; Zapata, Miguel Angel; Raya, Angel; Ruberte, Jesus; Veiga, Anna; Garcia-Arumi, Jose

2016-01-01

Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD. PMID:27006969

DeepPap: Deep Convolutional Networks for Cervical Cell Classification.

PubMed

Zhang, Ling; Le Lu; Nogues, Isabella; Summers, Ronald M; Liu, Shaoxiong; Yao, Jianhua

2017-11-01

Automation-assisted cervical screening via Pap smear or liquid-based cytology (LBC) is a highly effective cell imaging based cancer detection tool, where cells are partitioned into "abnormal" and "normal" categories. However, the success of most traditional classification methods relies on the presence of accurate cell segmentations. Despite sixty years of research in this field, accurate segmentation remains a challenge in the presence of cell clusters and pathologies. Moreover, previous classification methods are only built upon the extraction of hand-crafted features, such as morphology and texture. This paper addresses these limitations by proposing a method to directly classify cervical cells-without prior segmentation-based on deep features, using convolutional neural networks (ConvNets). First, the ConvNet is pretrained on a natural image dataset. It is subsequently fine-tuned on a cervical cell dataset consisting of adaptively resampled image patches coarsely centered on the nuclei. In the testing phase, aggregation is used to average the prediction scores of a similar set of image patches. The proposed method is evaluated on both Pap smear and LBC datasets. Results show that our method outperforms previous algorithms in classification accuracy (98.3%), area under the curve (0.99) values, and especially specificity (98.3%), when applied to the Herlev benchmark Pap smear dataset and evaluated using five-fold cross validation. Similar superior performances are also achieved on the HEMLBC (H&E stained manual LBC) dataset. Our method is promising for the development of automation-assisted reading systems in primary cervical screening.

Measuring topology of low-intensity DNA methylation sites for high-throughput assessment of epigenetic drug-induced effects in cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gertych, Arkadiusz, E-mail: gertycha@cshs.org; Bioinformatics, Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA; Farkas, Daniel L., E-mail: dlfarkas@gmail.com

2010-11-15

Epigenetic anti-cancer drugs with demethylating effects have shown to alter genome organization in mammalian cell nuclei. The interest in the development of novel epigenetic drugs has increased the demand for cell-based assays to evaluate drug performance in pre-clinical studies. An imaging-based cytometrical approach that can measure demethylation effects as changes in the spatial nuclear distributions of methylated cytosine and global DNA in cancer cells is introduced in this paper. The cells were studied by immunofluorescence with a specific antibody against 5-methylcytosine (MeC), and 4,6-diamidino-2-phenylindole (DAPI) for delineation of methylated sites and global DNA in nuclei. In the preprocessing step themore » segmentation of nuclei in three-dimensional images (3-D) is followed by an automated assessment of nuclear DAPI/MeC patterns to exclude dissimilar entities. Next, low-intensity MeC (LIM) and low-intensity DNA (LID) sites of similar nuclei are localized and processed to obtain specific nuclear density profiles. These profiles sampled at half of the total nuclear volume yielded two parameters: LIM{sub 0.5} and LID{sub 0.5}. The analysis shows that zebularine and 5-azacytidine-the two tested epigenetic drugs introduce changes in the spatial distribution of low-intensity DNA and MeC signals. LIM{sub 0.5} and LID{sub 0.5} were significantly different (p < 0.001) in 5-azacytidine treated (n = 660) and zebularine treated (n = 496) vs. untreated (n = 649) DU145 human prostate cancer cells. In the latter case the LIM sites were predominantly found at the nuclear border, whereas treated populations showed different degrees of increase in LIMs towards the interior nuclear space, in which a large portion of heterochromatin is located. The cell-by-cell evaluation of changes in the spatial reorganization of MeC/DAPI signals revealed that zebularine is a more gentle demethylating agent than 5-azacytidine. Measuring changes in the topology of low-intensity sites can potentially be a valuable component in the high-throughput assessment of demethylation and risk of chromatin reorganization in epigenetic-drug screening tasks.« less

Fast segmentation of stained nuclei in terabyte-scale, time resolved 3D microscopy image stacks.

PubMed

Stegmaier, Johannes; Otte, Jens C; Kobitski, Andrei; Bartschat, Andreas; Garcia, Ariel; Nienhaus, G Ulrich; Strähle, Uwe; Mikut, Ralf

2014-01-01

Automated analysis of multi-dimensional microscopy images has become an integral part of modern research in life science. Most available algorithms that provide sufficient segmentation quality, however, are infeasible for a large amount of data due to their high complexity. In this contribution we present a fast parallelized segmentation method that is especially suited for the extraction of stained nuclei from microscopy images, e.g., of developing zebrafish embryos. The idea is to transform the input image based on gradient and normal directions in the proximity of detected seed points such that it can be handled by straightforward global thresholding like Otsu's method. We evaluate the quality of the obtained segmentation results on a set of real and simulated benchmark images in 2D and 3D and show the algorithm's superior performance compared to other state-of-the-art algorithms. We achieve an up to ten-fold decrease in processing times, allowing us to process large data sets while still providing reasonable segmentation results.

A Dataset and a Technique for Generalized Nuclear Segmentation for Computational Pathology.

PubMed

Kumar, Neeraj; Verma, Ruchika; Sharma, Sanuj; Bhargava, Surabhi; Vahadane, Abhishek; Sethi, Amit

2017-07-01

Nuclear segmentation in digital microscopic tissue images can enable extraction of high-quality features for nuclear morphometrics and other analysis in computational pathology. Conventional image processing techniques, such as Otsu thresholding and watershed segmentation, do not work effectively on challenging cases, such as chromatin-sparse and crowded nuclei. In contrast, machine learning-based segmentation can generalize across various nuclear appearances. However, training machine learning algorithms requires data sets of images, in which a vast number of nuclei have been annotated. Publicly accessible and annotated data sets, along with widely agreed upon metrics to compare techniques, have catalyzed tremendous innovation and progress on other image classification problems, particularly in object recognition. Inspired by their success, we introduce a large publicly accessible data set of hematoxylin and eosin (H&E)-stained tissue images with more than 21000 painstakingly annotated nuclear boundaries, whose quality was validated by a medical doctor. Because our data set is taken from multiple hospitals and includes a diversity of nuclear appearances from several patients, disease states, and organs, techniques trained on it are likely to generalize well and work right out-of-the-box on other H&E-stained images. We also propose a new metric to evaluate nuclear segmentation results that penalizes object- and pixel-level errors in a unified manner, unlike previous metrics that penalize only one type of error. We also propose a segmentation technique based on deep learning that lays a special emphasis on identifying the nuclear boundaries, including those between the touching or overlapping nuclei, and works well on a diverse set of test images.

Recognition of acute lymphoblastic leukemia cells in microscopic images using k-means clustering and support vector machine classifier.

PubMed

Amin, Morteza Moradi; Kermani, Saeed; Talebi, Ardeshir; Oghli, Mostafa Ghelich

2015-01-01

Acute lymphoblastic leukemia is the most common form of pediatric cancer which is categorized into three L1, L2, and L3 and could be detected through screening of blood and bone marrow smears by pathologists. Due to being time-consuming and tediousness of the procedure, a computer-based system is acquired for convenient detection of Acute lymphoblastic leukemia. Microscopic images are acquired from blood and bone marrow smears of patients with Acute lymphoblastic leukemia and normal cases. After applying image preprocessing, cells nuclei are segmented by k-means algorithm. Then geometric and statistical features are extracted from nuclei and finally these cells are classified to cancerous and noncancerous cells by means of support vector machine classifier with 10-fold cross validation. These cells are also classified into their sub-types by multi-Support vector machine classifier. Classifier is evaluated by these parameters: Sensitivity, specificity, and accuracy which values for cancerous and noncancerous cells 98%, 95%, and 97%, respectively. These parameters are also used for evaluation of cell sub-types which values in mean 84.3%, 97.3%, and 95.6%, respectively. The results show that proposed algorithm could achieve an acceptable performance for the diagnosis of Acute lymphoblastic leukemia and its sub-types and can be used as an assistant diagnostic tool for pathologists.

Hotspot detection in pancreatic neuroendocrine tumors: density approximation by α-shape maps

NASA Astrophysics Data System (ADS)

Niazi, M. K. K.; Hartman, Douglas J.; Pantanowitz, Liron; Gurcan, Metin N.

2016-03-01

The grading of neuroendocrine tumors of the digestive system is dependent on accurate and reproducible assessment of the proliferation with the tumor, either by counting mitotic figures or counting Ki-67 positive nuclei. At the moment, most pathologists manually identify the hotspots, a practice which is tedious and irreproducible. To better help pathologists, we present an automatic method to detect all potential hotspots in neuroendocrine tumors of the digestive system. The method starts by segmenting Ki-67 positive nuclei by entropy based thresholding, followed by detection of centroids for all Ki-67 positive nuclei. Based on geodesic distance, approximated by the nuclei centroids, we compute two maps: an amoeba map and a weighted amoeba map. These maps are later combined to generate the heat map, the segmentation of which results in the hotspots. The method was trained on three and tested on nine whole slide images of neuroendocrine tumors. When evaluated by two expert pathologists, the method reached an accuracy of 92.6%. The current method does not discriminate between tumor, stromal and inflammatory nuclei. The results show that α-shape maps may represent how hotspots are perceived.

Detection of mitotic nuclei in breast histopathology images using localized ACM and Random Kitchen Sink based classifier.

PubMed

Beevi, K Sabeena; Nair, Madhu S; Bindu, G R

2016-08-01

The exact measure of mitotic nuclei is a crucial parameter in breast cancer grading and prognosis. This can be achieved by improving the mitotic detection accuracy by careful design of segmentation and classification techniques. In this paper, segmentation of nuclei from breast histopathology images are carried out by Localized Active Contour Model (LACM) utilizing bio-inspired optimization techniques in the detection stage, in order to handle diffused intensities present along object boundaries. Further, the application of a new optimal machine learning algorithm capable of classifying strong non-linear data such as Random Kitchen Sink (RKS), shows improved classification performance. The proposed method has been tested on Mitosis detection in breast cancer histological images (MITOS) dataset provided for MITOS-ATYPIA CONTEST 2014. The proposed framework achieved 95% recall, 98% precision and 96% F-score.

Fast Segmentation of Stained Nuclei in Terabyte-Scale, Time Resolved 3D Microscopy Image Stacks

PubMed Central

Stegmaier, Johannes; Otte, Jens C.; Kobitski, Andrei; Bartschat, Andreas; Garcia, Ariel; Nienhaus, G. Ulrich; Strähle, Uwe; Mikut, Ralf

2014-01-01

Automated analysis of multi-dimensional microscopy images has become an integral part of modern research in life science. Most available algorithms that provide sufficient segmentation quality, however, are infeasible for a large amount of data due to their high complexity. In this contribution we present a fast parallelized segmentation method that is especially suited for the extraction of stained nuclei from microscopy images, e.g., of developing zebrafish embryos. The idea is to transform the input image based on gradient and normal directions in the proximity of detected seed points such that it can be handled by straightforward global thresholding like Otsu’s method. We evaluate the quality of the obtained segmentation results on a set of real and simulated benchmark images in 2D and 3D and show the algorithm’s superior performance compared to other state-of-the-art algorithms. We achieve an up to ten-fold decrease in processing times, allowing us to process large data sets while still providing reasonable segmentation results. PMID:24587204

Multi-Atlas Based Segmentation of Brainstem Nuclei from MR Images by Deep Hyper-Graph Learning.

PubMed

Dong, Pei; Guo, Yangrong; Gao, Yue; Liang, Peipeng; Shi, Yonghong; Wang, Qian; Shen, Dinggang; Wu, Guorong

2016-10-01

Accurate segmentation of brainstem nuclei (red nucleus and substantia nigra) is very important in various neuroimaging applications such as deep brain stimulation and the investigation of imaging biomarkers for Parkinson's disease (PD). Due to iron deposition during aging, image contrast in the brainstem is very low in Magnetic Resonance (MR) images. Hence, the ambiguity of patch-wise similarity makes the recently successful multi-atlas patch-based label fusion methods have difficulty to perform as competitive as segmenting cortical and sub-cortical regions from MR images. To address this challenge, we propose a novel multi-atlas brainstem nuclei segmentation method using deep hyper-graph learning. Specifically, we achieve this goal in three-fold. First , we employ hyper-graph to combine the advantage of maintaining spatial coherence from graph-based segmentation approaches and the benefit of harnessing population priors from multi-atlas based framework. Second , besides using low-level image appearance, we also extract high-level context features to measure the complex patch-wise relationship. Since the context features are calculated on a tentatively estimated label probability map, we eventually turn our hyper-graph learning based label propagation into a deep and self-refining model. Third , since anatomical labels on some voxels (usually located in uniform regions) can be identified much more reliably than other voxels (usually located at the boundary between two regions), we allow these reliable voxels to propagate their labels to the nearby difficult-to-label voxels. Such hierarchical strategy makes our proposed label fusion method deep and dynamic. We evaluate our proposed label fusion method in segmenting substantia nigra (SN) and red nucleus (RN) from 3.0 T MR images, where our proposed method achieves significant improvement over the state-of-the-art label fusion methods.

Hierarchical nucleus segmentation in digital pathology images

NASA Astrophysics Data System (ADS)

Gao, Yi; Ratner, Vadim; Zhu, Liangjia; Diprima, Tammy; Kurc, Tahsin; Tannenbaum, Allen; Saltz, Joel

2016-03-01

Extracting nuclei is one of the most actively studied topic in the digital pathology researches. Most of the studies directly search the nuclei (or seeds for the nuclei) from the finest resolution available. While the richest information has been utilized by such approaches, it is sometimes difficult to address the heterogeneity of nuclei in different tissues. In this work, we propose a hierarchical approach which starts from the lower resolution level and adaptively adjusts the parameters while progressing into finer and finer resolution. The algorithm is tested on brain and lung cancers images from The Cancer Genome Atlas data set.

Associative image analysis: a method for automated quantification of 3D multi-parameter images of brain tissue

PubMed Central

Bjornsson, Christopher S; Lin, Gang; Al-Kofahi, Yousef; Narayanaswamy, Arunachalam; Smith, Karen L; Shain, William; Roysam, Badrinath

2009-01-01

Brain structural complexity has confounded prior efforts to extract quantitative image-based measurements. We present a systematic ‘divide and conquer’ methodology for analyzing three-dimensional (3D) multi-parameter images of brain tissue to delineate and classify key structures, and compute quantitative associations among them. To demonstrate the method, thick (~100 μm) slices of rat brain tissue were labeled using 3 – 5 fluorescent signals, and imaged using spectral confocal microscopy and unmixing algorithms. Automated 3D segmentation and tracing algorithms were used to delineate cell nuclei, vasculature, and cell processes. From these segmentations, a set of 23 intrinsic and 8 associative image-based measurements was computed for each cell. These features were used to classify astrocytes, microglia, neurons, and endothelial cells. Associations among cells and between cells and vasculature were computed and represented as graphical networks to enable further analysis. The automated results were validated using a graphical interface that permits investigator inspection and corrective editing of each cell in 3D. Nuclear counting accuracy was >89%, and cell classification accuracy ranged from 81–92% depending on cell type. We present a software system named FARSIGHT implementing our methodology. Its output is a detailed XML file containing measurements that may be used for diverse quantitative hypothesis-driven and exploratory studies of the central nervous system. PMID:18294697

Distinct structural and mechanical properties of the nuclear lamina in Hutchinson-Gilford progeria syndrome.

PubMed

Dahl, Kris Noel; Scaffidi, Paola; Islam, Mohammad F; Yodh, Arjun G; Wilson, Katherine L; Misteli, Tom

2006-07-05

The nuclear lamina is a network of structural filaments, the A and B type lamins, located at the nuclear envelope and throughout the nucleus. Lamin filaments provide the nucleus with mechanical stability and support many basic activities, including gene regulation. Mutations in LMNA, the gene encoding A type lamins, cause numerous human diseases, including the segmental premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here we show that structural and mechanical properties of the lamina are altered in HGPS cells. We demonstrate by live-cell imaging and biochemical analysis that lamins A and C become trapped at the nuclear periphery in HGPS patient cells. Using micropipette aspiration, we show that the lamina in HGPS cells has a significantly reduced ability to rearrange under mechanical stress. Based on polarization microscopy results, we suggest that the lamins are disordered in the healthy nuclei, whereas the lamins in HGPS nuclei form orientationally ordered microdomains. The reduced deformability of the HGPS nuclear lamina possibly could be due to the inability of these orientationally ordered microdomains to dissipate mechanical stress. Surprisingly, intact HGPS cells exhibited a degree of resistance to acute mechanical stress similar to that of cells from healthy individuals. Thus, in contrast to the nuclear fragility seen in lmna null cells, the lamina network in HGPS cells has unique mechanical properties that might contribute to disease phenotypes by affecting responses to mechanical force and misregulation of mechanosensitive gene expression.

Automatic extraction of nuclei centroids of mouse embryonic cells from fluorescence microscopy images.

PubMed

Bashar, Md Khayrul; Komatsu, Koji; Fujimori, Toshihiko; Kobayashi, Tetsuya J

2012-01-01

Accurate identification of cell nuclei and their tracking using three dimensional (3D) microscopic images is a demanding task in many biological studies. Manual identification of nuclei centroids from images is an error-prone task, sometimes impossible to accomplish due to low contrast and the presence of noise. Nonetheless, only a few methods are available for 3D bioimaging applications, which sharply contrast with 2D analysis, where many methods already exist. In addition, most methods essentially adopt segmentation for which a reliable solution is still unknown, especially for 3D bio-images having juxtaposed cells. In this work, we propose a new method that can directly extract nuclei centroids from fluorescence microscopy images. This method involves three steps: (i) Pre-processing, (ii) Local enhancement, and (iii) Centroid extraction. The first step includes two variations: first variation (Variant-1) uses the whole 3D pre-processed image, whereas the second one (Variant-2) modifies the preprocessed image to the candidate regions or the candidate hybrid image for further processing. At the second step, a multiscale cube filtering is employed in order to locally enhance the pre-processed image. Centroid extraction in the third step consists of three stages. In Stage-1, we compute a local characteristic ratio at every voxel and extract local maxima regions as candidate centroids using a ratio threshold. Stage-2 processing removes spurious centroids from Stage-1 results by analyzing shapes of intensity profiles from the enhanced image. An iterative procedure based on the nearest neighborhood principle is then proposed to combine if there are fragmented nuclei. Both qualitative and quantitative analyses on a set of 100 images of 3D mouse embryo are performed. Investigations reveal a promising achievement of the technique presented in terms of average sensitivity and precision (i.e., 88.04% and 91.30% for Variant-1; 86.19% and 95.00% for Variant-2), when compared with an existing method (86.06% and 90.11%), originally developed for analyzing C. elegans images.

Cytologic appearance of retinal cells included in a fine-needle aspirate of a meningioma around the optic nerve of a dog.

PubMed

Tvedten, Harold; Hillström, Anna

2013-06-01

A 6-year-old Wirehair Dachshund had a meningioma around the optic nerve that caused exophthalmos. A benign mesenchymal tumor was suspected based on the cytologic pattern of a fine-needle aspirate, and a meningioma was diagnosed by histopathologic examination. In addition to the meningioma cells, the cytologic smears included groups of cells from apparently 4 layers of normal retina. In particular, uniform rod-shaped structures in the cytologic sample could suggest rod-shaped bacteria, but these structures were identified as cylindrical outer segments of photoreceptor rod cells. Other retinal structures recognized included pigmented epithelial layer cells with their uniquely formed pigment granules, the characteristic bi-lobed, cleaved nuclei from the outer nuclear layer, and nerve tissue likely from the outer plexiform layer of the retina. © 2013 American Society for Veterinary Clinical Pathology.

Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images.

PubMed

Zhang, Zhiqing; Kuzmin, Nikolay V; Groot, Marie Louise; de Munck, Jan C

2018-01-01

Third harmonic generation (THG) microscopy is a label-free imaging technique that shows great potential for rapid pathology of brain tissue during brain tumor surgery. However, the interpretation of THG brain images should be quantitatively linked to images of more standard imaging techniques, which so far has been done qualitatively only. We establish here such a quantitative link between THG images of mouse brain tissue and all-nuclei-highlighted fluorescence images, acquired simultaneously from the same tissue area. For quantitative comparison of a substantial pair of images, we present here a segmentation workflow that is applicable for both THG and fluorescence images, with a precision of 91.3 % and 95.8 % achieved respectively. We find that the correspondence between the main features of the two imaging modalities amounts to 88.9 %, providing quantitative evidence of the interpretation of dark holes as brain cells. Moreover, 80 % bright objects in THG images overlap with nuclei highlighted in the fluorescence images, and they are 2 times smaller than the dark holes, showing that cells of different morphologies can be recognized in THG images. We expect that the described quantitative comparison is applicable to other types of brain tissue and with more specific staining experiments for cell type identification. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Morphological feature extraction for the classification of digital images of cancerous tissues.

PubMed

Thiran, J P; Macq, B

1996-10-01

This paper presents a new method for automatic recognition of cancerous tissues from an image of a microscopic section. Based on the shape and the size analysis of the observed cells, this method provides the physician with nonsubjective numerical values for four criteria of malignancy. This automatic approach is based on mathematical morphology, and more specifically on the use of Geodesy. This technique is used first to remove the background noise from the image and then to operate a segmentation of the nuclei of the cells and an analysis of their shape, their size, and their texture. From the values of the extracted criteria, an automatic classification of the image (cancerous or not) is finally operated.

Multicell migration tracking within angiogenic networks by deep learning-based segmentation and augmented Bayesian filtering.

PubMed

Wang, Mengmeng; Ong, Lee-Ling Sharon; Dauwels, Justin; Asada, H Harry

2018-04-01

Cell migration is a key feature for living organisms. Image analysis tools are useful in studying cell migration in three-dimensional (3-D) in vitro environments. We consider angiogenic vessels formed in 3-D microfluidic devices (MFDs) and develop an image analysis system to extract cell behaviors from experimental phase-contrast microscopy image sequences. The proposed system initializes tracks with the end-point confocal nuclei coordinates. We apply convolutional neural networks to detect cell candidates and combine backward Kalman filtering with multiple hypothesis tracking to link the cell candidates at each time step. These hypotheses incorporate prior knowledge on vessel formation and cell proliferation rates. The association accuracy reaches 86.4% for the proposed algorithm, indicating that the proposed system is able to associate cells more accurately than existing approaches. Cell culture experiments in 3-D MFDs have shown considerable promise for improving biology research. The proposed system is expected to be a useful quantitative tool for potential microscopy problems of MFDs.

Radiation-induced association of beta-glucuronidase with purified nuclei from irradiated MOLT-4 and HeLa cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

McClain, D.E.; Kalinich, J.F.; Poplack, J.K.

1989-02-01

Beta-glucuronidase, a lysosomal marker enzyme, associates with purified nuclei from HeLa and MOLT-4 cell lines in a radiation dose-dependent manner, up to 300 cGy in MOLT-4 cells, and 1000 cGy in HeLa cells. In MOLT-4 cells (200-cGy exposure), there is a significant increase in beta-glucuronidase activity detected in the nuclear fraction 24 h postirradiation with a maximum association occurring at 72 h. In HeLa cells (1000-cGy exposure), a significant association is first detected 24 h postirradiation with a maximum association at 48 h. The association is not the result of nonspecific contamination occurring during nuclei purification since nuclei from irradiatedmore » cells show no greater levels of plasma membrane marker and mitochondrial marker than controls. The nature of the association remains unclear, but activity is not removed by detergents used in the nuclei isolation procedure, and incubation of the nuclei with EDTA reverses the association only modestly. Exposure of nuclei from irradiated cells to anisotonic buffers also results in only a small decrease in beta-glucuronidase activity associated with the nuclei. These observations suggest that lysosomal hydrolases become intimately associated with the nuclei of irradiated cells.« less

Development of tectal connectivity across metamorphosis in the bullfrog (Rana catesbeiana).

PubMed

Horowitz, Seth S; Simmons, Andrea Megela

2010-01-01

In the bullfrog (Rana catesbeiana), the process of metamorphosis culminates in the appearance of new visual and visuomotor behaviors reflective of the emergence of binocular vision and visually-guided prey capture behaviors as the animal transitions to life on land. Using several different neuroanatomical tracers, we examined the substrates that may underlie these behavioral changes by tracing the afferent and efferent connectivity of the midbrain optic tectum across metamorphic development. Intratectal, tectotoral, tectotegmental, tectobulbar, and tecto-thalamic tracts exhibit similar trajectories of neurobiotin fiber label across the developmental span from early larval tadpoles to adults. Developmental variability was apparent primarily in intensity and distribution of cell and puncta label in target nuclei. Combined injections of cholera toxin subunit β and Phaseolus vulgaris leucoagglutinin consistently label cell bodies, puncta, or fiber segments bilaterally in midbrain targets including the pretectal gray, laminar nucleus of the torus semicircularis, and the nucleus of the medial longitudinal fasciculus. Developmentally stable label was observed bilaterally in medullary targets including the medial vestibular nucleus, lateral vestibular nucleus, and reticular gray, and in forebrain targets including the posterior and ventromedial nuclei of the thalamus. The nucleus isthmi, cerebellum, lateral line nuclei, medial septum, ventral striatum, and medial pallium show more developmentally variable patterns of connectivity. Our results suggest that even during larval development, the optic tectum contains substrates for integration of visual with auditory, vestibular, and somatosensory cues, as well as for guidance of motivated behaviors. Copyright © 2011 S. Karger AG, Basel.

The pelvic kidney of male Ambystoma maculatum (Amphibia, urodela, ambystomatidae) with special reference to the sexual collecting ducts.

PubMed

Siegel, Dustin S; Sever, David M; Aldridge, Robert D

2010-12-01

This study details the gross and microscopic anatomy of the pelvic kidney in male Ambystoma maculatum. The nephron of male Ambystoma maculatum is divided into six distinct regions leading sequentially away from a renal corpuscle: (1) neck segment, which communicates with the coelomic cavity via a ventrally positioned pleuroperitoneal funnel, (2) proximal tubule, (3) intermediate segment, (4) distal tubule, (5) collecting tubule, and (6) collecting duct. The proximal tubule is divided into a vacuolated proximal region and a distal lysosomic region. The basal plasma membrane is modified into intertwining microvillus lamellae. The epithelium of the distal tubule varies little along its length and is demarcated by columns of mitochondria with their long axes oriented perpendicular to the basal lamina. The distal tubule possesses highly interdigitating microvillus lamellae from the lateral membranes and pronounced foot processes of the basal membrane that are not intertwined, but perpendicular to the basal lamina. The collecting tubule is lined by an epithelium with dark and light cells. Light cells are similar to those observed in the distal tuble except with less mitochondria and microvillus lamellae of the lateral and basal plasma membrane. Dark cells possess dark euchromatic nuclei and are filled with numerous small mitochondria. The epithelium of the neck segment, pleuroperitoneal funnel, and intermediate segment is composed entirely of ciliated cells with cilia protruding from only the central portion of the apical plasma membrane. The collecting duct is lined by a highly secretory epithelium that produces numerous membrane bound granules that stain positively for neutral carbohydrates and proteins. Apically positioned ciliated cells are intercalated between secretory cells. The collecting ducts anastomose caudally and unite with the Wolffian duct via a common collecting duct. The Wolffian duct is secretory, but not to the extent of the collecting duct, synthesizes neutral carbohydrates and proteins, and is also lined by apical ciliated cells intercalated between secretory cells. Although functional aspects associated with the morphological variation along the length of the proximal portions of the nephron have been investigated, the role of a highly secretory collecting duct has not. Historical data that implicated secretory activity concordant with mating activity, and similarity of structure and chemistry to sexual segments of the kidneys in other vertebrates, lead us to believe that the collecting duct functions as a secondary sexual organ in Ambystoma maculatum. © 2010 Wiley-Liss, Inc.

Differentiating invasive and pre-invasive lung cancer by quantitative analysis of histopathologic images

NASA Astrophysics Data System (ADS)

Zhou, Chuan; Sun, Hongliu; Chan, Heang-Ping; Chughtai, Aamer; Wei, Jun; Hadjiiski, Lubomir; Kazerooni, Ella

2018-02-01

We are developing automated radiopathomics method for diagnosis of lung nodule subtypes. In this study, we investigated the feasibility of using quantitative methods to analyze the tumor nuclei and cytoplasm in pathologic wholeslide images for the classification of pathologic subtypes of invasive nodules and pre-invasive nodules. We developed a multiscale blob detection method with watershed transform (MBD-WT) to segment the tumor cells. Pathomic features were extracted to characterize the size, morphology, sharpness, and gray level variation in each segmented nucleus and the heterogeneity patterns of tumor nuclei and cytoplasm. With permission of the National Lung Screening Trial (NLST) project, a data set containing 90 digital haematoxylin and eosin (HE) whole-slide images from 48 cases was used in this study. The 48 cases contain 77 regions of invasive subtypes and 43 regions of pre-invasive subtypes outlined by a pathologist on the HE images using the pathological tumor region description provided by NLST as reference. A logistic regression model (LRM) was built using leave-one-case-out resampling and receiver operating characteristic (ROC) analysis for classification of invasive and pre-invasive subtypes. With 11 selected features, the LRM achieved a test area under the ROC curve (AUC) value of 0.91+/-0.03. The results demonstrated that the pathologic invasiveness of lung adenocarcinomas could be categorized with high accuracy using pathomics analysis.

Release of cell-free ice nuclei from Halomonas elongata expressing the ice nucleation gene inaZ of Pseudomonas syringae.

PubMed

Tegos, G; Vargas, C; Perysinakis, A; Koukkou, A I; Christogianni, A; Nieto, J J; Ventosa, A; Drainas, C

2000-11-01

Release of ice nuclei in the growth medium of recombinant Halomonas elongata cells expressing the inaZ gene of Pseudomonas syringae was studied in an attempt to produce cell-free active ice nuclei for biotechnological applications. Cell-free ice nuclei were not retained by cellulose acetate filters of 0.2 microm pore size. Highest activity of cell-free ice nuclei was obtained when cells were grown in low salinity (0.5-5% NaCl, w/v). Freezing temperature threshold, estimated to be below -7 degrees C indicating class C nuclei, was not affected by medium salinity. Their density, as estimated by Percoll density centrifugation, was 1.018 +/- 0.002 gml(-1) and they were found to be free of lipids. Ice nuclei are released in the growth medium of recombinant H. elongata cells probably because of inefficient anchoring of the ice-nucleation protein aggregates in the outer membrane. The ice+ recombinant H. elongata cells could be useful for future use as a source of active cell-free ice nucleation protein.

Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori

PubMed Central

Sakai, Hiroki; Yokoyama, Takeshi; Abe, Hiroaki; Fujii, Tsuguru; Suzuki, Masataka G.

2013-01-01

In Bombyx mori, polar body nuclei are observed until 9 h after egg lying, however, the fate of polar body nuclei remains unclear. To examine the fate of polar body nuclei, we employed a mutation of serosal cell pigmentation, pink-eyed white egg (pe). The heterozygous pe/+pe females produced black serosal cells in white eggs, while pe/pe females did not produce black serosal cells in white eggs. These results suggest that the appearance of black serosal cells in white eggs depends on the genotype (pe/+pe) of the mother. Because the polar body nuclei had +pe genes in the white eggs laid by a pe/+pe female, polar body nuclei participate in development and differentiate into functional cell (serosal cells). Analyses of serosal cells pigmentation indicated that ~30% of the eggs contained polar-body-nucleus-derived cells. These results demonstrate that polar-body-nucleus-derived cells appeared at a high frequency under natural conditions. Approximately 80% of polar-body-nucleus-derived cells appeared near the anterior pole and the dorsal side, which is opposite to where embryogenesis occurs. The number of cells derived from the polar body nuclei was very low. Approximately 26% of these eggs contained only one black serosal cell. PCR-based analysis revealed that the polar-body-nucleus-derived cells disappeared in late embryonic stages (stage 25). Overall, polar-body-nuclei-derived cells were unlikely to contribute to embryos. PMID:24027530

Longitudinal atlas for normative human brain development and aging over the lifespan using quantitative susceptibility mapping.

PubMed

Zhang, Yuyao; Wei, Hongjiang; Cronin, Matthew J; He, Naying; Yan, Fuhua; Liu, Chunlei

2018-05-01

Longitudinal brain atlases play an important role in the study of human brain development and cognition. Existing atlases are mainly based on anatomical features derived from T1-and T2-weighted MRI. A 4D developmental quantitative susceptibility mapping (QSM) atlas may facilitate the estimation of age-related iron changes in deep gray matter nuclei and myelin changes in white matter. To this end, group-wise co-registered QSM templates were generated over various age intervals from age 1-83 years old. Registration was achieved by combining both T1-weighted and QSM images. Based on the proposed template, we created an accurate deep gray matter nuclei parcellation map (DGM map). Notably, we segmented thalamus into 5 sub-regions, i.e. the anterior nuclei, the median nuclei, the lateral nuclei, the pulvinar and the internal medullary lamina. Furthermore, we built a "whole brain QSM parcellation map" by combining existing cortical parcellation and white-matter atlases with the proposed DGM map. Based on the proposed QSM atlas, the segmentation accuracy of iron-rich nuclei using QSM is significantly improved, especially for children and adolescent subjects. The age-related progression of magnetic susceptibility in each of the deep gray matter nuclei, the hippocampus, and the amygdala was estimated. Our automated atlas-based analysis provided a systematic confirmation of previous findings on susceptibility progression with age resulting from manual ROI drawings in deep gray matter nuclei. The susceptibility development in the hippocampus and the amygdala follow an iron accumulation model; while in the thalamus sub-regions, the susceptibility development exhibits a variety of trends. It is envisioned that the newly developed 4D QSM atlas will serve as a template for studying brain iron deposition and myelination/demyelination in both normal aging and various brain diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

In vivo imaging of cell nuclei by photoacoustic microscopy without staining

NASA Astrophysics Data System (ADS)

Yao, Da-Kang; Chen, Ruimin; Maslov, Konstantin; Zhou, Qifa; Wang, Lihong V.

2012-02-01

Ultraviolet photoacoustic microscopy (UVPAM) can image cell nuclei in vivo with high contrast and resolution noninvasively without staining. Here, we used UV light at wavelengths of 210-310 nm for excitation of DNA and RNA to produce photoacoustic waves. We applied the UVPAM to in vivo imaging of cell nuclei in mouse skin, and obtained UVPAM images of the unstained cell nuclei at wavelengths of 245-282 nm as ultrasound gel was used for acoustic coupling. The largest ratio of contrast to noise was found for the images of cell nuclei at a 250 nm wavelength.

A High-Content Live-Cell Viability Assay and Its Validation on a Diverse 12K Compound Screen.

PubMed

Chiaravalli, Jeanne; Glickman, J Fraser

2017-08-01

We have developed a new high-content cytotoxicity assay using live cells, called "ImageTOX." We used a high-throughput fluorescence microscope system, image segmentation software, and the combination of Hoechst 33342 and SYTO 17 to simultaneously score the relative size and the intensity of the nuclei, the nuclear membrane permeability, and the cell number in a 384-well microplate format. We then performed a screen of 12,668 diverse compounds and compared the results to a standard cytotoxicity assay. The ImageTOX assay identified similar sets of compounds to the standard cytotoxicity assay, while identifying more compounds having adverse effects on cell structure, earlier in treatment time. The ImageTOX assay uses inexpensive commercially available reagents and facilitates the use of live cells in toxicity screens. Furthermore, we show that we can measure the kinetic profile of compound toxicity in a high-content, high-throughput format, following the same set of cells over an extended period of time.

An image processing approach to analyze morphological features of microscopic images of muscle fibers.

PubMed

Comin, Cesar Henrique; Xu, Xiaoyin; Wang, Yaming; Costa, Luciano da Fontoura; Yang, Zhong

2014-12-01

We present an image processing approach to automatically analyze duo-channel microscopic images of muscular fiber nuclei and cytoplasm. Nuclei and cytoplasm play a critical role in determining the health and functioning of muscular fibers as changes of nuclei and cytoplasm manifest in many diseases such as muscular dystrophy and hypertrophy. Quantitative evaluation of muscle fiber nuclei and cytoplasm thus is of great importance to researchers in musculoskeletal studies. The proposed computational approach consists of steps of image processing to segment and delineate cytoplasm and identify nuclei in two-channel images. Morphological operations like skeletonization is applied to extract the length of cytoplasm for quantification. We tested the approach on real images and found that it can achieve high accuracy, objectivity, and robustness. Copyright © 2014 Elsevier Ltd. All rights reserved.

The developmental basis for germline mosaicism in mouse and Drosophila melanogaster.

PubMed

Drost, J B; Lee, W R

1998-01-01

Data involving germline mosaics in Drosophila melanogaster and mouse are reconciled with developmental observations. Mutations that become fixed in the early embryo before separation of soma from the germline may, by the sampling process of development, continue as part of germline and/or differentiate into any somatic tissue. The cuticle of adult D. melanogaster, because of segmental development, can be used to estimate the proportion of mutant nuclei in the early embryo, but most somatic tissues and the germlines of both species continue from samples too small to be representative of the early embryo. Because of the small sample of cells/nuclei that remain in the germline after separation of soma in both species, mosaic germlines have percentages of mutant cells that vary widely, with a mean of 50% and an unusual platykurtic, flat-topped distribution. While the sampling process leads to similar statistical results for both species, their patterns of development are very different. In D. melanogaster the first differentiation is the separation of soma from germline with the germline continuing from a sample of only two to four nuclei, whereas the adult cuticle is a representative sample of cleavage nuclei. The presence of mosaicism in D. melanogaster germline is independent of mosaicism in the eye, head, and thorax. This independence was used to determine that mutations can occur at any of the early embryonic cell divisions and still average 50% mutant germ cells when the germline is mosaic; however, the later the mutation occurs, the higher the proportion of completely nonmutant germlines. In contrast to D. melanogaster, the first differentiation in the mouse does not separate soma from germline but produces the inner cell mass that is representative of the cleavage nuclei. Following formation of the primitive streak, the primordial germ cells develop at the base of the allantois and among a clonally related sample of cells, providing the same statistical distribution in the mouse germlines as in D. melanogaster. The proportion of mutations that are fixed during early embryonic development is greatly underestimated. For example, a DNA lesion in a postmeiotic gamete that becomes fixed as a dominant mutation during early embryonic development of the F1 may produce an individual completely mutant in the germ line and relevant somatic tissue or, alternatively, the F1 germline may be completely mutant but with no relevant somatic tissues for detecting the mutation until the F2. In both cases the mutation would be classified as complete in the F1 and F2, respectively, and not recognized as embryonic in origin. Because germ cells differentiate later in mammalian development, there are more opportunities for correlation between germline and soma in the mammal than Drosophila. However, because the germ cells and any somatic tissue, like blood, are derived from small samples, there may be many individuals that test negative in blood but have germlines that are either mosaic or entirely mutant.

Roles of endothelial A-type lamins in migration of T cells on and under endothelial layers

NASA Astrophysics Data System (ADS)

Song, Kwang Hoon; Lee, Jaehyun; Park, Hyoungjun; Kim, Hye Mi; Park, Jeehun; Kwon, Keon Woo; Doh, Junsang

2016-03-01

Stiff nuclei in cell-dense microenvironments may serve as distinct biomechanical cues for cell migration, but such a possibility has not been tested experimentally. As a first step addressing this question, we altered nuclear stiffness of endothelial cells (ECs) by reducing the expression of A-type lamins using siRNA, and investigated the migration of T cells on and under EC layers. While most T cells crawling on control EC layers avoided crossing over EC nuclei, a significantly higher fraction of T cells on EC layers with reduced expression of A-type lamins crossed over EC nuclei. This result suggests that stiff EC nuclei underlying T cells may serve as “duro-repulsive” cues to direct T cell migration toward less stiff EC cytoplasm. During subendothelial migration under EC layers with reduced expression of A-type lamins, T cells made prolonged contact and substantially deformed EC nuclei, resulting in reduced speed and directional persistence. This result suggests that EC nuclear stiffness promotes fast and directionally persistent subendothelial migration of T cells by allowing minimum interaction between T cells and EC nuclei.

Early development and segment formation in the centipede, Strigamia maritima (Geophilomorpha).

PubMed

Chipman, Ariel D; Arthur, Wallace; Akam, Michael

2004-01-01

Geophilomorph centipedes exhibit a number of unique characteristics that make them of particular developmental and evolutionary interest. Segment numbers in geophilomorphs are higher than in any other centipedes, ranging from 27 to 191. They may be constant within a species, presenting in extreme form the "counting" problem in development, or they may vary--a situation that provides us with the opportunity to study naturally occurring variation in segment numbers. All their segments are generated during embryogenesis, a situation unlike that in the more basal centipede orders, which generate only a fraction of their 15 trunk segments in the embryo and develop the rest postembryonically. Here we provide a foundation for further developmental studies of the Geophilomorpha, building on the one study that has been conducted to date, on the coastal species Strigamia maritima. Development begins with the migration of nuclei to the surface of the egg, which then condense to form an embryonic rudiment of more than 20,000 cells, covering an entire hemisphere. During early development, the embryo can be divided into two distinct areas: a large terminal disc of apparently undifferentiated tissue and the germ-band, which has a clear anteroposterior axis and differentiated segments. The germ-band forms from the anterior of the terminal disc and extends anteriorly as the disc contracts. New segments are formed at the posterior margin of the germ-band. Once the process of segmentation ends, the germ-band folds and sinks into the yolk. We note that the classic description of centipede development, by Heymons more than a century ago, contains a fundamental error in the identification of the axes and hence in the interpretation of early segmentation.

Transition from one revolving cluster to two revolving clusters in the ground-state rotational bands of nuclei in the lanthanon region.

PubMed

Pauling, L

1991-02-01

Whereas 234(92)U142 and other actinon nuclei have ground-state bands that indicate that each nucleus consists of a sphere and a single revolving cluster with constant composition and with only a steady increase in the moment of inertia with increase in J, the angular-momentum quantum number, many of the lanthanon ground-state bands show discontinuities, usually with an initial slightly or strongly curved segment followed by one or two nearly straight segments. The transition to nearly straight segments is interpreted as a change in structure from one revolving cluster to two revolving clusters. The proton-neutron compositions of the clusters and the central sphere are assigned, leading to values of the radius of revolution. The approximation of the two-cluster sequences to linearity is attributed to the very small values of the quadrupole polarizability of the central sphere. Values of the nucleon numbers of clusters and spheres, of the radius of revolution, and of promotion energy are discussed.

Objective automated quantification of fluorescence signal in histological sections of rat lens.

PubMed

Talebizadeh, Nooshin; Hagström, Nanna Zhou; Yu, Zhaohua; Kronschläger, Martin; Söderberg, Per; Wählby, Carolina

2017-08-01

Visual quantification and classification of fluorescent signals is the gold standard in microscopy. The purpose of this study was to develop an automated method to delineate cells and to quantify expression of fluorescent signal of biomarkers in each nucleus and cytoplasm of lens epithelial cells in a histological section. A region of interest representing the lens epithelium was manually demarcated in each input image. Thereafter, individual cell nuclei within the region of interest were automatically delineated based on watershed segmentation and thresholding with an algorithm developed in Matlab™. Fluorescence signal was quantified within nuclei, cytoplasms and juxtaposed backgrounds. The classification of cells as labelled or not labelled was based on comparison of the fluorescence signal within cells with local background. The classification rule was thereafter optimized as compared with visual classification of a limited dataset. The performance of the automated classification was evaluated by asking 11 independent blinded observers to classify all cells (n = 395) in one lens image. Time consumed by the automatic algorithm and visual classification of cells was recorded. On an average, 77% of the cells were correctly classified as compared with the majority vote of the visual observers. The average agreement among visual observers was 83%. However, variation among visual observers was high, and agreement between two visual observers was as low as 71% in the worst case. Automated classification was on average 10 times faster than visual scoring. The presented method enables objective and fast detection of lens epithelial cells and quantification of expression of fluorescent signal with an accuracy comparable with the variability among visual observers. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Induction of micronuclei and nuclear abnormalities in tadpoles of the common toad (Rhinella arenarum) treated with the herbicides Liberty® and glufosinate-ammonium.

PubMed

Lajmanovich, Rafael C; Cabagna-Zenklusen, Mariana C; Attademo, Andrés M; Junges, Celina M; Peltzer, Paola M; Bassó, Agustín; Lorenzatti, Eduardo

2014-07-15

The assessment of micronucleated erythrocytes (ME) in blood represents a widely used method for the detection of chromosomal damage by chemical agents, such as herbicides that may occur as water contaminants. We investigated the changes in some circulating blood-cell parameters of tadpoles of the common toad (Rhinella arenarum) that were exposed during 48 or 96 h to three sub-lethal concentrations (3.75, 7.5, and 15 mg/L) of a commercial formulation of a glufosinate-ammonium (GLA)-based herbicide (Liberty(®), LY(®)) as well as to the corresponding active ingredient GLA. The frequency of ME and other erythrocyte nuclear abnormalities (ENA, i.e., lobed nuclei, binucleates or segmented nuclei, kidney-shaped nuclei, notched nuclei, and picnotic nuclei) were evaluated and compared with positive (cyclophosphamide, CP, 40 mg/L) and negative (de-chlorinated tap water) controls. The results indicate that the exposure of R. arenarum tadpoles to LY(®) induces a concentration-dependent increase in ME frequency. The ENA frequency at 48 h was also significantly higher than that in the negative control group for all the chemicals assayed (CP, LY(®) and GLA) whereas at 96 h, increases in ENA over the negative control group were found only for CP and GLA (7.5 mg/L). Our study demonstrates that the commercial formulation of a GLA-based herbicide induces micronucleus formation in R. arenarum tadpoles, in contrast to the active ingredient. According to these results, the inert ingredients of the commercial formulation played an important role in the production of genotoxic damage in erythrocytes of amphibian tadpoles. Copyright © 2014 Elsevier B.V. All rights reserved.

Filopodia-like Actin Cables Position Nuclei in Association with Perinuclear Actin in Drosophila Nurse Cells

PubMed Central

Huelsmann, Sven; Ylänne, Jari; Brown, Nicholas H.

2013-01-01

Summary Controlling the position of the nucleus is vital for a number of cellular processes from yeast to humans. In Drosophila nurse cells, nuclear positioning is crucial during dumping, when nurse cells contract and expel their contents into the oocyte. We provide evidence that in nurse cells, continuous filopodia-like actin cables, growing from the plasma membrane and extending to the nucleus, achieve nuclear positioning. These actin cables move nuclei away from ring canals. When nurse cells contract, actin cables associate laterally with the nuclei, in some cases inducing nuclear turning so that actin cables become partially wound around the nuclei. Our data suggest that a perinuclear actin meshwork connects actin cables to nuclei via actin-crosslinking proteins such as the filamin Cheerio. We provide a revised model for how actin structures position nuclei in nurse cells, employing evolutionary conserved machinery. PMID:24091012

Analysis of growth of tetraploid nuclei in roots of Vicia faba.

PubMed

Bansal, J; Davidson, D

1978-03-01

Growth of nuclei of a marked population of cells was determined from G1 to prophase in roots of Vicia faba. The cells were marked by inducing them to become tetraploid by treatment with 0.002% colchicine for 1 hr. Variation in nuclear volume is large; it is established in early G1 and maintained through interphase and into prophase. One consequence of this variation is that there is considerable overlap between volumes of nuclei of different ages in the cell cycle; nuclear volume, we suggest, cannot be used as an accurate indicator of the age of the cell in its growth cycle. Nuclei exhibit considerable variation in their growth rate through the cell cycle. Of the marked population of cells, about 65% had completed a cell cycle 14--15 hr after they were formed. These tetraploid nuclei have a cell cycle duration similar to that of fast cycling diploid cells of the same roots. Since they do complete a cell cycle, at least 65% of the nuclei studied must come from rapidly proliferating cells, showing that variability in nuclear volumes must be present in growing cells and cannot be attributed solely to the presence, in our samples, of non-cycling cells.

Novel computer-aided diagnosis of mesothelioma using nuclear structure of mesothelial cells in effusion cytology specimens

NASA Astrophysics Data System (ADS)

Tosun, Akif Burak; Yergiyev, Oleksandr; Kolouri, Soheil; Silverman, Jan F.; Rohde, Gustavo K.

2014-03-01

diagnostic standard is a pleural biopsy with subsequent histologic examination of the tissue demonstrating invasion by the tumor. The diagnostic tissue is obtained through thoracoscopy or open thoracotomy, both being highly invasive procedures. Thoracocenthesis, or removal of effusion fluid from the pleural space, is a far less invasive procedure that can provide material for cytological examination. However, it is insufficient to definitively confirm or exclude the diagnosis of malignant mesothelioma, since tissue invasion cannot be determined. In this study, we present a computerized method to detect and classify malignant mesothelioma based on the nuclear chromatin distribution from digital images of mesothelial cells in effusion cytology specimens. Our method aims at determining whether a set of nuclei belonging to a patient, obtained from effusion fluid images using image segmentation, is benign or malignant, and has a potential to eliminate the need for tissue biopsy. This method is performed by quantifying chromatin morphology of cells using the optimal transportation (Kantorovich-Wasserstein) metric in combination with the modified Fisher discriminant analysis, a k-nearest neighborhood classification, and a simple voting strategy. Our results show that we can classify the data of 10 different human cases with 100% accuracy after blind cross validation. We conclude that nuclear structure alone contains enough information to classify the malignant mesothelioma. We also conclude that the distribution of chromatin seems to be a discriminating feature between nuclei of benign and malignant mesothelioma cells.

Evaluation of nucleus segmentation in digital pathology images through large scale image synthesis

NASA Astrophysics Data System (ADS)

Zhou, Naiyun; Yu, Xiaxia; Zhao, Tianhao; Wen, Si; Wang, Fusheng; Zhu, Wei; Kurc, Tahsin; Tannenbaum, Allen; Saltz, Joel; Gao, Yi

2017-03-01

Digital histopathology images with more than 1 Gigapixel are drawing more and more attention in clinical, biomedical research, and computer vision fields. Among the multiple observable features spanning multiple scales in the pathology images, the nuclear morphology is one of the central criteria for diagnosis and grading. As a result it is also the mostly studied target in image computing. Large amount of research papers have devoted to the problem of extracting nuclei from digital pathology images, which is the foundation of any further correlation study. However, the validation and evaluation of nucleus extraction have yet been formulated rigorously and systematically. Some researches report a human verified segmentation with thousands of nuclei, whereas a single whole slide image may contain up to million. The main obstacle lies in the difficulty of obtaining such a large number of validated nuclei, which is essentially an impossible task for pathologist. We propose a systematic validation and evaluation approach based on large scale image synthesis. This could facilitate a more quantitatively validated study for current and future histopathology image analysis field.

On the occurrence of nuclei in mature sieve elements.

PubMed

Event, R F; Davis, J D; Tucker, C M; Alfieri, F J

1970-12-01

The secondary phloem of 3 species of the Taxodiaceae and 13 species of woody dicotyledons was examined for the occurrence of nuclei in mature sieve elements. Nuclei were found in all mature sieve cells of Metasequoia glyptostroboides, Sequoia sempervirens and Taxodium distichum, and in some mature sieve-tube members in 12 of the 13 species of woody dicotyledons. Except for nuclei of sieve cells undergoing cessation of function, the nuclei in mature sieve cells of M. glyptostroboides, S. sempervirens and T. distichum were normal in appearance. The occurrence and morphology of nuclei in mature sieve-tube members of the woody dicotyledons were quite variable. Only 3 species, Robinia pseudoacacia, Ulmus americana and Vitis riparia, contained some mature sieve elements with apparently normal nuclei.

Hepatocyte Ploidy Is a Diversity Factor for Liver Homeostasis

PubMed Central

Kreutz, Clemens; MacNelly, Sabine; Follo, Marie; Wäldin, Astrid; Binninger-Lacour, Petra; Timmer, Jens; Bartolomé-Rodríguez, María M.

2017-01-01

Polyploidy, the existence of cells containing more than one pair of chromosomes, is a well-known feature of mammalian hepatocytes. Polyploid hepatocytes are found either as cells with a single polyploid nucleus or as multinucleated cells with diploid or even polyploid nuclei. In this study, we evaluate the degree of polyploidy in the murine liver by accounting both DNA content and number of nuclei per cell. We demonstrate that mouse hepatocytes with diploid nuclei have distinct metabolic characteristics compared to cells with polyploid nuclei. In addition to strong differential gene expression, comprising metabolic as well as signaling compounds, we found a strongly decreased insulin binding of nuclear polyploid cells. Our observations were associated with nuclear ploidy but not with total ploidy within a cell. We therefore suggest ploidy of the nuclei as an new diversity factor of hepatocytes and hypothesize that hepatocytes with polyploid nuclei may have distinct biological functions than mono-nuclear ones. This diversity is independent from the well-known heterogeneity related to the cells' position along the porto-central liver-axis. PMID:29163206

A new model of sperm nuclear architecture following assessment of the organization of centromeres and telomeres in three-dimensions.

PubMed

Ioannou, Dimitrios; Millan, Nicole M; Jordan, Elizabeth; Tempest, Helen G

2017-01-31

The organization of chromosomes in sperm nuclei has been proposed to possess a unique "hairpin-loop" arrangement, which is hypothesized to aid in the ordered exodus of the paternal genome following fertilization. This study simultaneously assessed the 3D and 2D radial and longitudinal organization of telomeres, centromeres, and investigated whether chromosomes formed the same centromere clusters in sperm cells. Reproducible radial and longitudinal non-random organization was observed for all investigated loci using both 3D and 2D approaches in multiple subjects. We report novel findings, with telomeres and centromeres being localized throughout the nucleus but demonstrating roughly a 1:1 distribution in the nuclear periphery and the intermediate regions with <15% occupying the nuclear interior. Telomeres and centromeres were observed to aggregate in sperm nuclei, forming an average of 20 and 7 clusters, respectively. Reproducible longitudinal organization demonstrated preferential localization of telomeres and centromeres in the mid region of the sperm cell. Preliminary evidence is also provided to support the hypothesis that specific chromosomes preferentially form the same centromere clusters. The more segmental distribution of telomeres and centromeres as described in this study could more readily accommodate and facilitate the sequential exodus of paternal chromosomes following fertilization.

A new model of sperm nuclear architecture following assessment of the organization of centromeres and telomeres in three-dimensions

PubMed Central

Ioannou, Dimitrios; Millan, Nicole M.; Jordan, Elizabeth; Tempest, Helen G.

2017-01-01

The organization of chromosomes in sperm nuclei has been proposed to possess a unique “hairpin-loop” arrangement, which is hypothesized to aid in the ordered exodus of the paternal genome following fertilization. This study simultaneously assessed the 3D and 2D radial and longitudinal organization of telomeres, centromeres, and investigated whether chromosomes formed the same centromere clusters in sperm cells. Reproducible radial and longitudinal non-random organization was observed for all investigated loci using both 3D and 2D approaches in multiple subjects. We report novel findings, with telomeres and centromeres being localized throughout the nucleus but demonstrating roughly a 1:1 distribution in the nuclear periphery and the intermediate regions with <15% occupying the nuclear interior. Telomeres and centromeres were observed to aggregate in sperm nuclei, forming an average of 20 and 7 clusters, respectively. Reproducible longitudinal organization demonstrated preferential localization of telomeres and centromeres in the mid region of the sperm cell. Preliminary evidence is also provided to support the hypothesis that specific chromosomes preferentially form the same centromere clusters. The more segmental distribution of telomeres and centromeres as described in this study could more readily accommodate and facilitate the sequential exodus of paternal chromosomes following fertilization. PMID:28139771

Classification of glioblastoma and metastasis for neuropathology intraoperative diagnosis: a multi-resolution textural approach to model the background

NASA Astrophysics Data System (ADS)

Ahmad Fauzi, Mohammad Faizal; Gokozan, Hamza Numan; Elder, Brad; Puduvalli, Vinay K.; Otero, Jose J.; Gurcan, Metin N.

2014-03-01

Brain cancer surgery requires intraoperative consultation by neuropathology to guide surgical decisions regarding the extent to which the tumor undergoes gross total resection. In this context, the differential diagnosis between glioblastoma and metastatic cancer is challenging as the decision must be made during surgery in a short time-frame (typically 30 minutes). We propose a method to classify glioblastoma versus metastatic cancer based on extracting textural features from the non-nuclei region of cytologic preparations. For glioblastoma, these regions of interest are filled with glial processes between the nuclei, which appear as anisotropic thin linear structures. For metastasis, these regions correspond to a more homogeneous appearance, thus suitable texture features can be extracted from these regions to distinguish between the two tissue types. In our work, we use the Discrete Wavelet Frames to characterize the underlying texture due to its multi-resolution capability in modeling underlying texture. The textural characterization is carried out in primarily the non-nuclei regions after nuclei regions are segmented by adapting our visually meaningful decomposition segmentation algorithm to this problem. k-nearest neighbor method was then used to classify the features into glioblastoma or metastasis cancer class. Experiment on 53 images (29 glioblastomas and 24 metastases) resulted in average accuracy as high as 89.7% for glioblastoma, 87.5% for metastasis and 88.7% overall. Further studies are underway to incorporate nuclei region features into classification on an expanded dataset, as well as expanding the classification to more types of cancers.

Mechanical positioning of multiple nuclei in muscle cells.

PubMed

Manhart, Angelika; Windner, Stefanie; Baylies, Mary; Mogilner, Alex

2018-06-01

Many types of large cells have multiple nuclei. In skeletal muscle fibers, the nuclei are distributed along the cell to maximize their internuclear distances. This myonuclear positioning is crucial for cell function. Although microtubules, microtubule associated proteins, and motors have been implicated, mechanisms responsible for myonuclear positioning remain unclear. We used a combination of rough interacting particle and detailed agent-based modeling to examine computationally the hypothesis that a force balance generated by microtubules positions the muscle nuclei. Rather than assuming the nature and identity of the forces, we simulated various types of forces between the pairs of nuclei and between the nuclei and cell boundary to position the myonuclei according to the laws of mechanics. We started with a large number of potential interacting particle models and computationally screened these models for their ability to fit biological data on nuclear positions in hundreds of Drosophila larval muscle cells. This reverse engineering approach resulted in a small number of feasible models, the one with the best fit suggests that the nuclei repel each other and the cell boundary with forces that decrease with distance. The model makes nontrivial predictions about the increased nuclear density near the cell poles, the zigzag patterns of the nuclear positions in wider cells, and about correlations between the cell width and elongated nuclear shapes, all of which we confirm by image analysis of the biological data. We support the predictions of the interacting particle model with simulations of an agent-based mechanical model. Taken together, our data suggest that microtubules growing from nuclear envelopes push on the neighboring nuclei and the cell boundaries, which is sufficient to establish the nearly-uniform nuclear spreading observed in muscle fibers.

Laser microbeam irradiation and renucleation of mouse eggs. Final progress report, July 1, 1979-December 31, 1983

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, T.P.

1983-01-01

We have combined laser microbeam irradiation of mouse egg nuclei with fusion to donor cell nuclei in order to develop a new procedure for transferring nuclei into mammalian eggs. We have been using virus-treated cells injected into the perivitelline for fusion with egg cells. Binucleate cells inside the zona pellucida were often produced indicating nuclear transfer between cells had occurred. To prevent the formation of such abortive polyploidy, host nuclei were inactivated with a laser microbeam. The subsequent cleavage of the microirradiated eggs has been studied.

A workflow to process 3D+time microscopy images of developing organisms and reconstruct their cell lineage

PubMed Central

Faure, Emmanuel; Savy, Thierry; Rizzi, Barbara; Melani, Camilo; Stašová, Olga; Fabrèges, Dimitri; Špir, Róbert; Hammons, Mark; Čúnderlík, Róbert; Recher, Gaëlle; Lombardot, Benoît; Duloquin, Louise; Colin, Ingrid; Kollár, Jozef; Desnoulez, Sophie; Affaticati, Pierre; Maury, Benoît; Boyreau, Adeline; Nief, Jean-Yves; Calvat, Pascal; Vernier, Philippe; Frain, Monique; Lutfalla, Georges; Kergosien, Yannick; Suret, Pierre; Remešíková, Mariana; Doursat, René; Sarti, Alessandro; Mikula, Karol; Peyriéras, Nadine; Bourgine, Paul

2016-01-01

The quantitative and systematic analysis of embryonic cell dynamics from in vivo 3D+time image data sets is a major challenge at the forefront of developmental biology. Despite recent breakthroughs in the microscopy imaging of living systems, producing an accurate cell lineage tree for any developing organism remains a difficult task. We present here the BioEmergences workflow integrating all reconstruction steps from image acquisition and processing to the interactive visualization of reconstructed data. Original mathematical methods and algorithms underlie image filtering, nucleus centre detection, nucleus and membrane segmentation, and cell tracking. They are demonstrated on zebrafish, ascidian and sea urchin embryos with stained nuclei and membranes. Subsequent validation and annotations are carried out using Mov-IT, a custom-made graphical interface. Compared with eight other software tools, our workflow achieved the best lineage score. Delivered in standalone or web service mode, BioEmergences and Mov-IT offer a unique set of tools for in silico experimental embryology. PMID:26912388

Automated analysis and classification of melanocytic tumor on skin whole slide images.

PubMed

Xu, Hongming; Lu, Cheng; Berendt, Richard; Jha, Naresh; Mandal, Mrinal

2018-06-01

This paper presents a computer-aided technique for automated analysis and classification of melanocytic tumor on skin whole slide biopsy images. The proposed technique consists of four main modules. First, skin epidermis and dermis regions are segmented by a multi-resolution framework. Next, epidermis analysis is performed, where a set of epidermis features reflecting nuclear morphologies and spatial distributions is computed. In parallel with epidermis analysis, dermis analysis is also performed, where dermal cell nuclei are segmented and a set of textural and cytological features are computed. Finally, the skin melanocytic image is classified into different categories such as melanoma, nevus or normal tissue by using a multi-class support vector machine (mSVM) with extracted epidermis and dermis features. Experimental results on 66 skin whole slide images indicate that the proposed technique achieves more than 95% classification accuracy, which suggests that the technique has the potential to be used for assisting pathologists on skin biopsy image analysis and classification. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes – A method for quantitative transcriptional reprogramming

PubMed Central

Halley-Stott, R.P.; Pasque, V.; Astrand, C.; Miyamoto, K.; Simeoni, I.; Jullien, J.; Gurdon, J.B.

2010-01-01

Full-grown Xenopus oocytes in first meiotic prophase contain an immensely enlarged nucleus, the Germinal Vesicle (GV), that can be injected with several hundred somatic cell nuclei. When the nuclei of mammalian somatic cells or cultured cell lines are injected into a GV, a wide range of genes that are not transcribed in the donor cells, including pluripotency genes, start to be transcriptionally activated, and synthesize primary transcripts continuously for several days. Because of the large size and abundance of Xenopus laevis oocytes, this experimental system offers an opportunity to understand the mechanisms by which somatic cell nuclei can be reprogrammed to transcribe genes characteristic of oocytes and early embryos. The use of mammalian nuclei ensures that there is no background of endogenous maternal transcripts of the kind that are induced. The induced gene transcription takes place in the absence of cell division or DNA synthesis and does not require protein synthesis. Here we summarize new as well as established results that characterize this experimental system. In particular, we describe optimal conditions for transplanting somatic nuclei to oocytes and for the efficient activation of transcription by transplanted nuclei. We make a quantitative determination of transcript numbers for pluripotency and housekeeping genes, comparing cultured somatic cell nuclei with those of embryonic stem cells. Surprisingly we find that the transcriptional activation of somatic nuclei differs substantially from one donor cell-type to another and in respect of different pluripotency genes. We also determine the efficiency of an injected mRNA translation into protein. PMID:20123126

Light scattering properties of kidney epithelial cells and nuclei

NASA Astrophysics Data System (ADS)

Vitol, Elina A.; Kurzweg, Timothy P.; Nabet, Bahram

2006-02-01

Enlargement of mammalian cells nuclei due to the cancerous inflammation can be detected early through noninvasive optical techniques. We report on the results of cellular experiments, aimed towards the development of a fiber optic endoscopic probe used for precancerous detection of Barrett's esophagus. We previously presented white light scattering results from tissue phantoms (polystyrene polybead microspheres). In this paper, we discuss light scattering properties of epithelial MDCK (Madine-Darby Canine Kidney) cells and cell nuclei suspensions. A bifurcated optical fiber is used for experimental illumination and signal detection. The resulting scattering spectra from the cells do not exhibit the predicted Mie theory oscillatory behavior inherent to ideally spherical scatterers, such as polystyrene microspheres. However, we are able to demonstrate that the Fourier transform spectra of the cell suspensions are well correlated with the Fourier transform spectra of cell nuclei, concluding that the dominate scatterer in the backscattering region is the nucleus. This correlation experimentally illustrates that in the backscattering region, the cell nuclei are the main scatterer in the cells of the incident light.

Regeneration of the digestive system in the crinoid Himerometra robustipinna occurs by transdifferentiation of neurosecretory-like cells

PubMed Central

Kalacheva, Nadezhda V.; Eliseikina, Marina G.; Frolova, Lidia T.

2017-01-01

The structure and regeneration of the digestive system in the crinoid Himerometra robustipinna (Carpenter, 1881) were studied. The gut comprises a spiral tube forming radial lateral processes, which gives it a five-lobed shape. The digestive tube consists of three segments: esophagus, intestine, and rectum. The epithelia of these segments have different cell compositions. Regeneration of the gut after autotomy of the visceral mass progresses very rapidly. Within 6 h after autotomy, an aggregation consisting of amoebocytes, coelomic epithelial cells and juxtaligamental cells (neurosecretory neurons) forms on the inner surface of the skeletal calyx. At 12 h post-autotomy, transdifferentiation of the juxtaligamental cells starts. At 24 h post-autotomy these cells undergo a mesenchymal-epithelial-like transition, resulting in the formation of the luminal epithelium of the gut. Specialization of the intestinal epithelial cells begins on day 2 post-autotomy. At this stage animals acquire the mouth and anal opening. On day 4 post-autotomy the height of both the enterocytes and the visceral mass gradually increases. Proliferation does not play any noticeable role in gut regeneration. The immersion of animals in a 10−7 M solution of colchicine neither stopped formation of the lost structures nor caused accumulation of mitoses in tissues. Weakly EdU-labeled nuclei were observed in the gut only on day 2 post-autotomy and were not detected at later regeneration stages. Single mitotically dividing cells were recorded during the same period. It is concluded that juxtaligamental cells play a major role in gut regeneration in H. robustipinna. The main mechanisms of morphogenesis are cell migration and transdifferentiation. PMID:28753616

Regeneration of the digestive system in the crinoid Himerometra robustipinna occurs by transdifferentiation of neurosecretory-like cells.

PubMed

Kalacheva, Nadezhda V; Eliseikina, Marina G; Frolova, Lidia T; Dolmatov, Igor Yu

2017-01-01

The structure and regeneration of the digestive system in the crinoid Himerometra robustipinna (Carpenter, 1881) were studied. The gut comprises a spiral tube forming radial lateral processes, which gives it a five-lobed shape. The digestive tube consists of three segments: esophagus, intestine, and rectum. The epithelia of these segments have different cell compositions. Regeneration of the gut after autotomy of the visceral mass progresses very rapidly. Within 6 h after autotomy, an aggregation consisting of amoebocytes, coelomic epithelial cells and juxtaligamental cells (neurosecretory neurons) forms on the inner surface of the skeletal calyx. At 12 h post-autotomy, transdifferentiation of the juxtaligamental cells starts. At 24 h post-autotomy these cells undergo a mesenchymal-epithelial-like transition, resulting in the formation of the luminal epithelium of the gut. Specialization of the intestinal epithelial cells begins on day 2 post-autotomy. At this stage animals acquire the mouth and anal opening. On day 4 post-autotomy the height of both the enterocytes and the visceral mass gradually increases. Proliferation does not play any noticeable role in gut regeneration. The immersion of animals in a 10-7 M solution of colchicine neither stopped formation of the lost structures nor caused accumulation of mitoses in tissues. Weakly EdU-labeled nuclei were observed in the gut only on day 2 post-autotomy and were not detected at later regeneration stages. Single mitotically dividing cells were recorded during the same period. It is concluded that juxtaligamental cells play a major role in gut regeneration in H. robustipinna. The main mechanisms of morphogenesis are cell migration and transdifferentiation.

The nucleus is irreversibly shaped by motion of cell boundaries in cancer and non-cancer cells.

PubMed

Tocco, Vincent J; Li, Yuan; Christopher, Keith G; Matthews, James H; Aggarwal, Varun; Paschall, Lauren; Luesch, Hendrik; Licht, Jonathan D; Dickinson, Richard B; Lele, Tanmay P

2018-02-01

Actomyosin stress fibers impinge on the nucleus and can exert compressive forces on it. These compressive forces have been proposed to elongate nuclei in fibroblasts, and lead to abnormally shaped nuclei in cancer cells. In these models, the elongated or flattened nuclear shape is proposed to store elastic energy. However, we found that deformed shapes of nuclei are unchanged even after removal of the cell with micro-dissection, both for smooth, elongated nuclei in fibroblasts and abnormally shaped nuclei in breast cancer cells. The lack of shape relaxation implies that the nuclear shape in spread cells does not store any elastic energy, and the cellular stresses that deform the nucleus are dissipative, not static. During cell spreading, the deviation of the nucleus from a convex shape increased in MDA-MB-231 cancer cells, but decreased in MCF-10A cells. Tracking changes of nuclear and cellular shape on micropatterned substrata revealed that fibroblast nuclei deform only during deformations in cell shape and only in the direction of nearby moving cell boundaries. We propose that motion of cell boundaries exert a stress on the nucleus, which allows the nucleus to mimic cell shape. The lack of elastic energy in the nuclear shape suggests that nuclear shape changes in cells occur at constant surface area and volume. © 2017 Wiley Periodicals, Inc.

An improved FSL-FIRST pipeline for subcortical gray matter segmentation to study abnormal brain anatomy using quantitative susceptibility mapping (QSM).

PubMed

Feng, Xiang; Deistung, Andreas; Dwyer, Michael G; Hagemeier, Jesper; Polak, Paul; Lebenberg, Jessica; Frouin, Frédérique; Zivadinov, Robert; Reichenbach, Jürgen R; Schweser, Ferdinand

2017-06-01

Accurate and robust segmentation of subcortical gray matter (SGM) nuclei is required in many neuroimaging applications. FMRIB's Integrated Registration and Segmentation Tool (FIRST) is one of the most popular software tools for automated subcortical segmentation based on T 1 -weighted (T1w) images. In this work, we demonstrate that FIRST tends to produce inaccurate SGM segmentation results in the case of abnormal brain anatomy, such as present in atrophied brains, due to a poor spatial match of the subcortical structures with the training data in the MNI space as well as due to insufficient contrast of SGM structures on T1w images. Consequently, such deviations from the average brain anatomy may introduce analysis bias in clinical studies, which may not always be obvious and potentially remain unidentified. To improve the segmentation of subcortical nuclei, we propose to use FIRST in combination with a special Hybrid image Contrast (HC) and Non-Linear (nl) registration module (HC-nlFIRST), where the hybrid image contrast is derived from T1w images and magnetic susceptibility maps to create subcortical contrast that is similar to that in the Montreal Neurological Institute (MNI) template. In our approach, a nonlinear registration replaces FIRST's default linear registration, yielding a more accurate alignment of the input data to the MNI template. We evaluated our method on 82 subjects with particularly abnormal brain anatomy, selected from a database of >2000 clinical cases. Qualitative and quantitative analyses revealed that HC-nlFIRST provides improved segmentation compared to the default FIRST method. Copyright © 2017 Elsevier Inc. All rights reserved.

[Tyrosine hydroxylase in telencephalon and diencephalon of Rhodeus sericeus (Cyprinidae)].

PubMed

Pushchina, E V

2009-01-01

Immunohistochemical labeling of tyrosine hydroxylase was used to demonstrate catecholaminergic neuronal populations in the telencephalon and diencephalonof adult cypryniform fish Rhodeus sericeus. Various immunoreactive cell populations have been found in the telencephalon (ventral, central and lateral nuclei of ventral telencephalic area). Immunoreactive cells and fibers were discovered in dorsal nucleus of ventral telencephalic area and supracomissural nucleus in the caudal part of the telencephalon. In the diencephalon, periventricular nuclei (preoptic, periventricular nucleus of posterior tuberculum and periventricular organ) contained considerable TH-ergic cells. High activity of tyrosine hydroxylase was revealed in the pretectal, ventro-medial, ventro-lateral and suprachiasmatic nuclei. Periventricular hypothalamic nuclei also displayed high activity of tyrosine hydroxylase. Pseudounipolar neurons prevailed in all TH-immunereactive structures of the telencephalon and diencephalon: numerous bipolar liquor-contacting cells were discovered in the periventricular nuclei. Large pear-shaped cells and bipolar TH-ergic cells were found in posterior tuberculum. These cells may be functionally related to the dopamine-acquiring system.

Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen

PubMed Central

2011-01-01

Background Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei. Findings Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates. Conclusions We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls. PMID:22088094

Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen.

PubMed

Givens, Robert M; Mesner, Larry D; Hamlin, Joyce L; Buck, Michael J; Huberman, Joel A

2011-11-16

Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei. Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates. We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.

Analyzing the spatial positioning of nuclei in polynuclear giant cells

NASA Astrophysics Data System (ADS)

Stange, Maike; Hintsche, Marius; Sachse, Kirsten; Gerhardt, Matthias; Valleriani, Angelo; Beta, Carsten

2017-11-01

How cells establish and maintain a well-defined size is a fundamental question of cell biology. Here we investigated to what extent the microtubule cytoskeleton can set a predefined cell size, independent of an enclosing cell membrane. We used electropulse-induced cell fusion to form giant multinuclear cells of the social amoeba Dictyostelium discoideum. Based on dual-color confocal imaging of cells that expressed fluorescent markers for the cell nucleus and the microtubules, we determined the subcellular distributions of nuclei and centrosomes in the giant cells. Our two- and three-dimensional imaging results showed that the positions of nuclei in giant cells do not fall onto a regular lattice. However, a comparison with model predictions for random positioning showed that the subcellular arrangement of nuclei maintains a low but still detectable degree of ordering. This can be explained by the steric requirements of the microtubule cytoskeleton, as confirmed by the effect of a microtubule degrading drug.

Early Events in Retinal Degeneration Caused by Rhodopsin Mutation or Pigment Epithelium Malfunction: Differences and Similarities

PubMed Central

Di Pierdomenico, Johnny; García-Ayuso, Diego; Pinilla, Isabel; Cuenca, Nicolás; Vidal-Sanz, Manuel; Agudo-Barriuso, Marta; Villegas-Pérez, María P.

2017-01-01

To study the course of photoreceptor cell death and macro and microglial reactivity in two rat models of retinal degeneration with different etiologies. Retinas from P23H-1 (rhodopsin mutation) and Royal College of Surgeon (RCS, pigment epithelium malfunction) rats and age-matched control animals (Sprague-Dawley and Pievald Viro Glaxo, respectively) were cross-sectioned at different postnatal ages (from P10 to P60) and rhodopsin, L/M- and S-opsin, ionized calcium-binding adapter molecule 1 (Iba1), glial fibrillary acid protein (GFAP), and proliferating cell nuclear antigen (PCNA) proteins were immunodetected. Photoreceptor nuclei rows and microglial cells in the different retinal layers were quantified. Photoreceptor degeneration starts earlier and progresses quicker in P23H-1 than in RCS rats. In both models, microglial cell activation occurs simultaneously with the initiation of photoreceptor death while GFAP over-expression starts later. As degeneration progresses, the numbers of microglial cells increase in the retina, but decreasing in the inner retina and increasing in the outer retina, more markedly in RCS rats. Interestingly, and in contrast with healthy animals, microglial cells reach the outer nuclei and outer segment layers. The higher number of microglial cells in dystrophic retinas cannot be fully accounted by intraretinal migration and PCNA immunodetection revealed microglial proliferation in both models but more importantly in RCS rats. The etiology of retinal degeneration determines the initiation and pattern of photoreceptor cell death and simultaneously there is microglial activation and migration, while the macroglial response is delayed. The actions of microglial cells in the degeneration cannot be explained only in the basis of photoreceptor death because they participate more actively in the RCS model. Thus, the retinal degeneration caused by pigment epithelium malfunction is more inflammatory and would probably respond better to interventions by inhibiting microglial cells. PMID:28321183

A probabilistic template of human mesopontine tegmental nuclei from in vivo 7T MRI.

PubMed

Bianciardi, Marta; Strong, Christian; Toschi, Nicola; Edlow, Brian L; Fischl, Bruce; Brown, Emery N; Rosen, Bruce R; Wald, Lawrence L

2018-04-15

Mesopontine tegmental nuclei such as the cuneiform, pedunculotegmental, oral pontine reticular, paramedian raphe and caudal linear raphe nuclei, are deep brain structures involved in arousal and motor function. Dysfunction of these nuclei is implicated in the pathogenesis of disorders of consciousness and sleep, as well as in neurodegenerative diseases. However, their localization in conventional neuroimages of living humans is difficult due to limited image sensitivity and contrast, and a stereotaxic probabilistic neuroimaging template of these nuclei in humans does not exist. We used semi-automatic segmentation of single-subject 1.1mm-isotropic 7T diffusion-fractional-anisotropy and T 2 -weighted images in healthy adults to generate an in vivo probabilistic neuroimaging structural template of these nuclei in standard stereotaxic (Montreal Neurological Institute, MNI) space. The template was validated through independent manual delineation, as well as leave-one-out validation and evaluation of nuclei volumes. This template can enable localization of five mesopontine tegmental nuclei in conventional images (e.g. 1.5T, 3T) in future studies of arousal and motor physiology (e.g. sleep, anesthesia, locomotion) and pathology (e.g. disorders of consciousness, sleep disorders, Parkinson's disease). The 7T magnetic resonance imaging procedure for single-subject delineation of these nuclei may also prove useful for future 7T studies of arousal and motor mechanisms. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins

PubMed Central

Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

2013-01-01

Purpose To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features. Materials and Methods Tissue excised from a genetically engineered mouse model of sarcoma was imaged using a subcellular resolution microendoscope after topical application of a fluorescent anatomical contrast agent: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma. Results Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach. Conclusion The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue. PMID:23824589

Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins.

PubMed

Mueller, Jenna L; Harmany, Zachary T; Mito, Jeffrey K; Kennedy, Stephanie A; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G; Willett, Rebecca M; Brown, J Quincy; Ramanujam, Nimmi

2013-01-01

To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features. TISSUE EXCISED FROM A GENETICALLY ENGINEERED MOUSE MODEL OF SARCOMA WAS IMAGED USING A SUBCELLULAR RESOLUTION MICROENDOSCOPE AFTER TOPICAL APPLICATION OF A FLUORESCENT ANATOMICAL CONTRAST AGENT: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma. Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach. The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.

Global gray-level thresholding based on object size.

PubMed

Ranefall, Petter; Wählby, Carolina

2016-04-01

In this article, we propose a fast and robust global gray-level thresholding method based on object size, where the selection of threshold level is based on recall and maximum precision with regard to objects within a given size interval. The method relies on the component tree representation, which can be computed in quasi-linear time. Feature-based segmentation is especially suitable for biomedical microscopy applications where objects often vary in number, but have limited variation in size. We show that for real images of cell nuclei and synthetic data sets mimicking fluorescent spots the proposed method is more robust than all standard global thresholding methods available for microscopy applications in ImageJ and CellProfiler. The proposed method, provided as ImageJ and CellProfiler plugins, is simple to use and the only required input is an interval of the expected object sizes. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

Mie-type scattering and non-Beer-Lambert absorption behavior of human cells in infrared microspectroscopy.

PubMed

Mohlenhoff, Brian; Romeo, Melissa; Diem, Max; Wood, Bayden R

2005-05-01

We report infrared microspectral features of nuclei in a completely inactive and contracted (pyknotic) state, and of nuclei of actively dividing cells. For pyknotic nuclei, the very high local concentration of DNA leads to opaqueness of the chromatin and, consequently, the absence of DNA signals in the IR spectra of very small nuclei. However, these nuclei can be detected by their scattering properties, which can be described by the Mie theory of scattering from dielectric spheres. This scattering depends on the size of the nucleus; consequently, quite different scattering cross-sections are calculated and observed for pyknotic and mitotic nuclei.

Retinal pigment epithelial dystrophy in Briard dogs.

PubMed

Lightfoot, R M; Cabral, L; Gooch, L; Bedford, P G; Boulton, M E

1996-01-01

The eyes of normal Briard dogs, Briards affected with inherited retinal pigment epithelial dystrophy (RPED) and a range of normal crossbred and beagle dogs were examined and the histopathology of RPED in the Briard was compared with the histopathological features of ageing in the normal canine retina. RPED was characterised by the accumulation of auto-fluorescent lipofuscin-like inclusions in the retinal pigment epithelium (RPE), which initially involved only non-pigmented RPE cells overlying the tapetum but subsequently spread to all pigmented RPE cells. Secondary neuro-retinal degeneration was characterised by a gradual loss of the outer nuclear layer and the subsequent atrophy and degeneration of the inner retina. The loss of primary photoreceptors in the peripheral retina was accompanied by the migration of photoreceptor nuclei and appeared to resemble severe changes due to ageing. Intra-vitreal radiolabelled leucine was used to examine the rate of turnover of the outer segments of the rods in some Briards, but no significant variations were found. The activity of acid phosphatase in RPE was assayed in vitro and showed comparable regional variations in Briard and crossbred dogs. The results suggest that RPED in the Briard is unlikely to be due either to an increased rate of turnover of rod outer segments (and thus an increased phagocytic load) or to a primary insufficiency of lysosomal enzyme.

Rapid alkaline methylene blue supravital staining for assessment of anterior segment infections.

PubMed

Kiuchi, Katsuji

2016-01-01

To present the Löffler's alkaline methylene blue technique of staining eye discharges in eyes with anterior segment infections. The Löffler's alkaline methylene blue staining method is a simple staining technique that can be used to differentiate bacterial, viral, and fungal infections. It is a cationic dye that stains cells blue because the positively charged dye is attracted to negatively charged particles such as polyphosphates, DNAs, and RNAs. Specimens collected from patients by swabbing are smeared onto microscope slides and the methylene blue solution is dropped on the slide. The slide is covered with a glass cover slip and examined under a microscope. The entire time from the collection to the viewing is about 30 seconds. Histopathological images of the conjunctival epithelial cells and neutrophils in eye discharges were dyed blue and the nuclei were stained more intensely blue. Bacterial infections consisted mainly of neutrophils, and viral infections consisted mainly of lymphocytes. Löffler's alkaline methylene blue staining can be done in about 30 seconds for diagnosis. Even though this is a one color stain, it is possible to infer the cause of the infection by detection of the absence of bacteria and/or fungi in context of the differential distribution of neutrophils and lymphocytes.

Proflavine Hemisulfate as a Fluorescent Contrast Agent for Point-of-Care Cytology

PubMed Central

Prieto, Sandra P.; Powless, Amy J.; Boice, Jackson W.; Sharma, Shree G.; Muldoon, Timothy J.

2015-01-01

Proflavine hemisulfate, an acridine-derived fluorescent dye, can be used as a rapid stain for cytologic examination of biological specimens. Proflavine fluorescently stains cell nuclei and cytoplasmic structures, owing to its small amphipathic structure and ability to intercalate DNA. In this manuscript, we demonstrated the use of proflavine as a rapid cytologic dye on a number of specimens, including normal exfoliated oral squamous cells, cultured human oral squamous carcinoma cells, and leukocytes derived from whole blood specimens using a custom-built, portable, LED-illuminated fluorescence microscope. No incubation time was needed after suspending cells in 0.01% (w/v) proflavine diluted in saline. Images of proflavine stained oral cells had clearly visible nuclei as well as granular cytoplasm, while stained leukocytes exhibited bright nuclei, and highlighted the multilobar nature of nuclei in neutrophils. We also demonstrated the utility of quantitative analysis of digital images of proflavine stained cells, which can be used to detect significant morphological differences between different cell types. Proflavine stained oral cells have well-defined nuclei and cell membranes which allowed for quantitative analysis of nuclear to cytoplasmic ratios, as well as image texture analysis to extract quantitative image features. PMID:25962131

Proflavine Hemisulfate as a Fluorescent Contrast Agent for Point-of-Care Cytology.

PubMed

Prieto, Sandra P; Powless, Amy J; Boice, Jackson W; Sharma, Shree G; Muldoon, Timothy J

2015-01-01

Proflavine hemisulfate, an acridine-derived fluorescent dye, can be used as a rapid stain for cytologic examination of biological specimens. Proflavine fluorescently stains cell nuclei and cytoplasmic structures, owing to its small amphipathic structure and ability to intercalate DNA. In this manuscript, we demonstrated the use of proflavine as a rapid cytologic dye on a number of specimens, including normal exfoliated oral squamous cells, cultured human oral squamous carcinoma cells, and leukocytes derived from whole blood specimens using a custom-built, portable, LED-illuminated fluorescence microscope. No incubation time was needed after suspending cells in 0.01% (w/v) proflavine diluted in saline. Images of proflavine stained oral cells had clearly visible nuclei as well as granular cytoplasm, while stained leukocytes exhibited bright nuclei, and highlighted the multilobar nature of nuclei in neutrophils. We also demonstrated the utility of quantitative analysis of digital images of proflavine stained cells, which can be used to detect significant morphological differences between different cell types. Proflavine stained oral cells have well-defined nuclei and cell membranes which allowed for quantitative analysis of nuclear to cytoplasmic ratios, as well as image texture analysis to extract quantitative image features.

A general system for automatic biomedical image segmentation using intensity neighborhoods.

PubMed

Chen, Cheng; Ozolek, John A; Wang, Wei; Rohde, Gustavo K

2011-01-01

Image segmentation is important with applications to several problems in biology and medicine. While extensively researched, generally, current segmentation methods perform adequately in the applications for which they were designed, but often require extensive modifications or calibrations before being used in a different application. We describe an approach that, with few modifications, can be used in a variety of image segmentation problems. The approach is based on a supervised learning strategy that utilizes intensity neighborhoods to assign each pixel in a test image its correct class based on training data. We describe methods for modeling rotations and variations in scales as well as a subset selection for training the classifiers. We show that the performance of our approach in tissue segmentation tasks in magnetic resonance and histopathology microscopy images, as well as nuclei segmentation from fluorescence microscopy images, is similar to or better than several algorithms specifically designed for each of these applications.

Higher Levels of Organization in the Interphase Nucleus of Cycling and Differentiated Cells

PubMed Central

Leitch, Andrew R.

2000-01-01

The review examines the structured organization of interphase nuclei using a range of examples from the plants, animals, and fungi. Nuclear organization is shown to be an important phenomenon in cell differentiation and development. The review commences by examining nuclei in dividing cells and shows that the organization patterns can be dynamic within the time frame of the cell cycle. When cells stop dividing, derived differentiated cells often show quite different nuclear organizations. The developmental fate of nuclei is divided into three categories. (i) The first includes nuclei that undergo one of several forms of polyploidy and can themselves change in structure during the course of development. Possible function roles of polyploidy is given. (ii) The second is nuclear reorganization without polyploidy, where nuclei reorganize their structure to form novel arrangements of proteins and chromosomes. (iii) The third is nuclear disintegration linked to programmed cell death. The role of the nucleus in this process is described. The review demonstrates that recent methods to probe nuclei for nucleic acids and proteins, as well as to examine their intranuclear distribution in vivo, has revealed much about nuclear structure. It is clear that nuclear organization can influence or be influenced by cell activity and development. However, the full functional role of many of the observed phenomena has still to be fully realized. PMID:10704477

Rational Variety Mapping for Contrast-Enhanced Nonlinear Unsupervised Segmentation of Multispectral Images of Unstained Specimen

PubMed Central

Kopriva, Ivica; Hadžija, Mirko; Popović Hadžija, Marijana; Korolija, Marina; Cichocki, Andrzej

2011-01-01

A methodology is proposed for nonlinear contrast-enhanced unsupervised segmentation of multispectral (color) microscopy images of principally unstained specimens. The methodology exploits spectral diversity and spatial sparseness to find anatomical differences between materials (cells, nuclei, and background) present in the image. It consists of rth-order rational variety mapping (RVM) followed by matrix/tensor factorization. Sparseness constraint implies duality between nonlinear unsupervised segmentation and multiclass pattern assignment problems. Classes not linearly separable in the original input space become separable with high probability in the higher-dimensional mapped space. Hence, RVM mapping has two advantages: it takes implicitly into account nonlinearities present in the image (ie, they are not required to be known) and it increases spectral diversity (ie, contrast) between materials, due to increased dimensionality of the mapped space. This is expected to improve performance of systems for automated classification and analysis of microscopic histopathological images. The methodology was validated using RVM of the second and third orders of the experimental multispectral microscopy images of unstained sciatic nerve fibers (nervus ischiadicus) and of unstained white pulp in the spleen tissue, compared with a manually defined ground truth labeled by two trained pathophysiologists. The methodology can also be useful for additional contrast enhancement of images of stained specimens. PMID:21708116

Nuclear characteristics of the endometrial cytology: liquid-based versus conventional preparation.

PubMed

Norimatsu, Yoshiaki; Shigematsu, Yumie; Sakamoto, Shingo; Ohsaki, Hiroyuki; Yanoh, Kenji; Kawanishi, Namiki; Kobayashi, Tadao K

2013-02-01

The aim of this study was to assess the utility of liquid-based cytologic preparation (LP) compared with conventional preparation (CP) for the assessment of nuclear findings in endometrial glandular and stromal breakdown (EGBD) which may be misdiagnosed as carcinoma in EGBD cases. The material consists of cytologic smears including 20 cases of proliferative endometrium (PE), 20 cases of EGBD, and 20 cases of endometrioid adenocarcinoma grade1 (G1) for which histopathological diagnosis was obtained by endometrial curettage at the JA Suzuka General Hospital. Nuclear findings were examined in PE cells, EGBD-stromal cells, EGBD-metaplastic cells, and G1 cells, respectively. It was examined about the following items; (1) nuclear shape; (2) A long/minor axis ratio in cell nuclei; (3) an area of cell nuclei; (4) overlapping nuclei. Results are as follows: (1) nuclear shape; as for the reniform shape of EGBD-stromal cells and spindle shape of EGBD-metaplastic cells, the ratio of the LP method was a higher value than the CP method. (2) The long axis and area of cell nuclei; LP in all groups was a recognizable tendency for nuclear shrinkage. (3) The long/minor axis ratio in cell nuclei; only EGBD-metaplastic cells recognize a significant difference between CP and LP. (4) Overlapping nuclei; LP was a higher value in comparison with CP in the other groups except PE cells, and the degree of overlapping nuclei was enhanced about three times. Therefore, although a cell of LP has a shrinking tendency, (1) it is excellent that LP preserves a characteristic of nuclear shape than CP; (2) a cellular characteristic becomes clearer, because three-dimensional architecture of LP is preserved of than CP. As for the standard preparation method for endometrial cytology samples, we considered that a concrete introduction of the LP method poses no problems. Copyright © 2011 Wiley Periodicals, Inc.

Preparing nuclei from cells in monolayer cultures suitable for counting and for following synchronized cells through the cell cycle.

PubMed

Butler, W B

1984-08-15

A procedure is described for preparing nuclei from cells in monolayer culture so that they may be counted using an electronic particle counter. It takes only 10 to 15 min, and consists of swelling the cells in hypotonic buffer and then lysing them with the quaternary ammonium salt, ethylhexadecyldimethylammonium bromide. The cells are completely lysed, yielding a suspension of clean single nuclei which is stable, free of debris, and easily counted. The method was developed for a cell line of epithelial origin (MCF-7), which is often difficult to trypsinize to single cells. It works equally well at all cell densities up to and beyond confluence, and has been used with a variety of cells in culture, including 3T3 cells, bovine macrophages, rat mammary epithelial cells, mouse mammary tumor cell lines, and human fibroblasts. The size of the nuclei produced by this procedure is related to their DNA content, and the method is thus suitable for following cultures of synchronized cells through the cell cycle, and for performing differential counts of cells with substantial differences in DNA content.

Polyploidization on SK-N-MC human neuroblastoma cells infected with herpes simplex virus 1.

PubMed

Karalyan, Zaven; Izmailyan, Roza; Karalova, Elena; Abroyan, Liana; Hakobyan, Lina; Avetisyan, Aida; Semerjyan, Zara

2016-01-01

Polyploidization is one of the most dramatic changes occurring within cell genome owing to various reasons including under many viral infections. We examined the impact of herpes simplex virus-1 (HSV-1) on SK-N-MC human neuroblastoma cell line. The infected cells were followed from 6 hours up to 96 hours post infection (hpi). A large number of polyploid cells with giant nuclei was observed under the influence of HSV-1 at 24 hpi with the DNA content of 32c to 64c or more, in comparison with control SK-N-MC cells that were characterized by relatively moderate values of ploidy, i.e. 8с to 16с (where 1c is the haploid amount of nuclear DNA found in normal diploid populations in G0/G1). After 48-96 hpi, the population of polyploid cells with giant nuclei decreased to the benchmark level. The SK-NMC cells infected with HSV-1 for 24 hours were stained with gallocyanine and monitored for cytological features. The infected cells underwent virus induced cellcell and nuclei fusion with the formation of dense nuclei syncytium. The metabolic activity of HSV-1 infected cells was higher in both nuclei and nucleoli when compared to control cells.

Absorbed dose in target cell nuclei and dose conversion coefficient of radon progeny in the human lung.

PubMed

Nikezic, D; Lau, B M F; Stevanovic, N; Yu, K N

2006-01-01

To calculate the absorbed dose in the human lung due to inhaled radon progeny, ICRP focussed on the layers containing the target cells, i.e., the basal and secretory cells. Such an approach did not consider details of the sensitive cells in the layers. The present work uses the microdosimetric approach and determines the absorbed alpha-particle energy in non-spherical nuclei of target cells (basal and secretory cells). The absorbed energy for alpha particles emitted by radon progeny in the human respiratory tract was calculated in basal- and secretory-cell nuclei, assuming conical and ellipsoidal forms for these cells. Distributions of specific energy for different combinations of alpha-particle sources, energies and targets are calculated and shown. The dose conversion coefficient for radon progeny is reduced for about 2mSv/WLM when conical and ellipsoidal cell nuclei are considered instead of the layers. While changes in the geometry of secretory-cell nuclei do not have significant effects on their absorbed dose, changes from spherical to conical basal-cell nuclei have significantly reduced their absorbed dose from approximately 4 to approximately 3mGy/WLM. This is expected because basal cells are situated close to the end of the range of 6MeV alpha particles. This also underlines the significance of better and more precise information on targets in the T-B tree. A further change in the dose conversion coefficient can be achieved if a different weighting scheme is adopted for the doses for the cells. The results demonstrate the necessity for better information on the target cells for more accurate dosimetry for radon progeny.

Inheritance of gene density–related higher order chromatin arrangements in normal and tumor cell nuclei

PubMed Central

Cremer, Marion; Küpper, Katrin; Wagler, Babett; Wizelman, Leah; Hase, Johann v.; Weiland, Yanina; Kreja, Ludwika; Diebold, Joachim; Speicher, Michael R.; Cremer, Thomas

2003-01-01

A gene density–related difference in the radial arrangement of chromosome territories (CTs) was previously described for human lymphocyte nuclei with gene-poor CT #18 located toward the nuclear periphery and gene-dense CT #19 in the nuclear interior (Croft, J.A., J.M. Bridger, S. Boyle, P. Perry, P. Teague, and W.A. Bickmore. 1999. J. Cell Biol. 145:1119–1131). Here, we analyzed the radial distribution of chromosome 18 and 19 chromatin in six normal cell types and in eight tumor cell lines, some of them with imbalances and rearrangements of the two chromosomes. Our findings demonstrate that a significant difference in the radial distribution of #18 and #19 chromatin is a common feature of higher order chromatin architecture in both normal and malignant cell types. However, in seven of eight tumor cell lines, the difference was less pronounced compared with normal cell nuclei due to a higher fraction of nuclei showing an inverted CT position, i.e., a CT #18 located more internally than a CT #19. This observation emphasizes a partial loss of radial chromatin order in tumor cell nuclei. PMID:12952935

Evidence for alternative pathways of granulosa cell death in healthy and slightly atretic bovine antral follicles.

PubMed

Van Wezel, I L; Dharmarajan, A M; Lavranos, T C; Rodgers, R J

1999-06-01

Granulosa cell death is an early feature of atresia; however, there are many apparent contradictions in the literature concerning the mode of granulosa cell death. We have therefore examined this process in bovine healthy and atretic antral follicles, using a variety of established techniques. Light and electron microscopic observations indicated the presence of pyknotic or shrunken nuclei in both the membrana granulosa and the antrum. In the membrana granulosa, these nuclei were frequently crescent shaped and uniformly electron dense and were approximately the same size as healthy nuclei, all of which are typical of early apoptosis. However, these nuclei were within the membranes of a healthy granulosa cell, suggesting that phagocytosis by a neighboring granulosa cell is an unusually early event in the apoptotic pathway of granulosa cells. In the membrana granulosa, pyknotic nuclei stained intensely with hematoxylin but weakly with the DNA-intercalating stain propidium iodide. A percentage of these pyknotic nuclei stained by TUNEL (terminal deoxy-UTP nick end-labeling). However, in the antrum, the pyknotic nuclei and larger globules of DNA stained intensely with both hematoxylin and propidium iodide, but were not TUNEL positive. The comet assay of cell death produced a streak tail of randomly nicked DNA, rather than the plume of low mol wt apoptotic DNA. Globules collected from fresh follicular fluid stained intensely with propidium iodide and were shown by PAGE to contain DNA, the majority of which was high mol wt. In conclusion, granulosa cells within the membrana granulosa die by apoptosis, with phagocytosis by a neighboring cell preceding any potential budding of the nucleus or cell itself. Granulosa cells near the antrum are sloughed off into the antrum, and their death has features more consistent with that of other cell types that undergo death as a result of terminal differentiation.

Relationship between Endopolyploidy and Cell Size in Epidermal Tissue of Arabidopsis.

PubMed Central

Melaragno, JE; Mehrotra, B; Coleman, AW

1993-01-01

Relative quantities of DNA in individual nuclei of stem and leaf epidermal cells of Arabidopsis were measured microspectrofluorometrically using epidermal peels. The relative ploidy level in each nucleus was assessed by comparison to root tip mitotic nuclei. A clear pattern of regular endopolyploidy is evident in epidermal cells. Guard cell nuclei contain levels of DNA comparable to dividing root cells, the 2C level (i.e., one unreplicated copy of the nuclear DNA). Leaf trichome nuclei had elevated ploidy levels of 4C, 8C, 16C, 32C, and 64C, and their cytology suggested that the polyploidy represents a form of polyteny. The nuclei of epidermal pavement cells were 2C, 4C, and 8C in stem epidermis, and 2C, 4C, 8C, and 16C in leaf epidermis. Morphometry of epidermal pavement cells revealed a direct proportionality between nuclear DNA level and cell size. A consideration of the development process suggests that the cells of highest ploidy level are developmentally oldest; consequently, the developmental pattern of epidermal tissues can be read from the ploidy pattern of the cells. This observation is relevant to theories of stomate spacing and offers opportunities for genetic analysis of the endopolyploidy/polyteny phenomenon. PMID:12271050

Time-course of programmed cell death during leaf senescence in Eucommia ulmoides.

PubMed

Cao, Jing; Jiang, Feng; Sodmergen; Cui, Keming

2003-02-01

Leaves of Eucommia ulmoidesOliv. harvested between April to November were examined for programmed cell death (PCD) during growth and senescence. Leaves developed in April, becoming fully expanded in late May, remaining unchanged until November when they started to dehisce. Falling leaves retained a green color. Our results showed that (1) mesophyll cells gradually reduced their nuclei from September to November, (2) positive TUNEL signals appeared on the nuclei from August, (3) ladder-like DNA fragmentation occurred in September and October, and (4) a 20-kDa Ca(2+)-dependent DNase appeared in these same months. In fallen leaves, intact mesophyll cell nuclei could not be detected, but a few cells around the vascular bundle had nuclei. Therefore, (1) programmed cell death (PCD) of leaf cells occurred in the leaves of E. ulmoides, (2) the progress of mesophyll cell PCD lasted for more than 2 months, and (3) PCD of leaf cells was asynchronous in natural senescing leaves.

Refractometry of melanocyte cell nuclei using optical scatter images recorded by digital Fourier microscopy.

PubMed

Seet, Katrina Y T; Nieminen, Timo A; Zvyagin, Andrei V

2009-01-01

The cell nucleus is the dominant optical scatterer in the cell. Neoplastic cells are characterized by cell nucleus polymorphism and polychromism-i.e., the nuclei exhibits an increase in the distribution of both size and refractive index. The relative size parameter, and its distribution, is proportional to the product of the nucleus size and its relative refractive index and is a useful discriminant between normal and abnormal (cancerous) cells. We demonstrate a recently introduced holographic technique, digital Fourier microscopy (DFM), to provide a sensitive measure of this relative size parameter. Fourier holograms were recorded and optical scatter of individual scatterers were extracted and modeled with Mie theory to determine the relative size parameter. The relative size parameter of individual melanocyte cell nuclei were found to be 16.5+/-0.2, which gives a cell nucleus refractive index of 1.38+/-0.01 and is in good agreement with previously reported data. The relative size parameters of individual malignant melanocyte cell nuclei are expected to be greater than 16.5.

Inputs from regularly and irregularly discharging vestibular nerve afferents to secondary neurons in squirrel monkey vestibular nuclei. III. Correlation with vestibulospinal and vestibuloocular output pathways

NASA Technical Reports Server (NTRS)

Boyle, R.; Goldberg, J. M.; Highstein, S. M.

1992-01-01

1. A previous study measured the relative contributions made by regularly and irregularly discharging afferents to the monosynaptic vestibular nerve (Vi) input of individual secondary neurons located in and around the superior vestibular nucleus of barbiturate-anesthetized squirrel monkeys. Here, the analysis is extended to more caudal regions of the vestibular nuclei, which are a major source of both vestibuloocular and vestibulospinal pathways. As in the previous study, antidromic stimulation techniques are used to classify secondary neurons as oculomotor or spinal projecting. In addition, spinal-projecting neurons are distinguished by their descending pathways, their termination levels in the spinal cord, and their collateral projections to the IIIrd nucleus. 2. Monosynaptic excitatory postsynaptic potentials (EPSPs) were recorded intracellularly from secondary neurons as shocks of increasing strength were applied to Vi. Shocks were normalized in terms of the threshold (T) required to evoke field potentials in the vestibular nuclei. As shown previously, the relative contribution of irregular afferents to the total monosynaptic Vi input of each secondary neuron can be expressed as a %I index, the ratio (x100) of the relative sizes of the EPSPs evoked by shocks of 4 x T and 16 x T. 3. Antidromic stimulation was used to type secondary neurons as 1) medial vestibulospinal tract (MVST) cells projecting to spinal segments C1 or C6; 2) lateral vestibulospinal tract (LVST) cells projecting to C1, C6; or L1; 3) vestibulooculo-collic (VOC) cells projecting both to the IIIrd nucleus and by way of the MVST to C1 or C6; and 4) vestibuloocular (VOR) neurons projecting to the IIIrd nucleus but not to the spinal cord. Most of the neurons were located in the lateral vestibular nucleus (LV), including its dorsal (dLV) and ventral (vLV) divisions, and adjacent parts of the medial (MV) and descending nuclei (DV). Cells receiving quite different proportions of their direct inputs from regular and irregular afferents were intermingled in all regions explored. 4. LVST neurons are restricted to LV and DV and show a somatotopic organization. Those destined for the cervical and thoracic cord come from vLV, from a transition zone between vLV and DV, and to a lesser extent from dLV. Lumbar-projecting neurons are located more dorsally in dLV and more caudally in DV. MVST neurons reside in MV and in the vLV-DV transition zone.(ABSTRACT TRUNCATED AT 400 WORDS).

Evaluation of genetic damage induced by glyphosate isopropylamine salt using Tradescantia bioassays.

PubMed

Alvarez-Moya, Carlos; Silva, Mónica Reynoso; Arámbula, Alma Rosa Villalobos; Sandoval, Alfonso Islas; Vasquez, Hugo Castañeda; González Montes, Rosa María

2011-01-01

Glyphosate is noted for being non-toxic in fishes, birds and mammals (including humans). Nevertheless, the degree of genotoxicity is seriously controversial. In this work, various concentrations of a glyphosate isopropylamine salt were tested using two methods of genotoxicity assaying, viz., the pink mutation assay with Tradescantia (4430) and the comet assay with nuclei from staminal cells of the same plant. Staminal nuclei were studied in two different forms, namely nuclei from exposed plants, and nuclei exposed directly. Using the pink mutation assay, isopropylamine induced a total or partial loss of color in staminal cells, a fundamental criterion utilized in this test. Consequently, its use is not recommended when studying genotoxicity with agents that produce pallid staminal cells. The comet assay system detected statistically significant (p < 0.01) genotoxic activity by isopropylamine, when compared to the negative control in both the nuclei of treated plants and directly treated nuclei, but only the treated nuclei showed a dose-dependent increase. Average migration in the nuclei of treated plants increased, when compared to that in treated nuclei. This was probably due, either to the permanence of isopropylamine in inflorescences, or to the presence of secondary metabolites. In conclusion, isopropylamine possesses strong genotoxic activity, but its detection can vary depending on the test systems used.

Evaluation of genetic damage induced by glyphosate isopropylamine salt using Tradescantia bioassays

PubMed Central

Alvarez-Moya, Carlos; Silva, Mónica Reynoso; Arámbula, Alma Rosa Villalobos; Sandoval, Alfonso Islas; Vasquez, Hugo Castañeda; González Montes, Rosa María

2011-01-01

Glyphosate is noted for being non-toxic in fishes, birds and mammals (including humans). Nevertheless, the degree of genotoxicity is seriously controversial. In this work, various concentrations of a glyphosate isopropylamine salt were tested using two methods of genotoxicity assaying, viz., the pink mutation assay with Tradescantia (4430) and the comet assay with nuclei from staminal cells of the same plant. Staminal nuclei were studied in two different forms, namely nuclei from exposed plants, and nuclei exposed directly. Using the pink mutation assay, isopropylamine induced a total or partial loss of color in staminal cells, a fundamental criterion utilized in this test. Consequently, its use is not recommended when studying genotoxicity with agents that produce pallid staminal cells. The comet assay system detected statistically significant (p < 0.01) genotoxic activity by isopropylamine, when compared to the negative control in both the nuclei of treated plants and directly treated nuclei, but only the treated nuclei showed a dose-dependent increase. Average migration in the nuclei of treated plants increased, when compared to that in treated nuclei. This was probably due, either to the permanence of isopropylamine in inflorescences, or to the presence of secondary metabolites. In conclusion, isopropylamine possesses strong genotoxic activity, but its detection can vary depending on the test systems used. PMID:21637555

Aphidicolin-induced nuclear elongation in tobacco BY-2 cells.

PubMed

Yasuhara, Hiroki; Kitamoto, Kazuki

2014-05-01

Plant nuclei are known to differentiate into various shapes within a single plant. However, little is known about the mechanisms of nuclear morphogenesis. We found that nuclei of tobacco BY-2 cells were highly elongated on long-term treatment with 5 mg l⁻¹ aphidicolin, an inhibitor of DNA polymerase α. In aphidicolin-treated cells, the nuclear length was correlated with the cell length. During culture in the presence of aphidicolin, the nuclei were elongated in parallel with cell elongation. Nuclear elongation was inhibited by the inhibition of cell elongation with 2,6-dichlorobenzonitrile, a cellulose synthesis inhibitor. However, cell elongation induced in the auxin-depleted medium in the absence of aphidicolin did not cause nuclear elongation, indicating that cell elongation alone is not sufficient for nuclear elongation. Treatment with either latrunculin B or propyzamide inhibited the aphidicolin-induced nuclear elongation, indicating that both actin filaments and microtubules (MTs) are required for nuclear elongation. Observations using BY-YTHCLR2 cells, in which actin filaments, MTs and nuclei were simultaneously visualized, revealed that the longitudinally arranged MT bundles associated with the nucleus play an important role in nuclear elongation, and that actin filaments affect the formation of these MT bundles. In aphidicolin-treated cells, the nuclear DNA contents of the elongated nuclei exceeded 4C, and the nuclear length was highly correlated with the nuclear DNA content. In cells treated with 50 mg l⁻¹ aphidicolin, cells were elongated and nucleus-associated longitudinal MT bundles were formed, but the nuclear DNA contents did not exceed 4C and the nuclei did not elongate. These results indicate that an increase in the nuclear DNA content above 4C is also required for nuclear elongation.

[Morpho-functional parameters of nucleoli in polyploid mucous and albumen cells of salivary gland in the snail Succinea lauta].

PubMed

Anisimova, A A; Anisimov, A P

2005-01-01

Variation of some characteristics of nucleoli of polyploid mucous and albumen cells was examined in salivary glands of the snail Succinea lauta. The number, total area and Ag-protein content of nucleoli, and DNA content in each nucleus were estimated on squashed preparations incubated with AgNO3, decolorized and then Feulgen stained. The ultrastructure of nucleoli was studied by electron microscopy. Differentiated mucous cells had 4c-8c-16c-32c nuclei; albumen cells had 8c-16c-32c-64c-128c nuclei. The ultrastructure of nucleoli of the two cell types was essentially the same. Normally, a large fibrous to granular zone was observed in the nucleoli, without a clear distinction between fibrous and granular components. At the same time, aggregations of granular matter could be discerned at the periphery of nucleoli. No fibrous centers were observed. Occassionally, nucleolonema-like structures occurred. Normally each nucleolus contacted several chromosomes. On squashed preparations, the least size of nucleoli was 2-3 microm, and the largest size amounted to 14 microm in mucous cells, and to 50-80 microm in albumen cells. The number of nucleoli rose from 1-2 in tetraploid nuclei to 2-3 in 32c-nuclei, and to 5-7 in 128c-nuclei. The disparity between the ploidy levels of nuclei and the numbers of nucleoli may be due, presumably, to aggregation of chromosome NORs. The Ag-protein content in the nucleoli, and the total nucleolar area displayed a strong mutual correlation. Both parameters differed significantly by 1.5-2.2 times in mucous and albumen cells of the same ploidy level. Thus, in albumen and mucous cells the total Ag-protein content in octaploid nuclei was 3.3 and 2.2 relative units (r. u.), respectively. In 16c- and 32c-nuclei of albumen cells, it was 7.6 and 15.1 r. u.; and in the same nuclei of mucous cells--3.8 and 6.8 r. u., respectively. On the whole, in albumen cells, in the course of 4 endocycles (4c-128c), the total Ag-protein content increased by 17 times. Therefore, the mean multiplication factor for this parameter was found to be 2.05 per endocycle. In mucous cells, in the course of 3 endocycles (4c-32c), the total Ag-protein content increased by 5.2 times against 8 times expected, with the mean multiplication factor equal to 1.75 per endocycle. Thus, in the course of polyploidization of albumen and mucous cell nuclei, the gene dosage effect was fully pronounced in the former, and only partly in the latter. This differtence is due obviously to peculiarities of differentiation of the two cell types, in particular, to differences in the number of activated ribosomal genes.

Tissue mimicking simulations for temporal enhanced ultrasound-based tissue typing

NASA Astrophysics Data System (ADS)

Bayat, Sharareh; Imani, Farhad; Gerardo, Carlos D.; Nir, Guy; Azizi, Shekoofeh; Yan, Pingkun; Tahmasebi, Amir; Wilson, Storey; Iczkowski, Kenneth A.; Lucia, M. Scott; Goldenberg, Larry; Salcudean, Septimiu E.; Mousavi, Parvin; Abolmaesumi, Purang

2017-03-01

Temporal enhanced ultrasound (TeUS) is an imaging approach where a sequence of temporal ultrasound data is acquired and analyzed for tissue typing. Previously, in a series of in vivo and ex vivo studies we have demonstrated that, this approach is effective for detecting prostate and breast cancers. Evidences derived from our experiments suggest that both ultrasound-signal related factors such as induced heat and tissue-related factors such as the distribution and micro-vibration of scatterers lead to tissue typing information in TeUS. In this work, we simulate mechanical micro-vibrations of scatterers in tissue-mimicking phantoms that have various scatterer densities reflecting benign and cancerous tissue structures. Finite element modeling (FEM) is used for this purpose where the vertexes are scatterers representing cell nuclei. The initial positions of scatterers are determined by the distribution of nuclei segmented from actual digital histology scans of prostate cancer patients. Subsequently, we generate ultrasound images of the simulated tissue structure using the Field II package resulting in a temporal enhanced ultrasound. We demonstrate that the micro-vibrations of scatterers are captured by temporal ultrasound data and this information can be exploited for tissue typing.

Characterization of the Variability of Nucleoli in the Cells of Panax ginseng Meyer In Vivo and In Vitro.

PubMed

Khrolenko, Yuliya A; Burundukova, Olga L; Lauve, Lyudmila S; Muzarok, Tamara I; Makhan'kov, Vyacheslav V; Zhuravlev, Yuri N

2012-07-01

Results of karyological study of intact plants and some callus lines of Panax ginseng are presented. In the native plants of P. ginseng the nucleus with 1 nucleolus (90%) dominate, and nucleus with 2 nucleoli is rare. One nucleolar nucleus also dominate in interphase nuclei of cells of cultivated P. ginseng (from 2006), but we also found nucleus with 2 to 3 nucleoli in the same cell lines. Interphase nuclei of P. ginseng in long cultivated lines (from 1988) contain 1 to 9 nucleoli, with a predominance of nuclei containing from 3 to 4 nucleoli. It was shown that long-time cells (cultivated since 1988) had cytogenetic changes such as increase level of polyploid and aneuploid cells, increase of nucleoli number into interphase nucleus and decrease of nuclei/nucleoli ratio. These long-time cultivated cells had very low ginsenoside content.

Characterization of the Variability of Nucleoli in the Cells of Panax ginseng Meyer In Vivo and In Vitro

PubMed Central

Khrolenko, Yuliya A.; Burundukova, Olga L.; Lauve, Lyudmila S.; Muzarok, Tamara I.; Makhan’kov, Vyacheslav V.; Zhuravlev, Yuri N.

2012-01-01

Results of karyological study of intact plants and some callus lines of Panax ginseng are presented. In the native plants of P. ginseng the nucleus with 1 nucleolus (90%) dominate, and nucleus with 2 nucleoli is rare. One nucleolar nucleus also dominate in interphase nuclei of cells of cultivated P. ginseng (from 2006), but we also found nucleus with 2 to 3 nucleoli in the same cell lines. Interphase nuclei of P. ginseng in long cultivated lines (from 1988) contain 1 to 9 nucleoli, with a predominance of nuclei containing from 3 to 4 nucleoli. It was shown that long-time cells (cultivated since 1988) had cytogenetic changes such as increase level of polyploid and aneuploid cells, increase of nucleoli number into interphase nucleus and decrease of nuclei/nucleoli ratio. These long-time cultivated cells had very low ginsenoside content. PMID:23717134

Detection of nuclei in 4D Nomarski DIC microscope images of early Caenorhabditis elegans embryos using local image entropy and object tracking

PubMed Central

Hamahashi, Shugo; Onami, Shuichi; Kitano, Hiroaki

2005-01-01

Background The ability to detect nuclei in embryos is essential for studying the development of multicellular organisms. A system of automated nuclear detection has already been tested on a set of four-dimensional (4D) Nomarski differential interference contrast (DIC) microscope images of Caenorhabditis elegans embryos. However, the system needed laborious hand-tuning of its parameters every time a new image set was used. It could not detect nuclei in the process of cell division, and could detect nuclei only from the two- to eight-cell stages. Results We developed a system that automates the detection of nuclei in a set of 4D DIC microscope images of C. elegans embryos. Local image entropy is used to produce regions of the images that have the image texture of the nucleus. From these regions, those that actually detect nuclei are manually selected at the first and last time points of the image set, and an object-tracking algorithm then selects regions that detect nuclei in between the first and last time points. The use of local image entropy makes the system applicable to multiple image sets without the need to change its parameter values. The use of an object-tracking algorithm enables the system to detect nuclei in the process of cell division. The system detected nuclei with high sensitivity and specificity from the one- to 24-cell stages. Conclusion A combination of local image entropy and an object-tracking algorithm enabled highly objective and productive detection of nuclei in a set of 4D DIC microscope images of C. elegans embryos. The system will facilitate genomic and computational analyses of C. elegans embryos. PMID:15910690

Self-Affinity and Lacunarity of Chromatin Texture in Benign and Malignant Breast Epithelial Cell Nuclei

NASA Astrophysics Data System (ADS)

Einstein, Andrew J.; Wu, Hai-Shan; Gil, Joan

1998-01-01

Methods are presented for characterizing the self-affinity and lacunarity of arbitrarily shaped images. Chromatin appearance in breast epithelial cell nuclei is shown to be statistically self-affine. Spectral and Minkowski dimensions are lesser in nuclei of malignant cases than in nuclei of benign cases, and lacunarity further quantifies morphologic differences such as chromatin clumping and nucleoli. Fractal texture features are used as the basis for an accurate cytologic diagnosis of breast cancer.

Direct observation of light focusing by single photoreceptor cell nuclei.

PubMed

Błaszczak, Zuzanna; Kreysing, Moritz; Guck, Jochen

2014-05-05

The vertebrate retina is inverted with respect to its optical function, which requires light to pass through the entire tissue prior to detection. The last significant barrier for photons to overcome is the outer nuclear layer formed by photoreceptor cell (PRC) nuclei. Here we experimentally characterise the optical properties of PRC nuclei using bright-field defocusing microscopy to capture near-field intensity distributions behind individual nuclei. We find that some nuclei efficiently focus incident light confirming earlier predictions based on comparative studies of chromatin organisation in nocturnal and diurnal mammals. The emergence of light focusing during the development of mouse nuclei highlights the acquired nature of the observed lens-like behaviour. Optical characterisation of these nuclei is an important first step towards an improved understanding of how light transmission through the retina is influenced by its constituents.

Optical imaging modalities: From design to diagnosis of skin cancer

NASA Astrophysics Data System (ADS)

Korde, Vrushali Raj

This study investigates three high resolution optical imaging modalities to better detect and diagnose skin cancer. The ideal high resolution optical imaging system can visualize pre-malignant tissue growth non-invasively with resolution comparable to histology. I examined 3 modalities which approached this goal. The first method examined was high magnification microscopy of thin stained tissue sections, together with a statistical analysis of nuclear chromatin patterns termed Karyometry. This method has subcellular resolution, but it necessitates taking a biopsy at the desired tissue site and imaging the tissue ex-vivo. My part of this study was to develop an automated nuclear segmentation algorithm to segment cell nuclei in skin histology images for karyometric analysis. The results of this algorithm were compared to hand segmented cell nuclei in the same images, and it was concluded that the automated segmentations can be used for karyometric analysis. The second optical imaging modality I investigated was Optical Coherence Tomography (OCT). OCT is analogous to ultrasound, in which sound waves are delivered into the body and the echo time and reflected signal magnitude are measured. Due to the fast speed of light and detector temporal integration times, low coherence interferometry is needed to gate the backscattered light. OCT acquires cross sectional images, and has an axial resolution of 1-15 mum (depending on the source bandwidth) and a lateral resolution of 10-20 mum (depending on the sample arm optics). While it is not capable of achieving subcellular resolution, it is a non-invasive imaging modality. OCT was used in this study to evaluate skin along a continuum from normal to sun damaged to precancer. I developed algorithms to detect statistically significant differences between images of sun protected and sun damaged skin, as well as between undiseased and precancerous skin. An Optical Coherence Microscopy (OCM) endoscope was developed in the third portion of this study. OCM is a high resolution en-face imaging modality. It is a hybrid system that combines the principles of confocal microscopy with coherence gating to provide an increased imaging depth. It can also be described as an OCT system with a high NA objective. Similar to OCT, the axial resolution is determined by the source center wavelength and bandwidth. The NA of the sample arm optics determines the lateral resolution, usually on the order of 1-5 mum. My effort on this system was to develop a handheld endoscope. To my knowledge, an OCM endoscope has not been developed prior to this work. An image of skin was taken as a proof of concept. This rigid handheld OCM endoscope will be useful for applications ranging from minimally invasive surgical imaging to non-invasively assessing dysplasia and sun damage in skin.

An Image-Based Algorithm for Precise and Accurate High Throughput Assessment of Drug Activity against the Human Parasite Trypanosoma cruzi

PubMed Central

Moraes, Carolina Borsoi; Yang, Gyongseon; Kang, Myungjoo; Freitas-Junior, Lucio H.; Hansen, Michael A. E.

2014-01-01

We present a customized high content (image-based) and high throughput screening algorithm for the quantification of Trypanosoma cruzi infection in host cells. Based solely on DNA staining and single-channel images, the algorithm precisely segments and identifies the nuclei and cytoplasm of mammalian host cells as well as the intracellular parasites infecting the cells. The algorithm outputs statistical parameters including the total number of cells, number of infected cells and the total number of parasites per image, the average number of parasites per infected cell, and the infection ratio (defined as the number of infected cells divided by the total number of cells). Accurate and precise estimation of these parameters allow for both quantification of compound activity against parasites, as well as the compound cytotoxicity, thus eliminating the need for an additional toxicity-assay, hereby reducing screening costs significantly. We validate the performance of the algorithm using two known drugs against T.cruzi: Benznidazole and Nifurtimox. Also, we have checked the performance of the cell detection with manual inspection of the images. Finally, from the titration of the two compounds, we confirm that the algorithm provides the expected half maximal effective concentration (EC50) of the anti-T. cruzi activity. PMID:24503652

Outer nuclear membrane fusion of adjacent nuclei in varicella-zoster virus-induced syncytia.

PubMed

Wang, Wei; Yang, Lianwei; Huang, Xiumin; Fu, Wenkun; Pan, Dequan; Cai, Linli; Ye, Jianghui; Liu, Jian; Xia, Ningshao; Cheng, Tong; Zhu, Hua

2017-12-01

Syncytia formation has been considered important for cell-to-cell spread and pathogenesis of many viruses. As a syncytium forms, individual nuclei often congregate together, allowing close contact of nuclear membranes and possibly fusion to occur. However, there is currently no reported evidence of nuclear membrane fusion between adjacent nuclei in wild-type virus-induced syncytia. Varicella-zoster virus (VZV) is one typical syncytia-inducing virus that causes chickenpox and shingles in humans. Here, we report, for the first time, an interesting observation of apparent fusion of the outer nuclear membranes from juxtaposed nuclei that comprise VZV syncytia both in ARPE-19 human epithelial cells in vitro and in human skin xenografts in the SCID-hu mouse model in vivo. This work reveals a novel aspect of VZV-related cytopathic effect in the context of multinucleated syncytia. Additionally, the information provided by this study could be helpful for future studies on interactions of viruses with host cell nuclei. Copyright © 2017 Elsevier Inc. All rights reserved.

In-vivo cell tracking to quantify endothelial cell migration during zebrafish angiogenesis

NASA Astrophysics Data System (ADS)

Menon, Prahlad G.; Rochon, Elizabeth R.; Roman, Beth L.

2016-03-01

The mechanism of endothelial cell migration as individual cells or collectively while remaining an integral component of a functional blood vessel has not been well characterized. In this study, our overarching goal is to define an image processing workflow to facilitate quantification of how endothelial cells within the first aortic arch and are proximal to the zebrafish heart behave in response to the onset of flow (i.e. onset of heart beating). Endothelial cell imaging was conducted at this developmental time-point i.e. ~24-28 hours post fertilization (hpf) when flow first begins, using 3D+time two-photon confocal microscopy of a live, wild-type, transgenic, zebrafish expressing green fluorescent protein (GFP) in endothelial cell nuclei. An image processing pipeline comprised of image signal enhancement, median filtering for speckle noise reduction, automated identification of the nuclei positions, extraction of the relative movement of nuclei between consecutive time instances, and finally tracking of nuclei, was designed for achieving the tracking of endothelial cell nuclei and the identification of their movement towards or away from the heart. Pilot results lead to a hypothesis that upon the onset of heart beat and blood flow, endothelial cells migrate collectively towards the heart (by 21.51+/-10.35 μm) in opposition to blood flow (i.e. subtending 142.170+/-21.170 with the flow direction).

Nuclear traffic and peloton formation in fungal networks

NASA Astrophysics Data System (ADS)

Roper, Marcus; Hickey, Patrick; Lewkiewicz, Stephanie; Dressaire, Emilie; Read, Nick

2013-11-01

Hyphae, the network of microfluidic pipes that make up a growing fungal cell, must balance their function as conduits for the transport of nuclei with other cellular functions including secretion and growth. Constant flow of nuclei may interfere with the protein traffic that enables other functions to be performed. Live-cell imaging reveals that nuclear flows are anti-congestive; that groups of nuclei flow faster than single nuclei, and that nuclei sweep through the colony in dense clumps. We call these clumps pelotons, after the term used to describe groups of cycle racers slip-streaming off each other. Because of the pelotons, individual hyphae transport nuclei only intermittently, producing long intervals in which hyphae can perform their other functions. Modeling reveals how pelotons are created by interactions between nuclei and the hyphal cytoskeleton, and reveal the control that the fungus enjoys over peloton assembly and timing.

FACTORS INFLUENCING THE ABILITY OF ISOLATED CELL NUCLEI TO FORM GELS IN DILUTE ALKALI

PubMed Central

Dounce, Alexander L.; Monty, Kenneth J.

1955-01-01

1. Known methods for isolating cell nuclei are divided into two classes, depending on whether or not the nuclei are capable of forming gels in dilute alkali or strong saline solutions. Methods which produce nuclei that can form gels apparently prevent the action of an intramitochondrial enzyme capable of destroying the gel-forming capacity of the nuclei. Methods in the other class are believed to permit this enzyme to act on the nuclei during the isolation procedure, causing detachment of DNA from some nuclear constituent (probably protein). 2. It is shown that heating in alkaline solution and x-irradiation can destroy nuclear gels. Heating in acid or neutral solutions can destroy the capacity of isolated nuclei to form gels. 3. Chemical and biological evidence is summarized in favor of the hypothesis that DNA is normally bound firmly to some nuclear component by non-ionic linkages. PMID:14381437

Retinal adaptation to dim light vision in spectacled caimans (Caiman crocodilus fuscus): Analysis of retinal ultrastructure.

PubMed

Karl, Anett; Agte, Silke; Zayas-Santiago, Astrid; Makarov, Felix N; Rivera, Yomarie; Benedikt, Jan; Francke, Mike; Reichenbach, Andreas; Skatchkov, Serguei N; Bringmann, Andreas

2018-05-19

It has been shown that mammalian retinal glial (Müller) cells act as living optical fibers that guide the light through the retinal tissue to the photoreceptor cells (Agte et al., 2011; Franze et al., 2007). However, for nonmammalian species it is unclear whether Müller cells also improve the transretinal light transmission. Furthermore, for nonmammalian species there is a lack of ultrastructural data of the retinal cells, which, in general, delivers fundamental information of the retinal function, i.e. the vision of the species. A detailed study of the cellular ultrastructure provides a basic approach of the research. Thus, the aim of the present study was to investigate the retina of the spectacled caimans at electron and light microscopical levels to describe the structural features. For electron microscopy, we used a superfast microwave fixation procedure in order to achieve more precise ultrastructural information than common fixation techniques. As result, our detailed ultrastructural study of all retinal parts shows structural features which strongly indicate that the caiman retina is adapted to dim light and night vision. Various structural characteristics of Müller cells suppose that the Müller cell may increase the light intensity along the path of light through the neuroretina and, thus, increase the sensitivity of the scotopic vision of spectacled caimans. Müller cells traverse the whole thickness of the neuroretina and thus may guide the light from the inner retinal surface to the photoreceptor cell perikarya and the Müller cell microvilli between the photoreceptor segments. Thick Müller cell trunks/processes traverse the layers which contain light-scattering structures, i.e., nerve fibers and synapses. Large Müller cell somata run through the inner nuclear layer and contain flattened, elongated Müller cell nuclei which are arranged along the light path and, thus, may reduce the loss of the light intensity along the retinal light path. The oblique arrangement of many Müller cell trunks/processes in the inner plexiform layer and the large Müller cell somata in the inner nuclear layer may suggest that light guidance through Müller cells increases the visual sensitivity. Furthermore, an adaptation of the caiman retina to low light levels is strongly supported by detailed ultrastructural data of other retinal parts, e.g. by (i) the presence of a guanine-based retinal tapetum, (ii) the rod dominance of the retina, (iii) the presence of photoreceptor cell nuclei, which penetrate the outer limiting membrane, (iv) the relatively low densities of photoreceptor and neuronal cells which is compensated by (v) the presence of rods with long and thick outer segments, that may increase the probability of photon absorption. According to a cell number analysis, the central and temporal areas of the dorsal tapetal retina, which supports downward prey detection in darker water, are the sites of the highest diurnal contrast/color vision, i.e. cone vision and of the highest retinal light sensitivity, i.e. rod vision. Copyright © 2018 Elsevier Ltd. All rights reserved.

Color-coded Live Imaging of Heterokaryon Formation and Nuclear Fusion of Hybridizing Cancer Cells.

PubMed

Suetsugu, Atsushi; Matsumoto, Takuro; Hasegawa, Kosuke; Nakamura, Miki; Kunisada, Takahiro; Shimizu, Masahito; Saji, Shigetoyo; Moriwaki, Hisataka; Bouvet, Michael; Hoffman, Robert M

2016-08-01

Fusion of cancer cells has been studied for over half a century. However, the steps involved after initial fusion between cells, such as heterokaryon formation and nuclear fusion, have been difficult to observe in real time. In order to be able to visualize these steps, we have established cancer-cell sublines from the human HT-1080 fibrosarcoma, one expressing green fluorescent protein (GFP) linked to histone H2B in the nucleus and a red fluorescent protein (RFP) in the cytoplasm and the other subline expressing RFP in the nucleus (mCherry) linked to histone H2B and GFP in the cytoplasm. The two reciprocal color-coded sublines of HT-1080 cells were fused using the Sendai virus. The fused cells were cultured on plastic and observed using an Olympus FV1000 confocal microscope. Multi-nucleate (heterokaryotic) cancer cells, in addition to hybrid cancer cells with single-or multiple-fused nuclei, including fused mitotic nuclei, were observed among the fused cells. Heterokaryons with red, green, orange and yellow nuclei were observed by confocal imaging, even in single hybrid cells. The orange and yellow nuclei indicate nuclear fusion. Red and green nuclei remained unfused. Cell fusion with heterokaryon formation and subsequent nuclear fusion resulting in hybridization may be an important natural phenomenon between cancer cells that may make them more malignant. The ability to image the complex processes following cell fusion using reciprocal color-coded cancer cells will allow greater understanding of the genetic basis of malignancy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

The Long Adventurous Journey of Rhombic Lip Cells in Jawed Vertebrates: A Comparative Developmental Analysis

PubMed Central

Wullimann, Mario F.; Mueller, Thomas; Distel, Martin; Babaryka, Andreas; Grothe, Benedikt; Köster, Reinhard W.

2011-01-01

This review summarizes vertebrate rhombic lip and early cerebellar development covering classic approaches up to modern developmental genetics which identifies the relevant differential gene expression domains and their progeny. Most of this information is derived from amniotes. However, progress in anamniotes, particularly in the zebrafish, has recently been made. The current picture suggests that rhombic lip and cerebellar development in jawed vertebrates (gnathostomes) share many characteristics. Regarding cerebellar development, these include a ptf1a expressing ventral cerebellar proliferation (VCP) giving rise to Purkinje cells and other inhibitory cerebellar cell types, and an atoh1 expressing upper rhombic lip giving rise to an external granular layer (EGL, i.e., excitatory granule cells) and an early ventral migration into the anterior rhombencephalon (cholinergic nuclei). As for the lower rhombic lip (LRL), gnathostome commonalities likely include the formation of precerebellar nuclei (mossy fiber origins) and partially primary auditory nuclei (likely convergently evolved) from the atoh1 expressing dorsal zone. The fate of the ptf1a expressing ventral LRL zone which gives rise to (excitatory cells of) the inferior olive (climbing fiber origin) and (inhibitory cells of ) cochlear nuclei in amniotes, has not been determined in anamniotes. Special for the zebrafish in comparison to amniotes is the predominant origin of anamniote excitatory deep cerebellar nuclei homologs (i.e., eurydendroid cells) from ptf1a expressing VCP cells, the sequential activity of various atoh1 paralogs and the incomplete coverage of the subpial cerebellar plate with proliferative EGL cells. Nevertheless, the conclusion that a rhombic lip and its major derivatives evolved with gnathostome vertebrates only and are thus not an ancestral craniate character complex is supported by the absence of a cerebellum (and likely absence of its afferent and efferent nuclei) in jawless fishes PMID:21559349

Determination of ACC-induced cell-programmed death in roots of Vicia faba ssp. minor seedlings by acridine orange and ethidium bromide staining.

PubMed

Byczkowska, Anna; Kunikowska, Anita; Kaźmierczak, Andrzej

2013-02-01

Fluorescence staining with acridine orange (AO) and ethidium bromide (EB) showed that nuclei of cortex root cells of 1-aminocyclopropane-1-carboxylic acid (ACC)-treated Vicia faba ssp. minor seedlings differed in color. Measurement of resultant fluorescence intensity (RFI) showed that it increased when the color of nuclear chromatin was changed from green to red, indicating that EB moved to the nuclei via the cell membrane which lost its integrity and stained nuclei red. AO/EB staining showed that changes in color of the nuclear chromatin were accompanied by DNA condensation, nuclei fragmentation, and chromatin degradation which were also shown after 4,6-diamidino-2-phenylindol staining. These results indicate that ACC induced programmed cell death. The increasing values of RFI together with the corresponding morphological changes of nuclear chromatin were the basis to prepare the standard curve; cells with green unchanged nuclear chromatin were alive while those with dark orange and bright red nuclei were dead. The cells with nuclei with green-yellow, yellow-orange, and bright orange chromatin with or without their condensation and fragmentation chromatin were dying. The prepared curve has became the basis to draw up the digital method for detection and determination of the number of living, dying, and dead cells in an in planta system and revealed that ACC induced death in about 20% of root cortex cells. This process was accompanied by increase in ion leakage, shortening of cells and whole roots, as well as by increase in weight and width of the apical part of roots and appearance of few aerenchymatic spaces while not by internucleosomal DNA degradation.

High-Performance Computational Analysis of Glioblastoma Pathology Images with Database Support Identifies Molecular and Survival Correlates.

PubMed

Kong, Jun; Wang, Fusheng; Teodoro, George; Cooper, Lee; Moreno, Carlos S; Kurc, Tahsin; Pan, Tony; Saltz, Joel; Brat, Daniel

2013-12-01

In this paper, we present a novel framework for microscopic image analysis of nuclei, data management, and high performance computation to support translational research involving nuclear morphometry features, molecular data, and clinical outcomes. Our image analysis pipeline consists of nuclei segmentation and feature computation facilitated by high performance computing with coordinated execution in multi-core CPUs and Graphical Processor Units (GPUs). All data derived from image analysis are managed in a spatial relational database supporting highly efficient scientific queries. We applied our image analysis workflow to 159 glioblastomas (GBM) from The Cancer Genome Atlas dataset. With integrative studies, we found statistics of four specific nuclear features were significantly associated with patient survival. Additionally, we correlated nuclear features with molecular data and found interesting results that support pathologic domain knowledge. We found that Proneural subtype GBMs had the smallest mean of nuclear Eccentricity and the largest mean of nuclear Extent, and MinorAxisLength. We also found gene expressions of stem cell marker MYC and cell proliferation maker MKI67 were correlated with nuclear features. To complement and inform pathologists of relevant diagnostic features, we queried the most representative nuclear instances from each patient population based on genetic and transcriptional classes. Our results demonstrate that specific nuclear features carry prognostic significance and associations with transcriptional and genetic classes, highlighting the potential of high throughput pathology image analysis as a complementary approach to human-based review and translational research.

76 FR 63702 - In the Matter of the Designation of Conspiracy of Fire Nuclei, aka Conspiracy of the Nuclei of...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-13

... DEPARTMENT OF STATE [Public Notice: 7643] In the Matter of the Designation of Conspiracy of Fire Nuclei, aka Conspiracy of the Nuclei of Fire, aka Conspiracy of Cells of Fire, aka Synomosia of Pyrinon Tis Fotias, aka Thessaloniki-Athens Fire Nuclei Conspiracy, as a Specially Designated Global Terrorist...

Optimized Methods for the Isolation of Arabidopsis Female Central Cells and Their Nuclei

PubMed Central

Park, Kyunghyuk; Frost, Jennifer M.; Adair, Adam James; Kim, Dong Min; Yun, Hyein; Brooks, Janie S.; Fischer, Robert L.; Choi, Yeonhee

2016-01-01

The Arabidopsis female gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations of Arabidopsis central cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dissection followed by the derivation of central cell protoplasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75–90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights into Arabidopsis reproduction. PMID:27788573

A photoreceptor calcium binding protein is recognized by autoantibodies obtained from patients with cancer-associated retinopathy

PubMed Central

1991-01-01

Cancer-associated retinopathy (CAR), a paraneoplastic syndrome, is characterized by the degeneration of retinal photoreceptors under conditions where the tumor and its metastases have not invaded the eye. The retinopathy often is apparent before the diagnosis of cancer and may be associated with autoantibodies that react with specific sites in the retina. We have examined the sera from patients with CAR to further characterize the retinal antigen. Western blot analysis of human retinal proteins reveals a prominent band at 26 kD that is labeled by the CAR antisera. Antibodies to the 26-kD protein were affinity- purified from complex CAR antisera and used for EM-immunocytochemical localization of the protein to the nuclei, inner and outer segments of both rod and cone cells. Other antibodies obtained from the CAR sera did not label photoreceptors. Using the affinity-purified antibodies for detection, the 26-kD protein, designated p26, was purified to homogeneity from the outer segments of bovine rod photoreceptor cells by Phenyl-Sepharose and ion exchange chromatography. Partial amino acid sequence of p26 was determined by gas phase Edman degradation and revealed extensive homology with a cone-specific protein, visinin. Based upon structural relatedness, both the p26 rod protein and visinin are members of the calmodulin family and contain calcium binding domains of the E-F hand structure. PMID:1999465

Nuclear CD38 in retinoic acid-induced HL-60 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yalcintepe, Leman; Albeniz, Isil; Adin-Cinar, Suzan

2005-02-01

The cell surface antigen, CD38, is a 45-kDa transmembrane protein which is predominantly expressed on hematopoietic cells during differentiation. As a bifunctional ectoenzyme, it catalyzes the synthesis of cyclic ADP-ribose (cADPR) from NAD{sup +} and hydrolysis of either NAD{sup +} or cADPR to ADP-ribose. All-trans-retinoic acid (RA) is a potent and specific inducer of CD38 in myeloid cells. In this report, we demonstrate that the nuclei of RA-treated human HL-60 myeloblastic cells reveal enzymatic activities inherent to CD38. Thus, GDP-ribosyl cyclase and NAD{sup +} glycohydrolase activities in the nuclear fraction increased very significantly in response to incubation with RA. Withmore » Western blotting, we detected in the nuclear protein fraction from RA-treated cells a {approx}43-kDa protein band which was reactive with the CD38-specific monoclonal antibody OKT10. The expression of CD38 in HL-60 nuclei was also shown with FACScan analysis. RA treatment gave rise to an increase in in vitro ADP ribosylation of the {approx}43-kDa nuclear protein. Moreover, nuclei isolated from RA-treated HL-60 cells revealed calcium release in response to cADPR, whereas a similar response was not observed in control nuclei. These results suggest that CD38 is expressed in HL-60 cell nuclei during RA-induced differentiation.« less

Rational variety mapping for contrast-enhanced nonlinear unsupervised segmentation of multispectral images of unstained specimen.

PubMed

Kopriva, Ivica; Hadžija, Mirko; Popović Hadžija, Marijana; Korolija, Marina; Cichocki, Andrzej

2011-08-01

A methodology is proposed for nonlinear contrast-enhanced unsupervised segmentation of multispectral (color) microscopy images of principally unstained specimens. The methodology exploits spectral diversity and spatial sparseness to find anatomical differences between materials (cells, nuclei, and background) present in the image. It consists of rth-order rational variety mapping (RVM) followed by matrix/tensor factorization. Sparseness constraint implies duality between nonlinear unsupervised segmentation and multiclass pattern assignment problems. Classes not linearly separable in the original input space become separable with high probability in the higher-dimensional mapped space. Hence, RVM mapping has two advantages: it takes implicitly into account nonlinearities present in the image (ie, they are not required to be known) and it increases spectral diversity (ie, contrast) between materials, due to increased dimensionality of the mapped space. This is expected to improve performance of systems for automated classification and analysis of microscopic histopathological images. The methodology was validated using RVM of the second and third orders of the experimental multispectral microscopy images of unstained sciatic nerve fibers (nervus ischiadicus) and of unstained white pulp in the spleen tissue, compared with a manually defined ground truth labeled by two trained pathophysiologists. The methodology can also be useful for additional contrast enhancement of images of stained specimens. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

[Some morphometric parameters of nucleoli and nuclei in invasive ductal breast carcinomas in women].

PubMed

Karpinska-Kaczmarczyk, Katarzyna

2009-01-01

The purpose of this study was to correlate seven morphometric parameters of nucleoli and nuclei of invasive ductal cancer cells with some clinico-pathological factors such as age, tumor size, axillary lymph node status, MIB-1 proliferation index, and estrogen receptor expression in tumor cells. Methyl green-pyronin Y (MG-PY) was used for simultaneous staining of nuclei and nucleoli in histological sections of 150 invasive ductal breast carcinomas. Next, morphometric parameters of nucleoli and nuclei of tumor cells were measured with computerized image analysis. Nuclear area and number of nucleoli in breast tumor cells were greater in younger axillary node-negative patients. The number of nucleoli and nucleolar shape polymorphism were reduced in tumors measuring 20 mm or less or with lower histological grade. Nuclear area, nucleolar number, and nucleolar polymorphism in carcinomas with low proliferation index and estrogen receptor expression were smaller than in carcinomas with high proliferation index and no estrogen receptor expression. Nucleolar area in primary tumors without axillary node involvement was greater than in tumors with more than three axillary nodes positive. MG-PY selectively and simultaneously stains nucleoli and nuclei of tumor cells enabling standardized and reproducible examination of these structures with computerized image analysis. Univariate statistical analysis disclosed that some morphometric parameters of nucleoli and nuclei of tumor cells correlated with several established clinico-pathological prognostic factors. Therefore, the prognostic significance of these parameters should be studied in a larger group of patients with invasive ductal breast carcinomas.

Differentiation of sCJD and vCJD forms by automated analysis of basal ganglia intensity distribution in multisequence MRI of the brain--definition and evaluation of new MRI-based ratios.

PubMed

Linguraru, Marius George; Ayache, Nicholas; Bardinet, Eric; Ballester, Miguel Angel González; Galanaud, Damien; Haïk, Stéphane; Faucheux, Baptiste; Hauw, Jean-Jacques; Cozzone, Patrick; Dormont, Didier; Brandel, Jean-Philippe

2006-08-01

We present a method for the analysis of basal ganglia (including the thalamus) for accurate detection of human spongiform encephalopathy in multisequence magnetic resonance imaging (MRI) of the brain. One common feature of most forms of prion protein diseases is the appearance of hyperintensities in the deep grey matter area of the brain in T2-weighted magnetic resonance (MR) images. We employ T1, T2, and Flair-T2 MR sequences for the detection of intensity deviations in the internal nuclei. First, the MR data are registered to a probabilistic atlas and normalized in intensity. Then smoothing is applied with edge enhancement. The segmentation of hyperintensities is performed using a model of the human visual system. For more accurate results, a priori anatomical data from a segmented atlas are employed to refine the registration and remove false positives. The results are robust over the patient data and in accordance with the clinical ground truth. Our method further allows the quantification of intensity distributions in basal ganglia. The caudate nuclei are highlighted as main areas of diagnosis of sporadic Creutzfeldt-Jakob Disease (sCJD), in agreement with the histological data. The algorithm permitted the classification of the intensities of abnormal signals in sCJD patient FLAIR images with a higher hypersignal in caudate nuclei (10/10) and putamen (6/10) than in thalami. Defining normalized MRI measures of the intensity relations between the internal grey nuclei of patients, we robustly differentiate sCJD and variant CJD (vCJD) patients, in an attempt to create an automatic classification tool of human spongiform encephalopathies.

Spiral Ganglion Neuron Projection Development to the Hindbrain in Mice Lacking Peripheral and/or Central Target Differentiation

PubMed Central

Elliott, Karen L.; Kersigo, Jennifer; Pan, Ning; Jahan, Israt; Fritzsch, Bernd

2017-01-01

We investigate the importance of the degree of peripheral or central target differentiation for mouse auditory afferent navigation to the organ of Corti and auditory nuclei in three different mouse models: first, a mouse in which the differentiation of hair cells, but not central auditory nuclei neurons is compromised (Atoh1-cre; Atoh1f/f); second, a mouse in which hair cell defects are combined with a delayed defect in central auditory nuclei neurons (Pax2-cre; Atoh1f/f), and third, a mouse in which both hair cells and central auditory nuclei are absent (Atoh1−/−). Our results show that neither differentiated peripheral nor the central target cells of inner ear afferents are needed (hair cells, cochlear nucleus neurons) for segregation of vestibular and cochlear afferents within the hindbrain and some degree of base to apex segregation of cochlear afferents. These data suggest that inner ear spiral ganglion neuron processes may predominantly rely on temporally and spatially distinct molecular cues in the region of the targets rather than interaction with differentiated target cells for a crude topological organization. These developmental data imply that auditory neuron navigation properties may have evolved before auditory nuclei. PMID:28450830

Nuclear incorporation of iron during the eukaryotic cell cycle

DOE PAGES

Robinson, Ian; Yang, Yang; Zhang, Fucai; ...

2016-10-18

Scanning X-ray fluorescence microscopy has been used to probe the distribution of S, P and Fe within cell nuclei. Nuclei, which may have originated at different phases of the cell cycle, are found to show very different levels of Fe present with a strongly inhomogeneous distribution. P and S signals, presumably from DNA and associated nucleosomes, are high and relatively uniform across all the nuclei; these agree with X-ray phase contrast projection microscopy images of the same samples. Finally, we discuss possible reasons for the Fe incorporation.

[Effect of a glutamate and glutamine excess on the nucleic acid content of the spleen cell nuclei in rats].

PubMed

Vorontsova, E N; Okunev, V N

1976-01-01

In tests conducted with albino rats subject to investigation was the effect of sodium glutamate, or glutamine, daily introduced into the stomach in doses of 300 and 150 mg/kg, on the nucleic acids content in the splenic cell nuclei. All the animals taken in the experiment demonstrated a clearcut quantity of nucleonic RNA. By using a maximum dose of sodium glutamate and minimal one of glutamine a rise in the amount of DNA occurs in the nuclei of the splenic cells.

Fate of 3H-thymidine labelled myogenic cells in regeneration of muscle isografts.

PubMed

Gutmann, E; Mares, V; Stichová, J

1976-03-05

Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with 3H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later. In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The presen experiments provide a direct proof of utilization of donor satelite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process.

Role of Caspase-3 in acute light damage to retina of rats.

PubMed

Wang, Xiao; Hu, Shi-Xing; Li, Wei; Lin, Shao-Chun

2007-03-01

To investigate the role of Caspase-3 in retinal damage caused by light exposure in rats. Light injury to retina was induced by persistent exposure to illumination (intensity: 30 000 +/- 50 lux) of operating microscope for 30 minutes in the right eyes of Sprague-Dawley rats. The pathological changes of retina were observed under optical and electron microscopies at different time points, which were 6 hours, 1, 3, 7, and 15 days after the light exposure. Apoptosis of retinal cells was analyzed by flow cytometry. The activity of Caspase-3 was evaluated by using the Caspase-3 assay kit. At the same time, the expression of Caspase-3 protease was determined with Western blot analysis. The examination results of optical and transmission electron microscopes showed that edema of inner and outer segments of the retina, especially the chondriosome inside the inner segment, became obvious 6 hours after the light exposure. The change was deteriorated along with the increasing time. The structures of the discoidal valve dissociated in the outer segment simultaneously. Disorderly arranged nuclei, karyopycnosis, and thinning in the outer nuclear layer were observed. The retinal pigment epithelium almost disappeared during the later stage. The staining results of Annexin-V combined with PI demonstrated that the proportion of apoptotic cells increased with time. The proportion between 7th day (82.7%) and 15th day (80.4%), however, showed no significant difference. Caspase-3 became remarkably active with the lapse of time, which increased from 0.02 at 6th hour to the peak of 9.8 at 7th day before it started to descend. The Western blot detected a expression of the active form of Caspase-3 at 7th day and 15th day. Apoptosis of photoreceptor cells is markedly involved in the light damage and Caspase-3 protease may play an important role in the apoptotic process of the retina after light exposure in rats.

Distribution of CGRP in the minipig brainstem.

PubMed

Lisardo Sánchez, Manuel; Vecino, Elena; Coveñas, Rafael

2014-05-01

For the first time, an in-depth study has been made of the distribution of fibers and cell bodies containing calcitonin gene-related peptide (CGRP) in the minipig brainstem using an indirect immunoperoxidase technique. The animals studied were not treated with colchicine. Cell bodies containing CGRP were found in 20 nuclei/regions of the brainstem. These perikarya were located in somatomotor, brachiomotor and raphae nuclei, nucleus ambiguus, substantia nigra, nucleus reticularis tegmenti pontis, nucleus prepositus hypoglossi, nuclei olivaris inferior and superior, nuclei pontis, formatio reticularis, nucleus dorsalis tegmenti of Gudden, and in the nucleus reticularis lateralis. Fourteen of the 20 brainstem nuclei showed a high density of immunoreactive cell bodies. In comparison with other species, the minipig, together with the rat, show the most widespread distribution of cell bodies containing CGRP in the mammalian brainstem. Immunoreactive fibers were also observed in the brainstem. However, in the minipig brainstem the density of these fibers is low, as in many brainstem nuclei only single immunoreactive fibers were observed. A high density of immunoreactive fibers was only observed in the pars caudalis of the nucleus tractus spinalis nervi trigemini and in the nucleus ventralis tegmenti of Gudden. According to the observed anatomical distribution of the immunoreactive structures containing CGRP, the peptide could be involved in motor, somatosensory, gustative, and autonomic mechanisms. Copyright © 2014 Wiley Periodicals, Inc.

Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos.

PubMed

Giurumescu, Claudiu A; Kang, Sukryool; Planchon, Thomas A; Betzig, Eric; Bloomekatz, Joshua; Yelon, Deborah; Cosman, Pamela; Chisholm, Andrew D

2012-11-01

A quantitative understanding of tissue morphogenesis requires description of the movements of individual cells in space and over time. In transparent embryos, such as C. elegans, fluorescently labeled nuclei can be imaged in three-dimensional time-lapse (4D) movies and automatically tracked through early cleavage divisions up to ~350 nuclei. A similar analysis of later stages of C. elegans development has been challenging owing to the increased error rates of automated tracking of large numbers of densely packed nuclei. We present Nucleitracker4D, a freely available software solution for tracking nuclei in complex embryos that integrates automated tracking of nuclei in local searches with manual curation. Using these methods, we have been able to track >99% of all nuclei generated in the C. elegans embryo. Our analysis reveals that ventral enclosure of the epidermis is accompanied by complex coordinated migration of the neuronal substrate. We can efficiently track large numbers of migrating nuclei in 4D movies of zebrafish cardiac morphogenesis, suggesting that this approach is generally useful in situations in which the number, packing or dynamics of nuclei present challenges for automated tracking.

Automatic brain caudate nuclei segmentation and classification in diagnostic of Attention-Deficit/Hyperactivity Disorder.

PubMed

Igual, Laura; Soliva, Joan Carles; Escalera, Sergio; Gimeno, Roger; Vilarroya, Oscar; Radeva, Petia

2012-12-01

We present a fully automatic diagnostic imaging test for Attention-Deficit/Hyperactivity Disorder diagnosis assistance based on previously found evidences of caudate nucleus volumetric abnormalities. The proposed method consists of different steps: a new automatic method for external and internal segmentation of caudate based on Machine Learning methodologies; the definition of a set of new volume relation features, 3D Dissociated Dipoles, used for caudate representation and classification. We separately validate the contributions using real data from a pediatric population and show precise internal caudate segmentation and discrimination power of the diagnostic test, showing significant performance improvements in comparison to other state-of-the-art methods. Copyright © 2012 Elsevier Ltd. All rights reserved.

Pituitary adenylate cyclase-activating peptide in the rat central nervous system: an immunohistochemical and in situ hybridization study.

PubMed

Hannibal, Jens

2002-11-25

In the present study the localization of pituitary adenylate cyclase-activating peptide (PACAP)-expressing cell bodies and PACAP projections were mapped in the adult rat brain and spinal cord by using immunohistochemistry and in situ hybridization histochemistry. A widespread occurrence of PACAP-containing cell bodies was found, with the greatest accumulation in several hypothalamic nuclei and in several brainstem nuclei, especially the habenular nuclei, the pontine nucleus, the lateral parabrachial nucleus (LPB), and the vagal complex. PACAP was also present in cell bodies in the olfactory areas, in neocortical areas, in the hippocampus, in the vestibulo- and cochlear nuclei, in cell bodies of the intermediolateral cell column of the spinal cord and in Purkinje cells of the cerebellum, in the subfornical organ, and in the organum vasculosum of the lamina terminalis. An intense accumulation of PACAP-immunoreactive (-IR) nerve fibers was observed throughout the hypothalamus, in the amydaloid and extended amygdaloid complex, in the anterior and paraventricular thalamic nuclei, in the intergeniculate leaflet, in the pretectum, and in several brainstem nuclei, such as the parabrachial nucleus, the sensory trigeminal nucleus, and the nucleus of the solitary tract. PACAP-IR nerve fibers were also found in the area postrema, the posterior pituitary and the choroid plexus, and the dorsal and ventral horn of the spinal cord. The widespread distribution of PACAP in the brain and spinal cord suggests that PACAP is involved in the control of many autonomic and sensory functions as well as higher cortical processes. Copyright 2002 Wiley-Liss, Inc.

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells.

PubMed

Ono, Yukiko; Kono, Tomohiro

2006-08-01

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.

Does flat epithelial atypia have rounder nuclei than columnar cell change/hyperplasia? A morphometric approach to columnar cell lesions of the breast.

PubMed

Yamashita, Yoshiko; Ichihara, Shu; Moritani, Suzuko; Yoon, Han-Seung; Yamaguchi, Masahiro

2016-06-01

Columnar cell lesions of the breast encompass columnar cell change/hyperplasia (CCC/CCH) and flat epithelial atypia (FEA). These have attracted researchers because emerging data suggest that FEA may represent the earliest histologically detectable non-obligate precursor of breast cancer. However, it is occasionally difficult to distinguish FEA from CCC/CCH because of similar histology. Although the nuclei of FEA are frequently described as relatively round compared with those of CCC/CCH, there are few morphometric studies to support this statement. The aim of this study was to provide objective data as to the nuclear shape in columnar cell lesions. As a shape descriptor, we adopted ellipticity that is defined by the formula 2b/2a, where a is the length of the long axis of the ellipse and b is the length of the short axis. Contrary to circularity, ellipticity reflects the overall configuration of an ellipse irrespective of surface irregularity. Our image analysis included generating whole slide images, extracting glandular cell nuclei, measuring nuclear ellipticity, and superimposing graded colors based on execution of results on the captured images. A total of 7917 nuclei extracted from 22 FEA images and 5010 nuclei extracted from 13 CCC/CCH images were analyzed. There was a significant difference in nuclear roundness between FEA and CCC/CCH with mean ellipticity values of 0.723 and 0.679, respectively (p < 0.001, Welch's t test). Furthermore, FEA with malignancy had significantly rounder nuclei than FEA without malignancy (p < 0.001). Our preliminary results suggest that nuclear ellipticity is a key parameter in reproducibly classifying columnar cell lesions of the breast.

Computer vision approach to morphometric feature analysis of basal cell nuclei for evaluating malignant potentiality of oral submucous fibrosis.

PubMed

Muthu Rama Krishnan, M; Pal, Mousumi; Paul, Ranjan Rashmi; Chakraborty, Chandan; Chatterjee, Jyotirmoy; Ray, Ajoy K

2012-06-01

This research work presents a quantitative approach for analysis of histomorphometric features of the basal cell nuclei in respect to their size, shape and intensity of staining, from surface epithelium of Oral Submucous Fibrosis showing dysplasia (OSFD) to that of the Normal Oral Mucosa (NOM). For all biological activity, the basal cells of the surface epithelium form the proliferative compartment and therefore their morphometric changes will spell the intricate biological behavior pertaining to normal cellular functions as well as in premalignant and malignant status. In view of this, the changes in shape, size and intensity of staining of the nuclei in the basal cell layer of the NOM and OSFD have been studied. Geometric, Zernike moments and Fourier descriptor (FD) based as well as intensity based features are extracted for histomorphometric pattern analysis of the nuclei. All these features are statistically analyzed along with 3D visualization in order to discriminate the groups. Results showed increase in the dimensions (area and perimeter), shape parameters and decreasing mean nuclei intensity of the nuclei in OSFD in respect to NOM. Further, the selected features are fed to the Bayesian classifier to discriminate normal and OSFD. The morphometric and intensity features provide a good sensitivity of 100%, specificity of 98.53% and positive predicative accuracy of 97.35%. This comparative quantitative characterization of basal cell nuclei will be of immense help for oral onco-pathologists, researchers and clinicians to assess the biological behavior of OSFD, specially relating to their premalignant and malignant potentiality. As a future direction more extensive study involving more number of disease subjects is observed.

Identification of a nuclear-localized nuclease from wheat cells undergoing programmed cell death that is able to trigger DNA fragmentation and apoptotic morphology on nuclei from human cells

PubMed Central

Domínguez, Fernando; Cejudo, Francisco J.

2006-01-01

PCD (programmed cell death) in plants presents important morphological and biochemical differences compared with apoptosis in animal cells. This raises the question of whether PCD arose independently or from a common ancestor in plants and animals. In the present study we describe a cell-free system, using wheat grain nucellar cells undergoing PCD, to analyse nucleus dismantling, the final stage of PCD. We have identified a Ca2+/Mg2+ nuclease and a serine protease localized to the nucleus of dying nucellar cells. Nuclear extracts from nucellar cells undergoing PCD triggered DNA fragmentation and other apoptotic morphology in nuclei from different plant tissues. Inhibition of the serine protease did not affect DNA laddering. Furthermore, we show that the nuclear extracts from plant cells triggered DNA fragmentation and apoptotic morphology in nuclei from human cells. The inhibition of the nucleolytic activity with Zn2+ or EDTA blocked the morphological changes of the nucleus. Moreover, nuclear extracts from apoptotic human cells triggered DNA fragmentation and apoptotic morphology in nuclei from plant cells. These results show that degradation of the nucleus is morphologically and biochemically similar in plant and animal cells. The implication of this finding on the origin of PCD in plants and animals is discussed. PMID:16613587

Nigrothalamic projections in the monkey demonstrated by autoradiographic technics.

PubMed

Carpenter, M B; Nakano, K; Kim, R

1976-02-15

In spite of repeated demonstrations by degeneration technics, nigrothalamic fibers have been regarded with some skepticism. Attempts were made to trace nigral efferent projections in the monkey by autoradiographic technics. Tritiated amino acids (L-leucine, L-lysine and L-proline), injected into portions of the substantia nigra (SN), labeled cells in four regions, designated as, (1) rostrolateral, (2) caudolateral, (3) rostromedial and (4) central. Rostrolateral nigral neurons transported radioactive label preferentially and abundantly to thalamic nuclei; localized isotope was found in parts of three thalamic nuclei, the medial part of the ventral lateral nucleus (VLm), the magnocellular part of the ventral anterior nucleus (VAmc), and the paralaminar part of the dorsomedial nucleus (DMpl)9 Lateral neurons in the caudal half of the SN transmitted radioactive label to the same thalamic nuclei as rostrolateral nigral neuron. Isotope transported to portions of the striatum was modest and localized. Radioactive label taken up by large cells in the caudal third of the SN was transported to portions of the striatum, but not to thalamic nuclei. Labeled nigral neurons in the medial two-thirds of the rostral half of the SN, and in the middle third of the central part of the SN, transported the label mainly to parts of the caudate nucleus and putamen. In these animals modest radioactive label was seen in VLm and VAmc, but not in other thalamic nuclei. There was no evidence that nigral neurons project to the subthalamic nucleus. No radioactive transport from nigral neurons was detected in the superior colliculus, the midbrain tegmentum, or the red nucleus, and none was transported to more caudal brain stem nuclei. Nigrothalamic fibers arise particularly from cells in rostral and lateral parts of the substantia nigra. While some cells in other parts of the nigra project to thalamic nuclei, these appear scattered and less numerous. Large cells in caudal parts of the SN do not project to thalamic nuclei. These observations confirm nigrothalamic projections to VLm and VAmc, and identify a new nigral projection to part of the dorsomedial nucleus of the thalamus (DMpl). No nigral efferent fibers project to any of the intralaminar thalamic nuclei.

The synaptinemal complex in Rhoeo spathacea.

PubMed

McQuade, H A; Wells, B

1975-03-01

The synaptinemal complex in meiocytes of Rhoeo spathacea is described. Unpaired zygotene chromosomes do not exhibit well defined axial cores under the ordinary fixations of electron microscopy and appear diffuse. However, the axial core is defined by ethanolic phosphotungstic acid (PTA) although it does not respond to uranyl-EDTA-lead. Thus the core appears to contain histone but not RNA and presents a condition which is modified later in pairing when lateral elements of the synaptinemal complex respond positively to both tests. The total number of attachments of synaptinemal complexes to the nuclear envelope was determined in several nuclei from serial sections. Eleven of the twelve possible attachments were found in one nucleus. It thus seems certain that all must be so attached. In the same manner all chromosomes can be seen to have an attachment to a chromocentre. Chromocentres are often very large and compound in that two kinds of heterochromatin can be distinguished. These states of chromatin within the chromocentre are considered to be a function of the degree of condensation. Segments of synaptinemal complexes are distributed randomly through sections of pachytene nuclei and long uncoiled segments of complexes are frequently found in or near the centres of median nuclear sections. Synaptinemal complexes are also found in chromocentres. Our findings suggest that on completion of pairing, which begins distally, homologous chromosomes in Rhoeo are paired throughout their entire lengths, rather than in small terminal segments only.

Chromatin remodeling in somatic cells injected into mature pig oocytes.

PubMed

Bui, Hong-Thuy; Van Thuan, Nguyen; Wakayama, Teruhiko; Miyano, Takashi

2006-06-01

We examined the involvement of histone H3 modifications in the chromosome condensation and decondensation of somatic cell nuclei injected into mature pig oocytes. Nuclei of pig granulosa cells were transferred into in vitro matured intact pig oocytes, and histone H3 phosphorylation, acetylation, and methylation were examined by immunostaining with specific antibodies in relation to changes in chromosome morphology. In the condensed chromosomes of pig oocytes at metaphase II, histone H3 was phosphorylated at serine 10 (H3-S10) and serine 28 (H3-S28), and methylated at lysine 9 (H3-K9), but was not acetylated at lysine 9, 14 and 18 (H3-K9, H3-K14 and H3-K18). During the first 2 h after nuclear transfer, a series of events were observed in the somatic nuclei: nuclear membrane disassembly; chromosome condensation to form a metaphase-like configuration; an increase in histone H3 phosphorylation levels (H3-S10 and H3-S28). Next, pig oocytes injected with nuclei of somatic cells were electroactivated and the chromosome morphology of oocytes and somatic cells was examined along with histone modifications. Generally, chromosomes of the somatic cells showed a similar progression of cell cycle stage to that of oocytes, through anaphase II- and telophase II-like stages then formed pronucleus-like structures, although the morphology of the spindles differed from that of oocyte spindles. The chromosomes of somatic cells also showed changes in histone H3 dephosphorylation and reacetylation, similar to oocytes. In contrast, histone H3 methylation (H3-K9) of somatic cell nuclei did not show any significant change after injection and electroactivation of the oocytes. These results suggest that nuclear remodeling including histone H3 phosphorylation and acetylation of injected somatic nuclei took place in the oocytes under regulation by the oocyte cytoplasm.

Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus.

PubMed

Poncelet, Luc; Garigliany, Mutien; Ando, Kunie; Franssen, Mathieu; Desmecht, Daniel; Brion, Jean-Pierre

2016-12-16

The cell cycle-associated neuronal death hypothesis, which has been proposed as a common mechanism for most neurodegenerative diseases, is notably supported by evidencing cell cycle effectors in neurons. However, in naturally occurring nervous system diseases, these markers are not expressed in neuron nuclei but in cytoplasmic compartments. In other respects, the Feline Panleukopenia Virus (FPV) is able to complete its cycle in mature brain neurons in the feline species. As a parvovirus, the FPV is strictly dependent on its host cell reaching the cell cycle S phase to start its multiplication. In this retrospective study on the whole brain of 12 cats with naturally-occurring, FPV-associated cerebellar atrophy, VP2 capsid protein expression was detected by immunostaining not only in some brain neuronal nuclei but also in neuronal cytoplasm in 2 cats, suggesting that viral mRNA translation was still occurring. In these cats, double immunostainings demonstrated the expression of cell cycle S phase markers cyclin A, cdk2 and PCNA in neuronal nuclei. Parvoviruses are able to maintain their host cells in S phase by triggering the DNA damage response. S139 phospho H2A1, a key player in the cell cycle arrest, was detected in some neuronal nuclei, supporting that infected neurons were also blocked into the S phase. PCR studies did not support a co-infection with an adeno or herpes virus. ERK1/2 nuclear accumulation was observed in some neurons suggesting that the ERK signaling pathway might be involved as a mechanism driving these neurons far into the cell cycle.

Are gadolinium contrast agents suitable for gadolinium neutron capture therapy?

PubMed

De Stasio, Gelsomina; Rajesh, Deepika; Casalbore, Patrizia; Daniels, Matthew J; Erhardt, Robert J; Frazer, Bradley H; Wiese, Lisa M; Richter, Katherine L; Sonderegger, Brandon R; Gilbert, Benjamin; Schaub, Sebastien; Cannara, Rachel J; Crawford, John F; Gilles, Mary K; Tyliszczak, Tolek; Fowler, John F; Larocca, Luigi M; Howard, Steven P; Mercanti, Delio; Mehta, Minesh P; Pallini, Roberto

2005-06-01

Gadolinium neutron capture therapy (GdNCT) is a potential treatment for malignant tumors based on two steps: (1) injection of a tumor-specific (157)Gd compound; (2) tumor irradiation with thermal neutrons. The GdNC reaction can induce cell death provided that Gd is proximate to DNA. Here, we studied the nuclear uptake of Gd by glioblastoma (GBM) tumor cells after treatment with two Gd compounds commonly used for magnetic resonance imaging, to evaluate their potential as GdNCT agents. Using synchrotron X-ray spectromicroscopy, we analyzed the Gd distribution at the subcellular level in: (1) human cultured GBM cells exposed to Gd-DTPA or Gd-DOTA for 0-72 hours; (2) intracerebrally implanted C6 glioma tumors in rats injected with one or two doses of Gd-DOTA, and (3) tumor samples from GBM patients injected with Gd-DTPA. In cell cultures, Gd-DTPA and Gd-DOTA were found in 84% and 56% of the cell nuclei, respectively. In rat tumors, Gd penetrated the nuclei of 47% and 85% of the tumor cells, after single and double injection of Gd-DOTA, respectively. In contrast, in human GBM tumors 6.1% of the cell nuclei contained Gd-DTPA. Efficacy of Gd-DTPA and Gd-DOTA as GdNCT agents is predicted to be low, due to the insufficient number of tumor cell nuclei incorporating Gd. Although multiple administration schedules in vivo might induce Gd penetration into more tumor cell nuclei, a search for new Gd compounds with higher nuclear affinity is warranted before planning GdNCT in animal models or clinical trials.

Morphology and histology of the digestive system of the vector leafhopper Psammotettix striatus (L.) (Hemiptera: Cicadellidae).

PubMed

Zhang, Fangmei; Zhang, Chunni; Dai, Wu; Zhang, Yalin

2012-06-01

The vector leafhopper Psammotettix striatus (L.) (Hemiptera: Cicadellidae) is an important economic pest that is a serious threat to wheat in Northwest China, causing great losses to agricultural production by transmitting wheat blue dwarf (WBD) in a persistent circulative manner. Studies on morphology and ultrastructure of the digestive system were made using light, scanning and transmission electron microscopy. The gross morphology of the alimentary canal consists of esophagus (foregut), midgut, hindgut, and special filter chamber. The esophagus, a narrow and slender tube, runs through the whole thorax extending into the filter chamber and connecting with the anterior midgut. The midgut consists of three major regions, anterior, middle and posterior midgut. The anterior midgut is composed of exceptionally thick epithelial cells surrounding a large lumen. Numerous well-developed microvilli occur at the basal region of the epithelium. A large number of electron-lucent and lipid-like vesicles are observed under the microvilli. The posterior midgut is smaller than the anterior midgut in diameter. Numerous different concentric circular bodies are characteristic of the posterior midgut. The hindgut arises from the posterior midgut at the boundary of the filter chamber. It extends downward toward the anus where the hindgut enlarges to form the rectum. The rectum is formed by various cells typical of epithelium, whose nuclei are relatively smaller than those of the midgut. Relatively large muscle cells are present at the well-defined and thick basement membrane. There are two pairs of Malpighian tubules. Numerous mitochondria and lamellar rough endoplasmic reticulum in the cells of the anterior segment of the Malpighian tubules; the sub-anterior segment of the tubules resemble the distal segment which are wave-like, amount vesicles in the cells; numerous tightly packed large vesicles contain two types of brochosomes in the inflated segment of the Malpighian tubules. The salivary system of P. striatus contains one pair of salivary glands, which are made up of the principal and accessory gland. The principal glands are divided into anterior, mid- and posterior lobes. About nineteen acini are observed in the principal glands of P. striatus, and vary in structure and colour. Five cellular types are found in the principal glands by transmission electron microscopy, which are rich in secretory granules, modified in size, electron-dense and shape. Rough endoplasmic reticulum in the shape of vesicular and lamellar, mitochondria and Golgi complexes are observed. The accessory gland has only an oval or a rod-shaped acinus. Copyright © 2012 Elsevier Ltd. All rights reserved.

Estradiol-promoted accumulation of receptor in nuclei of porcine endometrium cells. Immunogold electron microscopy of resting and estradiol-stimulated cells.

PubMed

Sierralta, W D; Jakob, F; Thole, H; Engel, P; Jungblut, P W

1992-01-01

Endometrium was collected by curettage from castrated pigs, either untreated or exposed to estradiol in vivo by intrauterine injection, and processed for electron microscopy. The resin LR Gold was used for embedding, and sections were floated on droplets of 10 nm diameter gold particles, coated with the immunoglobulin-G1 (IgG1) fraction or its Fab2 fragment of a monospecific polyclonal antiserum raised in goats against the C-terminal half of the estradiol receptor. On average, only one gold particle per microns 2 became attached in the cytoplasmic area of untreated cells, whereas four were found over the nuclear area. These figures rose to 2-3/microns 2 and 15-26/microns 2, respectively, within 10 min after exposure to estradiol. The labeling intensities of nuclei in cell clusters and of coprocessed nuclei released from cells ruptured during curettage were identical in all situations. Nuclear pores were frequently tagged after estradiol treatment. The proportions of tagging densities in nuclei of untreated and estradiol-exposed cells corresponded to those of receptor contents measured in extracts of isolated nuclei by ligand binding. This correlation was not seen for the cytoplasmic compartment of untreated cells, the scarce tagging of which is interpreted by hidden antigenic determinants. Our morphological analyses support the conclusions drawn from biochemical data (Sierralta et al., 1992) of an estradiol-promoted translocation of receptor from the cytoplasm into the nucleus.

Automatic Detection of Mitosis and Nuclei From Cytogenetic Images by CellProfiler Software for Mitotic Index Estimation.

PubMed

González, Jorge Ernesto; Radl, Analía; Romero, Ivonne; Barquinero, Joan Francesc; García, Omar; Di Giorgio, Marina

2016-12-01

Mitotic Index (MI) estimation expressed as percentage of mitosis plays an important role as quality control endpoint. To this end, MI is applied to check the lot of media and reagents to be used throughout the assay and also to check cellular viability after blood sample shipping, indicating satisfactory/unsatisfactory conditions for the progression of cell culture. The objective of this paper was to apply the CellProfiler open-source software for automatic detection of mitotic and nuclei figures from digitized images of cultured human lymphocytes for MI assessment, and to compare its performance to that performed through semi-automatic and visual detection. Lymphocytes were irradiated and cultured for mitosis detection. Sets of images from cultures were analyzed visually and findings were compared with those using CellProfiler software. The CellProfiler pipeline includes the detection of nuclei and mitosis with 80% sensitivity and more than 99% specificity. We conclude that CellProfiler is a reliable tool for counting mitosis and nuclei from cytogenetic images, saves considerable time compared to manual operation and reduces the variability derived from the scoring criteria of different scorers. The CellProfiler automated pipeline achieves good agreement with visual counting workflow, i.e. it allows fully automated mitotic and nuclei scoring in cytogenetic images yielding reliable information with minimal user intervention. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Fine needle aspiration cytology of breast cancer in women aged 70 years and older.

PubMed

Tse, Gary M K; Somali, Anjali; Chan, Anthony W H; Chaiwun, Benjaporn; Lui, Philip C W; Moriya, Takuya; Hwang, Jacqueline S G; Chan, Norman H L; Tan, Puay Hoon

2008-10-01

Elderly breast cancers are associated with a more favourable biological marker profile and higher proportion of specific subtypes, some of which are of low histological grade. We reviewed the fine needle aspiration cytology (FNAC) to assess the cytological characteristics and any clues to assist in the diagnosis. The aspirates of 140 cancers of various histological types and grades and 39 benign lesions were evaluated for 13 cytological parameters including cellularity of the direct and cytospin smears, epithelial cell clusters, cellular atypism, cytoplasmic features, vacuoles, mitotic figures, presence of myoepithelial cells, single background epithelial cells, the presence of naked nuclei, stromal fragments and necrosis. We found that the presence of background single epithelial cells, atypism of such cells, absence of benign appearing epithelial fragments, nuclear atypism of the epithelial cells within the fragments, presence of moderate amount of cytoplasm of these cells, absence of myoepithelial cells within the cluster, and absence of bipolar nuclei in the background have a strong association with malignancy. Scoring only the presence of single cells in the background, single cell atypism and the absence of bipolar nuclei in a scoring system can differentiate between benign and malignant aspirates with high (>90%) sensitivity and specificity. Assessing the presence of single cells in the background, single cell atypism and the absence of bipolar nuclei facilitates identification of malignancy in the aspiration of breast lesions from elderly patients.

The Zebrafish G12 Gene is required for Nuclear Positioning and Cell Migrations during Early Development

NASA Technical Reports Server (NTRS)

Reinsch, S. S.; Conway, G. C.

2003-01-01

After fertilization Zebrafish embryos undergo synchronous cleavage to form a blastula of cells sitting upon a single multinucleate yolk cell. At the beginning of gastrulation these cells undergo extensive cell migrations to form the major body axes. We have discovered a gene, G12, which is required for cell migrations and positioning of nuclei in the large syncytial yolk cell. Overexpression of a G12-GFP fusion protein is not toxic and shows that the protein localizes inside the yolk cell to the yolk nuclei, microtubules, and to the margin between the blastomeres and the large yolk cell. Morpholino (MO) injection into the 1-cell embryo or into just the yolk syncytium conipletely inhibits cell migrations, doming of the yolk cell, and positioning of nuclei around the margin. This effect can be partially rescued by injection of G12-GFP encoding RNA. Given the known role of microtubules in nuclear positioning of yolk nuclei in Zebrafish, we investigated the microtubules in morpholiiio injected and rescued embryos. We find that microtubules are sparse and disorganized in MO-injected embryos and are restored to normal organization upon G12-GFP rescue. G12 plays a pivotal role in organization of inicrotubules during early development. G12 is highly conserved in vertebrates and two homologues exist in the human genome. One of the human hoinologues is amplified in aggressive breast tumors.

Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.

PubMed

Reipert, S; Reipert, B M; Allen, T D

1994-09-01

The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scanning electron microscope.

Nuclear inheritance and genetic exchange without meiosis in the binucleate parasite Giardia intestinalis

PubMed Central

Carpenter, Meredith L.; Assaf, Zoe June; Gourguechon, Stéphane; Cande, W. Zacheus

2012-01-01

The protozoan parasite Giardia intestinalis (also known as Giardia lamblia) is a major waterborne pathogen. During its life cycle, Giardia alternates between the actively growing trophozoite, which has two diploid nuclei with low levels of allelic heterozygosity, and the infectious cyst, which has four nuclei and a tough outer wall. Although the formation of the cyst wall has been studied extensively, we still lack basic knowledge about many fundamental aspects of the cyst, including the sources of the four nuclei and their distribution during the transformation from cyst into trophozoite. In this study, we tracked the identities of the nuclei in the trophozoite and cyst using integrated nuclear markers and immunofluorescence staining. We demonstrate that the cyst is formed from a single trophozoite by a mitotic division without cytokinesis and not by the fusion of two trophozoites. During excystation, the cell completes cytokinesis to form two daughter trophozoites. The non-identical nuclear pairs derived from the parent trophozoite remain associated in the cyst and are distributed to daughter cells during excystation as pairs. Thus, nuclear sorting (such that each daughter cell receives a pair of identical nuclei) does not appear to be a mechanism by which Giardia reduces heterozygosity between its nuclei. Rather, we show that the cyst nuclei exchange chromosomal genetic material, perhaps as a way to reduce heterozygosity in the absence of meiosis and sex, which have not been described in Giardia. These results shed light on fundamental aspects of the Giardia life cycle and have implications for our understanding of the population genetics and cell biology of this binucleate parasite. PMID:22366460

Blue two-photon fluorescence metal cluster probe precisely marking cell nuclei of two cell lines.

PubMed

Wang, Yaling; Cui, Yanyan; Liu, Ru; Wei, Yueteng; Jiang, Xinglu; Zhu, Huarui; Gao, Liang; Zhao, Yuliang; Chai, Zhifang; Gao, Xueyun

2013-11-25

A bifunctional peptide was designed to in situ reduce Cu ions and anchor a Cu cluster. The peptide-Cu cluster probe, mainly composed of Cu14, emitted blue two-photon fluorescence under femtosecond laser excitation. Most important, the probe can specifically mark the nuclei of HeLa and A549 cells, respectively.

Isolation of Nuclei and Nucleoli.

PubMed

Pendle, Alison F; Shaw, Peter J

2017-01-01

Here we describe methods for producing nuclei from Arabidopsis suspension cultures or root tips of Arabidopsis, wheat, or pea. These methods could be adapted for other species and cell types. The resulting nuclei can be further purified for use in biochemical or proteomic studies, or can be used for microscopy. We also describe how the nuclei can be used to obtain a preparation of nucleoli.

Quantitative semi-automated analysis of morphogenesis with single-cell resolution in complex embryos

PubMed Central

Giurumescu, Claudiu A.; Kang, Sukryool; Planchon, Thomas A.; Betzig, Eric; Bloomekatz, Joshua; Yelon, Deborah; Cosman, Pamela; Chisholm, Andrew D.

2012-01-01

A quantitative understanding of tissue morphogenesis requires description of the movements of individual cells in space and over time. In transparent embryos, such as C. elegans, fluorescently labeled nuclei can be imaged in three-dimensional time-lapse (4D) movies and automatically tracked through early cleavage divisions up to ~350 nuclei. A similar analysis of later stages of C. elegans development has been challenging owing to the increased error rates of automated tracking of large numbers of densely packed nuclei. We present Nucleitracker4D, a freely available software solution for tracking nuclei in complex embryos that integrates automated tracking of nuclei in local searches with manual curation. Using these methods, we have been able to track >99% of all nuclei generated in the C. elegans embryo. Our analysis reveals that ventral enclosure of the epidermis is accompanied by complex coordinated migration of the neuronal substrate. We can efficiently track large numbers of migrating nuclei in 4D movies of zebrafish cardiac morphogenesis, suggesting that this approach is generally useful in situations in which the number, packing or dynamics of nuclei present challenges for automated tracking. PMID:23052905

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feye-Treimer, U., E-mail: feye-treimer@helmholtz-berlin.de; Treimer, W.

Purpose: This theoretical work contains a detailed investigation of the potential and sensitivity of phase-based x-ray scattering for cancer detection in biopsies if cancer is in a very early stage of development. Methods: Cancer cells in their early stage of development differ from healthy ones mainly due to their faster growing cell nuclei and the enlargement of their densities. This growth is accompanied by an altered nucleus–plasma relation for the benefit of the cell nuclei, that changes the physical properties especially the index of refraction of the cell and the one of the cell nuclei. Interaction of radiation with mattermore » is known to be highly sensitive to small changes of the index of refraction of matter; therefore a detection of such changes of volume and density of cell nuclei by means of high angular resolved phase-based scattering of x rays might provide a technique to distinguish malignant cells from healthy ones ifthe cell–cell nucleus system is considered as a coherent phase shifting object. Then one can observe from a thin biopsy which represents a monolayer of cells (no multiple scattering) that phase-based x-ray scattering curves from healthy cells differ from those of cancer cells in their early stage of development. Results: Detailed calculations of x-ray scattering patterns from healthy and cancer cell nuclei yield graphs and numbers with which one can distinguish healthy cells from cancer ones, taking into account that both kinds of cells occur in a tissue within a range of size and density. One important result is the role and the influence of the (lateral) coherence width of the radiation on the scattering curves and the sensitivity of phase-based scattering for cancer detection. A major result is that a larger coherence width yields a larger sensitivity for cancer detection. Further import results are calculated limits for critical sizes and densities of cell nuclei in order to attribute the investigated tissue to be healthy or diseased. Conclusions: With this proposed method it should be in principle possible to detect cancer cells in apparently healthy tissues in biopsies and/or in samples of the far border region of abscised or excised tissues. Thus this method could support established methods in diagnostics of cancer-suspicious samples.« less

Time-Resolved Visualisation of Nearly-Native Influenza A Virus Progeny Ribonucleoproteins and Their Individual Components in Live Infected Cells

PubMed Central

Avilov, Sergiy; Magnus, Julie; Cusack, Stephen; Naffakh, Nadia

2016-01-01

Influenza viruses are a global health concern because of the permanent threat of novel emerging strains potentially capable of causing pandemics. Viral ribonucleoproteins (vRNPs) containing genomic RNA segments, nucleoprotein oligomers, and the viral polymerase, play a central role in the viral replication cycle. Our knowledge about critical events such as vRNP assembly and interactions with other viral and cellular proteins is poor and could be substantially improved by time lapse imaging of the infected cells. However, such studies are limited by the difficulty to achieve live-cell compatible labeling of active vRNPs. Previously we designed the first unimpaired recombinant influenza WSN-PB2-GFP11 virus allowing fluorescent labeling of the PB2 subunit of the viral polymerase (Avilov et al., J.Virol. 2012). Here, we simultaneously labeled the viral PB2 protein using the above-mentioned strategy, and virus-encoded progeny RNPs through spontaneous incorporation of transiently expressed NP-mCherry fusion proteins during RNP assembly in live infected cells. This dual labeling enabled us to visualize progeny vRNPs throughout the infection cycle and to characterize independently the mobility, oligomerization status and interactions of vRNP components in the nuclei of live infected cells. PMID:26978069

Computer-Aided Diagnosis Of Leukemic Blood Cells

NASA Astrophysics Data System (ADS)

Gunter, U.; Harms, H.; Haucke, M.; Aus, H. M.; ter Meulen, V.

1982-11-01

In a first clinical test, computer programs are being used to diagnose leukemias. The data collected include blood samples from patients suffering from acute myelomonocytic-, acute monocytic- and acute promyelocytic, myeloblastic, prolymphocytic, chronic lymphocytic leukemias and leukemic transformed immunocytoma. The proper differentiation of the leukemic cells is essential because the therapy depends on the type of leukemia. The algorithms analyse the fine chromatin texture and distribution in the nuclei as well as size and shape parameters from the cells and nuclei. Cells with similar nuclei from different leukemias can be distinguished from each other by analyzing the cell cytoplasm images. Recognition of these subtle differences in the cells require an image sampling rate of 15-30 pixel/micron. The results for the entire data set correlate directly to established hematological parameters and support the previously published initial training set .

Control of cell nucleus shapes via micropillar patterns.

PubMed

Pan, Zhen; Yan, Ce; Peng, Rong; Zhao, Yingchun; He, Yao; Ding, Jiandong

2012-02-01

We herein report a material technique to control the shapes of cell nuclei by the design of the microtopography of substrates to which the cells adhere. Poly(D,L-lactide-co-glycolide) (PLGA) micropillars or micropits of a series of height or depth were fabricated, and some surprising self deformation of the nuclei of bone marrow stromal cells (BMSCs) was found in the case of micropillars with a sufficient height. Despite severe nucleus deformation, BMSCs kept the ability of proliferation and differentiation. We further demonstrated that the shapes of cell nuclei could be regulated by the appropriate micropillar patterns. Besides circular and elliptoid shapes, some unusual nucleus shapes of BMSCs have been achieved, such as square, cross, dumbbell, and asymmetric sphere-protrusion. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

GEOPHYSICS, ASTRONOMY AND ASTROPHYSICS: Second-harmonic generation as a DNA malignancy indicator of prostate glandular epithelial cells

NASA Astrophysics Data System (ADS)

Zhuang, Zheng-Fei; Liu, Han-Ping; Guo, Zhou-Yi; Zhuo, Shuang-Mu; Yu, Bi-Ying; Deng, Xiao-Yuan

2010-04-01

This paper first demonstrates second-harmonic generation (SHG) in the intact cell nucleus, which acts as an optical indicator of DNA malignancy in prostate glandular epithelial cells. Within a scanning region of 2.7 μm×2.7 μm in cell nuclei, SHG signals produced from benign prostatic hyperplasia (BPH) and prostate carcinoma (PC) tissues (mouse model C57BL/6) have been investigated. Statistical analyses (t test) of a total of 405 measurements (204 nuclei from BPH and 201 nuclei from PC) show that SHG signals from BPH and PC have a distinct difference (p < 0.05), suggesting a potential optical method of revealing very early malignancy in prostate glandular epithelial cells based upon induced biochemical and/or biophysical modifications in DNA.

Nucleoprotein Changes in Plant Tumor Growth

PubMed Central

Rasch, Ellen; Swift, Hewson; Klein, Richard M.

1959-01-01

Tumor cell transformation and growth were studied in a plant neoplasm, crown gall of bean, induced by Agrobacterium rubi. Ribose nucleic acid (RNA), deoxyribose nucleic acid (DNA), histone, and total protein were estimated by microphotometry of nuclei, nucleoli, and cytoplasm in stained tissue sections. Transformation of normal cells to tumor cells was accompanied by marked increases in ribonucleoprotein content of affected tissues, reaching a maximum 2 to 3 days after inoculation with virulent bacteria. Increased DNA levels were in part associated with increased mitotic frequency, but also with progressive accumulation of nuclei in the higher DNA classes, formed by repeated DNA doubling without intervening reduction by mitosis. Some normal nuclei of the higher DNA classes (with 2, 4, or 8 times the DNA content of diploid nuclei) were reduced to diploid levels by successive cell divisions without intervening DNA synthesis. The normal relation between DNA synthesis and mitosis was thus disrupted in tumor tissue. Nevertheless, clearly defined DNA classes, as found in homologous normal tissues, were maintained in the tumor at all times. PMID:13673042

Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover.

PubMed

Bergmann, Olaf; Zdunek, Sofia; Alkass, Kanar; Druid, Henrik; Bernard, Samuel; Frisén, Jonas

2011-01-15

Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously ¹⁴C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that ¹⁴C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans. Copyright © 2010 Elsevier Inc. All rights reserved.

Activation of raphe nuclei triggers rapid and distinct effects on parallel olfactory bulb output channels

PubMed Central

Kapoor, Vikrant; Provost, Allison; Agarwal, Prateek; Murthy, Venkatesh N.

2015-01-01

The serotonergic raphe nuclei are involved in regulating brain states over time-scales of minutes and hours. We examined more rapid effects of serotonergic activation on two classes of principal neurons in the mouse olfactory bulb, mitral and tufted cells, which send olfactory information to distinct targets. Brief stimulation of the raphe nuclei led to excitation of tufted cells at rest and potentiation of their odor responses. While mitral cells at rest were also excited by raphe activation, their odor responses were bidirectionally modulated, leading to improved pattern separation of odors. In vitro whole-cell recordings revealed that specific optogenetic activation of raphe axons affected bulbar neurons through dual release of serotonin and glutamate. Therefore, the raphe nuclei, in addition to their role in neuromodulation of brain states, are also involved in fast, sub-second top-down modulation, similar to cortical feedback. This modulation can selectively and differentially sensitize or decorrelate distinct output channels. PMID:26752161

Modulation of Stem Cell Differentiation and Myostatin as an Approach to Counteract Fibrosis in Muscle Dystrophy and Regeneration After Injury. Addendum

DTIC Science & Technology

2012-03-01

considerable increase in central nuclei in the regenerating myofibers, and molsidomine supplementation appears to have upregulated the overall stem cell...the increase of central nuclei as indicator of muscle repair observed in the SC group in comparison to the UT group, in frozen tissue sections...treatments on myofiber repair will be better defined by the counting of central nuclei on hematoxylin/eosin stained frozen sections as in Fig 3, and the

Combined inhibition of autophagy and caspases fails to prevent developmental nurse cell death in the Drosophila melanogaster ovary.

PubMed

Peterson, Jeanne S; McCall, Kimberly

2013-01-01

During the final stages of Drosophila melanogaster oogenesis fifteen nurse cells, sister cells to the oocyte, degenerate as part of normal development. This process involves at least two cell death mechanisms, caspase-dependent cell death and autophagy, as indicated by apoptosis and autophagy markers. In addition, mutations affecting either caspases or autophagy partially reduce nurse cell removal, leaving behind end-stage egg chambers with persisting nurse cell nuclei. To determine whether apoptosis and autophagy work in parallel to degrade and remove these cells as is the case with salivary glands during pupariation, we generated mutants doubly affecting caspases and autophagy. We found no significant increase in either the number of late stage egg chambers containing persisting nuclei or in the number of persisting nuclei per egg chamber in the double mutants compared to single mutants. These findings suggest that there is another cell death mechanism functioning in the ovary to remove all nurse cell remnants from late stage egg chambers.

Segmentation of human brain using structural MRI.

PubMed

Helms, Gunther

2016-04-01

Segmentation of human brain using structural MRI is a key step of processing in imaging neuroscience. The methods have undergone a rapid development in the past two decades and are now widely available. This non-technical review aims at providing an overview and basic understanding of the most common software. Starting with the basis of structural MRI contrast in brain and imaging protocols, the concepts of voxel-based and surface-based segmentation are discussed. Special emphasis is given to the typical contrast features and morphological constraints of cortical and sub-cortical grey matter. In addition to the use for voxel-based morphometry, basic applications in quantitative MRI, cortical thickness estimations, and atrophy measurements as well as assignment of cortical regions and deep brain nuclei are briefly discussed. Finally, some fields for clinical applications are given.

Cell cycle synchronization of leukemia inhibitory factor (LIF)-dependent porcine-induced pluripotent stem cells and the generation of cloned embryos.

PubMed

Yuan, Ye; Lee, Kiho; Park, Kwang-Wook; Spate, Lee D; Prather, Randall S; Wells, Kevin D; Roberts, R Michael

2014-01-01

Nuclear transfer (NT) from porcine iPSC to create cloned piglets is unusually inefficient. Here we examined whether such failure might be related to the cell cycle stage of donor nuclei. Porcine iPSC, derived here from the inner cell mass of blastocysts, have a prolonged S phase and are highly sensitive to drugs normally used for synchronization. However, a double-blocking procedure with 0.3 μM aphidicolin for 10 h followed by 20 ng/ml nocodazole for 4 h arrested 94.3% of the cells at G2/M and, after release from the block, provided 70.1% cells in the subsequent G1 phase without causing any significant loss of cell viability or pluripotent phenotype. Nuclei from different cell cycle stages were used as donors for NT to in vitro-matured metaphase II oocytes. G2/M nuclei were more efficient than either G1 and S stage nuclei in undergoing first cleavage and in producing blastocysts, but all groups had a high incidence of chromosomal/nuclear abnormalities at 2 h and 6 h compared with non-synchronized NT controls from fetal fibroblasts. Many G2 embryos extruded a pseudo-second polar body soon after NT and, at blastocyst, tended to be either polyploid or diploid. By contrast, the few G1 blastocysts that developed were usually mosaic or aneuploid. The poor developmental potential of G1 nuclei may relate to lack of a G1/S check point, as the cells become active in DNA synthesis shortly after exit from mitosis. Together, these data provide at least a partial explanation for the almost complete failure to produce cloned piglets from piPSC.

The change is length and width of the Sertoli cell nuclei in cytologic smears of testes with depopulation of the seminiferous epithelium.

PubMed

Banek, L; Posinovec, J

1980-09-15

The appearance of the Sertoli cells in cytological smears of tests with depopulation of the seminiferous epithelium is described. The mean values of the lengths and widths of the Sertoli cell nuclei in smears differed significantly between the depopulation and the control group (p < 0.01).

A novel histological technique for distinguishing between epithelial cells in forensic casework.

PubMed

French, Claire E V; Jensen, Cynthia G; Vintiner, Susan K; Elliot, Douglas A; McGlashan, Susan R

2008-06-10

There are a number of forensic cases in which the identification of the epithelial cell type from which DNA originated would provide important probative evidence. This study aimed to develop a technique using histological staining of fixed cells to distinguish between skin, buccal and vaginal epithelium. First, 11 different stains were screened on formalin-fixed, wax-embedded cells from five women. Samples were analysed qualitatively by examining staining patterns (colour) and morphology (absence or presence of nuclei). Three of the staining methods--Dane's, Csaba's and Ayoub-Shklar--were successful in distinguishing skin epithelial cells from buccal and vaginal. Second, cells were smeared directly onto slides, fixed with one of five fixatives and stained with one of the three stains mentioned above. Methanol fixation, coupled with the Dane's staining method, specific to keratin, was the only technique that distinguished between all three cell types. Skin cells stained magenta, red and orange and lacked nuclei; buccal cells stained predominantly orange-pink with red nuclei; while vaginal cells stained bright orange with orange nuclei and a blue extracellular hue. This staining pattern in vaginal cells was consistent in samples collected from 50 women aged between 18 and 67. Identification of cell type from unlabelled micrographs by 10 trained observers showed a mean success rate of 95%. The results of this study demonstrate that histological staining may provide forensic scientists with a technique for distinguishing between skin, buccal and vaginal epithelial cells and thus would enable more conclusive analyses when investigating sexual assault cases.

Segmentation of the Clustered Cells with Optimized Boundary Detection in Negative Phase Contrast Images

PubMed Central

Wang, Yuliang; Zhang, Zaicheng; Wang, Huimin; Bi, Shusheng

2015-01-01

Cell image segmentation plays a central role in numerous biology studies and clinical applications. As a result, the development of cell image segmentation algorithms with high robustness and accuracy is attracting more and more attention. In this study, an automated cell image segmentation algorithm is developed to get improved cell image segmentation with respect to cell boundary detection and segmentation of the clustered cells for all cells in the field of view in negative phase contrast images. A new method which combines the thresholding method and edge based active contour method was proposed to optimize cell boundary detection. In order to segment clustered cells, the geographic peaks of cell light intensity were utilized to detect numbers and locations of the clustered cells. In this paper, the working principles of the algorithms are described. The influence of parameters in cell boundary detection and the selection of the threshold value on the final segmentation results are investigated. At last, the proposed algorithm is applied to the negative phase contrast images from different experiments. The performance of the proposed method is evaluated. Results show that the proposed method can achieve optimized cell boundary detection and highly accurate segmentation for clustered cells. PMID:26066315

A unique combination of anatomy and physiology in cells of the rat paralaminar thalamic nuclei adjacent to the medial geniculate body

PubMed Central

Smith, Philip H.; Bartlett, Edward L.; Kowalkowski, Anna

2010-01-01

The medial geniculate body (MGB) has three major subdivisions - ventral (MGV), dorsal (MGD) and medial (MGM). MGM is linked with paralaminar nuclei that are situated medial and ventral to MGV/MGD. Paralaminar nuclei have unique inputs and outputs when compared with MGV and MGD and have been linked to circuitry underlying some important functional roles. We recorded intracellularly from cells in the paralaminar nuclei in vitro. We found that they possess an unusual combination of anatomical and physiological features when compared to those reported for “standard” thalamic neurons seen in the MGV/MGD and elsewhere in the thalamus. Compared to MGV/MGD neurons, anatomically, 1) paralaminar cell dendrites can be long, branch sparingly and encompass a much larger area. 2) their dendrites may be smooth but can have well defined spines and 3) their axons can have collaterals that branch locally within the same or nearby paralaminar nuclei. When compared to MGV/MGD neurons physiologically 1) their spikes are larger in amplitude and can be shorter in duration and 2) can have dual afterhyperpolarizations with fast and slow components and 3) they can have a reduction or complete absence of the low threshold, voltage-sensitive calcium conductance that reduces or eliminates the voltage-dependent burst response. We also recorded from cells in the parafascicular nucleus, a nucleus of the posterior intralaminar nuclear group, because they have unusual anatomical features that are similar to some of our paralaminar cells. Like the labeled paralaminar cells, parafascicular cells had physiological features distinguishing them from typical thalamic neurons. PMID:16566009

Central projections of the lateral line and eighth nerves in the bowfin, Amia calva.

PubMed

McCormick, C A

1981-03-20

The first-order connections of the anterior and posterior lateral line nerves and of the eighth nerve were determined in the bowfin, Amia calva, using experimental degeneration and anterograde HRP transport techniques. The termination sites of these nerves define a dorsal lateralis cell column and a ventral octavus cell column. The anterior and posterior lateralis nerves distribute ipsilaterally to two medullary nuclei-nucleus medialis and nucleus caudalis. Nucleus medialis comprises the rostral two-thirds of the lateralis column and contains large, Purkinje-like cells dorsally and polygonal, granule, and fusiform cells ventrally. Nucleus caudalis is located posterior to nucleus medialis and consists of small, granule cells. Anterior lateralis fibers terminate ventrally to ventromedially in both nucleus medialis and nucleus caudalis. Posterior lateralis fibers terminate dorsally to dorsolaterally within these two nuclei. A sparse anterior lateralis input may also be present on the dendrites of one of the nuclei within the octavus cell column, nucleus magnocellularis. In contrast, the anterior and posterior rami of the eighth nerve each terminate within four medullary nuclei which comprise the octavus cell column: the anterior, magnocellular, descending, and posterior octavus nuclei. An eighth nerve projection to the medial reticular formation is also present. Some fibers of the lateralis and eighth nerves terminate within the ipsilateral eminentia granularis of the cerebellum. Lateralis fibers distribute to approximately the lateral half of this structure with posterior lateral line fibers terminating laterally and anterior lateral line fibers terminating medially. Eighth nerve fibers distribute to the medial half of the eminentia granularis.

Embryonic Origins of the Mouse Superior Olivary Complex

PubMed Central

Howell, David M.; Spirou, George A.; Mathers, Peter H.

2014-01-01

Many areas of the central nervous system are organized into clusters of cell groups, with component cell groups exhibiting diverse but related functions. One such cluster, the superior olivary complex (SOC), is located in the ventral auditory brainstem in mammals. The SOC is an obligatory contact point for most projection neurons of the ventral cochlear nucleus and plays central roles in many aspects of monaural and binaural information processing. Despite their important interrelated functions, little is known about the embryonic origins of SOC nuclei, due in part to a paucity of developmental markers to distinguish individual cell groups. In this report, we present a collection of novel markers for the developing SOC nuclei in mice, including the transcription factors FoxP1, MafB, and Sox2, and the lineage-marking transgenic line En1-Cre. We use these definitive markers to examine the rhombic lip and rhombomeric origins of SOC nuclei and demonstrate that they can serve to uniquely identify SOC nuclei and subnuclei in newborn pups. The markers are also useful in identifying distinct nuclear domains within the presumptive SOC as early as embryonic day (E) 14.5, well before morphological distinction of individual nuclei is evident. These findings indicate that the mediolateral and dorsoventral position of SOC nuclei characteristic of the adult brainstem is established during early neurogenesis. PMID:23303740

HAMLET interacts with histones and chromatin in tumor cell nuclei.

PubMed

Düringer, Caroline; Hamiche, Ali; Gustafsson, Lotta; Kimura, Hiroshi; Svanborg, Catharina

2003-10-24

HAMLET is a folding variant of human alpha-lactalbumin in an active complex with oleic acid. HAMLET selectively enters tumor cells, accumulates in their nuclei and induces apoptosis-like cell death. This study examined the interactions of HAMLET with nuclear constituents and identified histones as targets. HAMLET was found to bind histone H3 strongly and to lesser extent histones H4 and H2B. The specificity of these interactions was confirmed using BIAcore technology and chromatin assembly assays. In vivo in tumor cells, HAMLET co-localized with histones and perturbed the chromatin structure; HAMLET was found associated with chromatin in an insoluble nuclear fraction resistant to salt extraction. In vitro, HAMLET bound strongly to histones and impaired their deposition on DNA. We conclude that HAMLET interacts with histones and chromatin in tumor cell nuclei and propose that this interaction locks the cells into the death pathway by irreversibly disrupting chromatin organization.

Radioprotective Thiolamines WR-1065 and WR-33278 Selectively Denature Nonhistone Nuclear Proteins

NASA Technical Reports Server (NTRS)

Booth, Valerie K.; Roberts, Jeanette C.; Warters, Raymond L.; Wilmore, Britta H.; Lepock, James R.

2000-01-01

Differential scanning calorimetry was used to study the interactions of nuclei isolated from Chinese hamster V79 cells with the radioprotector WR-1065, other thiol compounds, and polyamines. Differential scanning calorimetry monitors denaturation of macromolecules and resolves the major nuclear components (e.g. constrained and relaxed DNA, nucleosome core, and nuclear matrix) of intact nuclei on the basis of thermal stability. WR-1065 treatment (0.5-10 mM) of isolated nuclei led to the irreversible denaturation of nuclear proteins, a fraction of which are nuclear matrix proteins. Denaturation of 50% of the total nonhistone nuclear protein content of isolated nuclei occurred after exposure to 4.7 mM WR-1065 for 20 min at 23 C. In addition, a 22% increase in the insoluble protein content of nuclei isolated from V79 cells that had been treated with 4 mM WR-1065 for 30 min at 37 C was observed, indicating that WR-1065-induced protein denaturation occurs not only in isolated nuclei but also in the nuclei of intact cells. From the extent of the increase in insoluble protein in the nucleus, protein denaturation by WR-1065 is expected to contribute to drug toxicity at concentrations greater than approximately 4 mM. WR-33278, the disulfide form of WR1065, was approximately twice as effective as the free thiol at denaturing nuclear proteins. The proposed mechanism for nucleoprotein denaturation is through direct interactions with protein cysteine groups with the formation of destabilizing protein-WR-1065 disulfides. In comparison to its effect on nuclear proteins in isolated nuclei, WR-1065 had only a very small effect on non-nuclear proteins of whole cells, isolated nuclear matrix, or the thiol-rich Ca (2+) ATPase of sarcoplasmic reticulum, indicating that WR-1065 can effectively denature protein only inside an intact nucleus, probably due to the increased concentration of the positively charged drug in the vicinity of DNA.

Nuclei pulposi formation from the embryonic notochord occurs normally in GDF-5-deficient mice.

PubMed

Maier, Jennifer A; Harfe, Brian D

2011-11-15

The transition of the mouse embryonic notochord into nuclei pulposi was determined ("fate mapped") in vivo in growth and differentiating factor-5 (GDF-5)-null mice using the Shhcre and R26R alleles. To determine whether abnormal nuclei pulposi formation from the embryonic notochord was responsible for defects present in adult nuclei pulposi of Gdf-5-null mice. The development, maintenance, and degeneration of the intervertebral disc are not understood. Previously, we demonstrated that all cells in the adult nucleus pulposus of normal mice are derived from the embryonic notochord. Gdf-5-null mice have been reported to contain intervertebral discs in which the nucleus pulposus is abnormal. It is currently unclear if disc defects in Gdf-5-null mice arise during the formation of nuclei pulposi from the notochord during embryogenesis or result from progressive postnatal degeneration of nuclei pulposi. Gdf-5 messenger RNA expression was examined in the discs of wild-type embryos by RNA in situ hybridization to determine when and where this gene was expressed. To examine nucleus pulposus formation in Gdf-5-null mice, intervertebral discs in which embryonic notochord cells were marked were analyzed in newborn and 24-week-old mice. Our Gdf-5 messenger RNA in situ experiments determined that this gene is localized to the annulus fibrosus and not the nucleus pulposus in mouse embryos. Notochord fate-mapping experiments revealed that notochord cells in Gdf-5-null mice correctly form nuclei pulposi. Our data suggest that the defects reported in the nucleus pulposus of adult Gdf-5-null mice do not result from abnormal patterning of the embryonic notochord. The use of mouse alleles to mark cells that produce all cell types that reside in the adult nucleus pulposus will allow for a detailed examination of disc formation in other mouse mutants that have been reported to contain disc defects.

Nuclei pulposi formation from the embryonic notochord occurs normally in GDF5-deficient mice

PubMed Central

Maier, Jennifer A.; Harfe, Brian D.

2011-01-01

Study Design The transition of the mouse embryonic notochord into nuclei pulposi was determined (“fate mapped”) in vivo in GDF-5 null mice using the Shhcre and R26R alleles. Objective To determine if abnormal nuclei pulposi formation from the embryonic notochord was responsible for defects present in adult nuclei pulposi of Gdf-5 null mice. Summary of Background Data The development, maintenance, and degeneration of the intervertebral disc are not understood. Previously, we demonstrated that all cells in the adult nucleus pulposus of normal mice are derived from the embryonic notochord. Gdf-5 null mice have been reported to contain intervertebral discs in which the nucleus pulposus is abnormal. It is currently unclear if disc defects in Gdf-5 null mice arise during the formation of nuclei pulposi from the notochord during embryogenesis or resulted from progressive postnatal degeneration of nuclei pulposi. Methods Gdf-5 mRNA expression was examined in the discs of wild-type embryos by RNA in situ hybridization to determine when and where this gene was expressed. To examine nucleus pulposus formation in Gdf-5 null mice, intervertebral discs in which embryonic notochord cells were marked were analyzed in newborn and 24 week old mice. Results Our Gdf-5 mRNA in situ experiments determined that this gene is localized to the annulus fibrosus and not the nucleus pulposus in mouse embryos. Notochord fate mapping experiments revealed that notochord cells in Gdf-5 null mice correctly form nuclei pulposi. Conclusion Our data suggest that the defects reported in the nucleus pulposus of adult Gdf-5 null mice do not result from abnormal patterning of the embryonic notochord. The use of mouse alleles to mark cells that produce all cell types that reside in the adult nucleus pulposus will allow for a detailed examination of disc formation in other mouse mutants that have been reported to contain disc defects. PMID:21278629

Neuronal nuclei isolation from human postmortem brain tissue.

PubMed

Matevossian, Anouch; Akbarian, Schahram

2008-10-01

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan. Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays.

Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes

PubMed Central

Bolzer, Andreas; Kreth, Gregor; Solovei, Irina; Koehler, Daniela; Saracoglu, Kaan; Fauth, Christine; Müller, Stefan; Eils, Roland; Cremer, Christoph; Speicher, Michael R

2005-01-01

Studies of higher-order chromatin arrangements are an essential part of ongoing attempts to explore changes in epigenome structure and their functional implications during development and cell differentiation. However, the extent and cell-type-specificity of three-dimensional (3D) chromosome arrangements has remained controversial. In order to overcome technical limitations of previous studies, we have developed tools that allow the quantitative 3D positional mapping of all chromosomes simultaneously. We present unequivocal evidence for a probabilistic 3D order of prometaphase chromosomes, as well as of chromosome territories (CTs) in nuclei of quiescent (G0) and cycling (early S-phase) human diploid fibroblasts (46, XY). Radial distance measurements showed a probabilistic, highly nonrandom correlation with chromosome size: small chromosomes—independently of their gene density—were distributed significantly closer to the center of the nucleus or prometaphase rosette, while large chromosomes were located closer to the nuclear or rosette rim. This arrangement was independently confirmed in both human fibroblast and amniotic fluid cell nuclei. Notably, these cell types exhibit flat-ellipsoidal cell nuclei, in contrast to the spherical nuclei of lymphocytes and several other human cell types, for which we and others previously demonstrated gene-density-correlated radial 3D CT arrangements. Modeling of 3D CT arrangements suggests that cell-type-specific differences in radial CT arrangements are not solely due to geometrical constraints that result from nuclear shape differences. We also found gene-density-correlated arrangements of higher-order chromatin shared by all human cell types studied so far. Chromatin domains, which are gene-poor, form a layer beneath the nuclear envelope, while gene-dense chromatin is enriched in the nuclear interior. We discuss the possible functional implications of this finding. PMID:15839726

Reconstruction of incomplete cell paths through a 3D-2D level set segmentation

NASA Astrophysics Data System (ADS)

Hariri, Maia; Wan, Justin W. L.

2012-02-01

Segmentation of fluorescent cell images has been a popular technique for tracking live cells. One challenge of segmenting cells from fluorescence microscopy is that cells in fluorescent images frequently disappear. When the images are stacked together to form a 3D image volume, the disappearance of the cells leads to broken cell paths. In this paper, we present a segmentation method that can reconstruct incomplete cell paths. The key idea of this model is to perform 2D segmentation in a 3D framework. The 2D segmentation captures the cells that appear in the image slices while the 3D segmentation connects the broken cell paths. The formulation is similar to the Chan-Vese level set segmentation which detects edges by comparing the intensity value at each voxel with the mean intensity values inside and outside of the level set surface. Our model, however, performs the comparison on each 2D slice with the means calculated by the 2D projected contour. The resulting effect is to segment the cells on each image slice. Unlike segmentation on each image frame individually, these 2D contours together form the 3D level set function. By enforcing minimum mean curvature on the level set surface, our segmentation model is able to extend the cell contours right before (and after) the cell disappears (and reappears) into the gaps, eventually connecting the broken paths. We will present segmentation results of C2C12 cells in fluorescent images to illustrate the effectiveness of our model qualitatively and quantitatively by different numerical examples.

[Diagnostic and prognostic value of p53 oncogene and the selected neoplastic markers (Ki67, PCNA, DNA ploidy) of the ultrastructure in patients with laryngeal cancer].

PubMed

Golusiński, W; Szmeja, Z; Olofsson, J; Biczysko, W; Krygier-Stojałowska, A; Majewski, P

1996-01-01

A comparison was performed of staining intensity of immunohistochemical proliferating antigens (p53, PCNA, Ki67), DNA flow cytometry and ultrastructure of the carcinoma cells in 120 cases of laryngeal cancer. Clinically very advanced tumors were in majority (T3 - 43%, T4 - 18%). A 5 graded scale was adapted to evaluate the level of immunohistochemical staining of the carcinoma cell nuclei. A positive staining was obtained in 70% for p53, 57% for Ki67 and in 80(2/3) for PCNA. 62% of the cases were DNA diploid and 38% DNA aneuploid. The DNA diploid carcinomas were accompanied by the enlargement of the cell nuclei, preserving of the nuclei's wide margins of heterochromatine, enlargement of the nuclear area and increase of the number of nuclei. In the aneuploid-polyploid cancer the nuclei had a substantial polymorphism with large cleaved nuclei and with significant variation in size, and with nuclear envelope. A frequent finding was euchromatization of chromatine. Dense chromatine appeared in the form of small clumps spread over the whole area of these irregular nuclei. Enlargement and activation of nucleoli occurred. There was a positive correlation (Chi-square) between T- and N-stage and immunohistochemical staining. There was also a positive correlation in staining intensity between p53, Ki67 and PCNA. There is also strong correlation between these markers of proliferative activity and the degree of aggressiveness of the tumour.

Organization of projections from the raphe nuclei to the vestibular nuclei in rats

NASA Technical Reports Server (NTRS)

Halberstadt, A. L.; Balaban, C. D.

2003-01-01

Previous anatomic and electrophysiological evidence suggests that serotonin modulates processing in the vestibular nuclei. This study examined the organization of projections from serotonergic raphe nuclei to the vestibular nuclei in rats. The distribution of serotonergic axons in the vestibular nuclei was visualized immunohistochemically in rat brain slices using antisera directed against the serotonin transporter. The density of serotonin transporter-immunopositive fibers is greatest in the superior vestibular nucleus and the medial vestibular nucleus, especially along the border of the fourth ventricle; it declines in more lateral and caudal regions of the vestibular nuclear complex. After unilateral iontophoretic injections of Fluoro-Gold into the vestibular nuclei, retrogradely labeled neurons were found in the dorsal raphe nucleus (including the dorsomedial, ventromedial and lateral subdivisions) and nucleus raphe obscurus, and to a minor extent in nucleus raphe pallidus and nucleus raphe magnus. The combination of retrograde tracing with serotonin immunohistofluorescence in additional experiments revealed that the vestibular nuclei receive both serotonergic and non-serotonergic projections from raphe nuclei. Tracer injections in densely innervated regions (especially the medial and superior vestibular nuclei) were associated with the largest numbers of Fluoro-Gold-labeled cells. Differences were observed in the termination patterns of projections from the individual raphe nuclei. Thus, the dorsal raphe nucleus sends projections that terminate predominantly in the rostral and medial aspects of the vestibular nuclear complex, while nucleus raphe obscurus projects relatively uniformly throughout the vestibular nuclei. Based on the topographical organization of raphe input to the vestibular nuclei, it appears that dense projections from raphe nuclei are colocalized with terminal fields of flocculo-nodular lobe and uvula Purkinje cells. It is hypothesized that raphe-vestibular connections are organized to selectively modulate processing in regions of the vestibular nuclear complex that receive input from specific cerebellar zones. This represents a potential mechanism whereby motor activity and behavioral arousal could influence the activity of cerebellovestibular circuits.

Impact of maternal diabetes type 1 on proliferative potential, differentiation and apoptotic activity in villous capillaries of term placenta.

PubMed

Jirkovská, Marie; Kučera, Tomáš; Dvořáková, Veronika; Jadrníček, Martin; Moravcová, Milena; Žižka, Zdeněk; Krejčí, Vratislav

2016-04-01

Maternal diabetes mellitus changes morphology and impairs function of placental capillaries. Here, quantitative parameters characterizing cell proliferation using detection of Ki67, differentiation reflected by nestin expression and apoptosis in placental capillary bed with active caspase 3 as a marker were compared in normal term placentas and placentas from pregnancies complicated by Type 1 maternal diabetes mellitus. Specimens of sixteen diabetic placentas and eight control placentas were collected by systematic uniform random sampling. Immunohistochemical detections of Ki67, nestin, and active caspase 3 were performed in histological sections of five haphazardly chosen blocks per placenta. Twenty fields of view per section, i.e. one hundred fields of view per placenta, were used for analysis of proliferation as well as of apoptosis, and in approximately 70 capillary cross-sections per placenta the nestin-positive segments of their circumference were measured. The percentage of Ki67-positive cells counted in the capillary wall was significantly lower in diabetic group. The counts of Ki67-labelled nuclei per villous area unit were significantly lower in cytotrophoblast and capillary wall of terminal villi in diabetic placenta. The proportion of nestin-labeled segments of capillary circumference was significantly higher in placentas of diabetic group. No differences in the numbers of apoptotic cells were found between studied groups. The results show that the term placenta in Type 1 diabetes has lower potential to enlarge the surface area of structures involved in maternofetal transport, and that the villous capillary bed displays delayed differentiation. Those factors may participate in decreased ability of diabetic placenta to comply with fetal requirements in the final stage of pregnancy. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Morphometric analysis of lymphocyte nuclei in chronic lymphocytic leukemia].

PubMed

Ostapenko, V A; Kruchinskiĭ, N G; Smirnova, L A; Cherednik, A B; Nesterov, V N; Tepliakov, A I

1994-01-01

This work is dedicated to the study of use of quantitative analysis of cell nucleus structure for the analysis of peripheral blood lymphocytes in patients with chronic lymphocytic leukaemia. The structure of lymphocytic nuclei of healthy donors was evaluated by means of staining by toluidine blue purified cell suspensions smears. The preparations were analysed on the television measuring system "omnicon" with measurements of the following parameters: square of the nucleus, euchromatin, heterochromatin, and the ratio of heterochromatin and euchromatin squares. Actuarial analysis and nuclei classification of the previously mentioned parameters showed, that in peripheral blood of patients with chronic lymphocytic leukemia a large amount of atypical lymphocytes is present with reduced nucleus sizes. Atypical cells retain the ratio of structural components of chromatine, characteristic to normal cells, which show their low proliferative activity.

Nuclear RNA quantification in protoplast cell-cycle phases.

PubMed

Bergounioux, C; Perennes, C; Brown, S C; Gadal, P

1988-01-01

Using acridine orange staining and flow cytometry the DNA and RNA levels (arbitrary units) of individual cells may be established. Here, this method has been applied to nuclei isolated from plant protoplasts during culture. The specificity of the technique has been validated for such plant material; ribonuclease markedly reduced nuclear staining without modifying the DNA histogram; ribonuclease inhibitor prevented the action of released cell nucleases; and protoplasts cultivated with actinomycin D did not synthesize RNA. First RNA synthesis was evident 18 h after Petunia hybrida protoplasts had been put into culture. An increase of RNA above a critical level was required for cells to be able to initiate DNA replication from G1, termed G1B. G2 nuclei had an RNA:DNA ratio similar to that of G1 nuclei.

Validation of a new classifier for the automated analysis of the human epidermal growth factor receptor 2 (HER2) gene amplification in breast cancer specimens

PubMed Central

2013-01-01

Amplification of the human epidermal growth factor receptor 2 (HER2) is a prognostic marker for poor clinical outcome and a predictive marker for therapeutic response to targeted therapies in breast cancer patients. With the introduction of anti-HER2 therapies, accurate assessment of HER2 status has become essential. Fluorescence in situ hybridization (FISH) is a widely used technique for the determination of HER2 status in breast cancer. However, the manual signal enumeration is time-consuming. Therefore, several companies like MetaSystem have developed automated image analysis software. Some of these signal enumeration software employ the so called “tile-sampling classifier”, a programming algorithm through which the software quantifies fluorescent signals in images on the basis of square tiles of fixed dimensions. Considering that the size of tile does not always correspond to the size of a single tumor cell nucleus, some users argue that this analysis method might not completely reflect the biology of cells. For that reason, MetaSystems has developed a new classifier which is able to recognize nuclei within tissue sections in order to determine the HER2 amplification status on nuclei basis. We call this new programming algorithm “nuclei-sampling classifier”. In this study, we evaluated the accuracy of the “nuclei-sampling classifier” in determining HER2 gene amplification by FISH in nuclei of breast cancer cells. To this aim, we randomly selected from our cohort 64 breast cancer specimens (32 nonamplified and 32 amplified) and we compared results obtained through manual scoring and through this new classifier. The new classifier automatically recognized individual nuclei. The automated analysis was followed by an optional human correction, during which the user interacted with the software in order to improve the selection of cell nuclei automatically selected. Overall concordance between manual scoring and automated nuclei-sampling analysis was 98.4% (100% for nonamplified cases and 96.9% for amplified cases). However, after human correction, concordance between the two methods was 100%. We conclude that the nuclei-based classifier is a new available tool for automated quantitative HER2 FISH signals analysis in nuclei in breast cancer specimen and it can be used for clinical purposes. PMID:23379971

In situ surface-enhanced Raman scattering spectroscopy exploring molecular changes of drug-treated cancer cell nucleus.

PubMed

Liang, Lijia; Huang, Dianshuai; Wang, Hailong; Li, Haibo; Xu, Shuping; Chang, Yixin; Li, Hui; Yang, Ying-Wei; Liang, Chongyang; Xu, Weiqing

2015-02-17

Investigating the molecular changes of cancer cell nucleus with drugs treatment is crucial for the design of new anticancer drugs, the development of novel diagnostic strategies, and the advancement of cancer therapy efficiency. In order to better understand the action effects of drugs, accurate location and in situ acquisition of the molecular information of the cell nuclei are necessary. In this work, we report a microspectroscopic technique called dark-field and fluorescence coimaging assisted surface-enhanced Raman scattering (SERS) spectroscopy, combined with nuclear targeting nanoprobes, to in situ study Soma Gastric Cancer (SGC-7901) cell nuclei treated with two model drugs, e.g., DNA binder (Hoechst33342) and anticancer drug (doxorubicin, Dox) via spectral analysis at the molecular level. Nuclear targeting nanoprobes with an assembly structure of thiol-modified polyethylene glycol polymers (PEG) and nuclear localizing signal peptides (NLS) around gold nanorods (AuNRs) were prepared to achieve the amplified SERS signals of biomolecules in the cell nuclei. With the assistance of dark field/fluorescence imaging with simultaneous location, in situ SERS spectra in one cell nucleus were measured and analyzed to disclose the effects of Hoechst33342 and Dox on main biomolecules in the cell nuclei. The experimental results show that this method possesses great potential to investigate the targets of new anticancer drugs and the real-time monitoring of the dynamic changes of cells caused by exogenous molecules.

Stacked Sparse Autoencoder (SSAE) for Nuclei Detection on Breast Cancer Histopathology Images.

PubMed

Xu, Jun; Xiang, Lei; Liu, Qingshan; Gilmore, Hannah; Wu, Jianzhong; Tang, Jinghai; Madabhushi, Anant

2016-01-01

Automated nuclear detection is a critical step for a number of computer assisted pathology related image analysis algorithms such as for automated grading of breast cancer tissue specimens. The Nottingham Histologic Score system is highly correlated with the shape and appearance of breast cancer nuclei in histopathological images. However, automated nucleus detection is complicated by 1) the large number of nuclei and the size of high resolution digitized pathology images, and 2) the variability in size, shape, appearance, and texture of the individual nuclei. Recently there has been interest in the application of "Deep Learning" strategies for classification and analysis of big image data. Histopathology, given its size and complexity, represents an excellent use case for application of deep learning strategies. In this paper, a Stacked Sparse Autoencoder (SSAE), an instance of a deep learning strategy, is presented for efficient nuclei detection on high-resolution histopathological images of breast cancer. The SSAE learns high-level features from just pixel intensities alone in order to identify distinguishing features of nuclei. A sliding window operation is applied to each image in order to represent image patches via high-level features obtained via the auto-encoder, which are then subsequently fed to a classifier which categorizes each image patch as nuclear or non-nuclear. Across a cohort of 500 histopathological images (2200 × 2200) and approximately 3500 manually segmented individual nuclei serving as the groundtruth, SSAE was shown to have an improved F-measure 84.49% and an average area under Precision-Recall curve (AveP) 78.83%. The SSAE approach also out-performed nine other state of the art nuclear detection strategies.

Spatial Statistics for Segmenting Histological Structures in H&E Stained Tissue Images.

PubMed

Nguyen, Luong; Tosun, Akif Burak; Fine, Jeffrey L; Lee, Adrian V; Taylor, D Lansing; Chennubhotla, S Chakra

2017-07-01

Segmenting a broad class of histological structures in transmitted light and/or fluorescence-based images is a prerequisite for determining the pathological basis of cancer, elucidating spatial interactions between histological structures in tumor microenvironments (e.g., tumor infiltrating lymphocytes), facilitating precision medicine studies with deep molecular profiling, and providing an exploratory tool for pathologists. This paper focuses on segmenting histological structures in hematoxylin- and eosin-stained images of breast tissues, e.g., invasive carcinoma, carcinoma in situ, atypical and normal ducts, adipose tissue, and lymphocytes. We propose two graph-theoretic segmentation methods based on local spatial color and nuclei neighborhood statistics. For benchmarking, we curated a data set of 232 high-power field breast tissue images together with expertly annotated ground truth. To accurately model the preference for histological structures (ducts, vessels, tumor nets, adipose, etc.) over the remaining connective tissue and non-tissue areas in ground truth annotations, we propose a new region-based score for evaluating segmentation algorithms. We demonstrate the improvement of our proposed methods over the state-of-the-art algorithms in both region- and boundary-based performance measures.

Quantitative microscopy uncovers ploidy changes during mitosis in live Drosophila embryos and their effect on nuclear size.

PubMed

Puah, Wee Choo; Chinta, Rambabu; Wasser, Martin

2017-03-15

Time-lapse microscopy is a powerful tool to investigate cellular and developmental dynamics. In Drosophila melanogaster , it can be used to study division cycles in embryogenesis. To obtain quantitative information from 3D time-lapse data and track proliferating nuclei from the syncytial stage until gastrulation, we developed an image analysis pipeline consisting of nuclear segmentation, tracking, annotation and quantification. Image analysis of maternal-haploid ( mh ) embryos revealed that a fraction of haploid syncytial nuclei fused to give rise to nuclei of higher ploidy (2n, 3n, 4n). Moreover, nuclear densities in mh embryos at the mid-blastula transition varied over threefold. By tracking synchronized nuclei of different karyotypes side-by-side, we show that DNA content determines nuclear growth rate and size in early interphase, while the nuclear to cytoplasmic ratio constrains nuclear growth during late interphase. mh encodes the Drosophila ortholog of human Spartan, a protein involved in DNA damage tolerance. To explore the link between mh and chromosome instability, we fluorescently tagged Mh protein to study its subcellular localization. We show Mh-mKO2 localizes to nuclear speckles that increase in numbers as nuclei expand in interphase. In summary, quantitative microscopy can provide new insights into well-studied genes and biological processes. © 2017. Published by The Company of Biologists Ltd.

Systematic Morphometry of Catecholamine Nuclei in the Brainstem.

PubMed

Bucci, Domenico; Busceti, Carla L; Calierno, Maria T; Di Pietro, Paola; Madonna, Michele; Biagioni, Francesca; Ryskalin, Larisa; Limanaqi, Fiona; Nicoletti, Ferdinando; Fornai, Francesco

2017-01-01

Catecholamine nuclei within the brainstem reticular formation (RF) play a pivotal role in a variety of brain functions. However, a systematic characterization of these nuclei in the very same experimental conditions is missing so far. Tyrosine hydroxylase (TH) immune-positive cells of the brainstem correspond to dopamine (DA)-, norepinephrine (NE)-, and epinephrine (E)-containing cells. Here, we report a systematic count of TH-positive neurons in the RF of the mouse brainstem by using stereological morphometry. All these nuclei were analyzed for anatomical localization, rostro-caudal extension, volume, neuron number, neuron density, and mean neuronal area for each nucleus. The present data apart from inherent informative value wish to represent a reference for neuronal mapping in those studies investigating the functional anatomy of the brainstem RF. These include: the sleep-wake cycle, movement control, muscle tone modulation, mood control, novelty orienting stimuli, attention, archaic responses to internal and external stressful stimuli, anxiety, breathing, blood pressure, and innumerable activities modulated by the archaic iso-dendritic hard core of the brainstem RF. Most TH-immune-positive cells fill the lateral part of the RF, which indeed possesses a high catecholamine content. A few nuclei are medial, although conventional nosography considers all these nuclei as part of the lateral column of the RF. Despite the key role of these nuclei in psychiatric and neurological disorders, only a few of them aspired a great attention in biomedical investigation, while most of them remain largely obscure although intense research is currently in progress. A simultaneous description of all these nuclei is not simply key to comprehend the variety of brainstem catecholamine reticular neurons, but probably represents an intrinsically key base for understanding brain physiology and physiopathology.

Systematic Morphometry of Catecholamine Nuclei in the Brainstem

PubMed Central

Bucci, Domenico; Busceti, Carla L.; Calierno, Maria T.; Di Pietro, Paola; Madonna, Michele; Biagioni, Francesca; Ryskalin, Larisa; Limanaqi, Fiona; Nicoletti, Ferdinando; Fornai, Francesco

2017-01-01

Catecholamine nuclei within the brainstem reticular formation (RF) play a pivotal role in a variety of brain functions. However, a systematic characterization of these nuclei in the very same experimental conditions is missing so far. Tyrosine hydroxylase (TH) immune-positive cells of the brainstem correspond to dopamine (DA)-, norepinephrine (NE)-, and epinephrine (E)-containing cells. Here, we report a systematic count of TH-positive neurons in the RF of the mouse brainstem by using stereological morphometry. All these nuclei were analyzed for anatomical localization, rostro-caudal extension, volume, neuron number, neuron density, and mean neuronal area for each nucleus. The present data apart from inherent informative value wish to represent a reference for neuronal mapping in those studies investigating the functional anatomy of the brainstem RF. These include: the sleep-wake cycle, movement control, muscle tone modulation, mood control, novelty orienting stimuli, attention, archaic responses to internal and external stressful stimuli, anxiety, breathing, blood pressure, and innumerable activities modulated by the archaic iso-dendritic hard core of the brainstem RF. Most TH-immune-positive cells fill the lateral part of the RF, which indeed possesses a high catecholamine content. A few nuclei are medial, although conventional nosography considers all these nuclei as part of the lateral column of the RF. Despite the key role of these nuclei in psychiatric and neurological disorders, only a few of them aspired a great attention in biomedical investigation, while most of them remain largely obscure although intense research is currently in progress. A simultaneous description of all these nuclei is not simply key to comprehend the variety of brainstem catecholamine reticular neurons, but probably represents an intrinsically key base for understanding brain physiology and physiopathology. PMID:29163071

[The polyploidization characteristics of the hepatocytes of the mouse-like hamster Calomyscus mystax].

PubMed

Anatskaia, O V; Malikov, V G; Meĭer, M N; Kudriavtsev, B N

1995-01-01

A cytophotometric measurement of DNA content in hepatocytes of maturing mouse-like hamsters was made. Cells belonging to ordinary mammalian ploidy classes 2c, 2c x 2, 4c, and 4c x 2 made about 90% of the hepatocyte population. The share of binucleated cells wa high (about 80%), the majority of these cells being 2c X 2 hepatocytes. Binucleated cells with tetraploid and diploid nuclei occur in almost every animal. An average hepatocyte ploidy level in mouse-like hamster is 4.6c. The main peculiarity of parenchymal liver cell populations is that up 5% of hepatocytes contain 3--11 nuclei of different ploidy classes. Multinucleated cells increase in number from 1.5% to 4% within the period from one year (the age of maturation) to two years. Later on their percentage does not change. It is found that in binucleated and multinucleated hepatocytes DNA synthesis can proceed asynchronously. Asynchrony in DNA synthesis elevates as the number of nuclei increases. Among the 2c x 2 and 2c x 3 cells an uneven distribution of 3H-thymidine label can occur, respectively, in 5 and in 50% cases, whereas all the cells with more than 3 nuclei display an uneven an uneven 3H-thymidin label distribution. The formation of multinucleated cells is supposed to be associated with asynchrony in DNA-synthesis in binucleated cells and with the restitution of mitosis.

Redox Changes During the Cell Cycle in the Embryonic Root Meristem of Arabidopsis thaliana.

PubMed

de Simone, Ambra; Hubbard, Rachel; de la Torre, Natanael Viñegra; Velappan, Yazhini; Wilson, Michael; Considine, Michael J; Soppe, Wim J J; Foyer, Christine H

2017-12-20

The aim of this study was to characterize redox changes in the nuclei and cytosol occurring during the mitotic cell cycle in the embryonic roots of germinating Arabidopsis seedlings, and to determine how redox cycling was modified in mutants with a decreased capacity for ascorbate synthesis. Using an in vivo reduction-oxidation (redox) reporter (roGFP2), we show that transient oxidation of the cytosol and the nuclei occurred at G1 in the synchronized dividing cells of the Arabidopsis root apical meristem, with reduction at G2 and mitosis. This redox cycle was absent from low ascorbate mutants in which nuclei were significantly more oxidized than controls. The cell cycle-dependent increase in nuclear size was impaired in the ascorbate-deficient mutants, which had fewer cells per unit area in the root proliferation zone. The transcript profile of the dry seeds and size of the imbibed seeds was strongly influenced by low ascorbate but germination, dormancy release and seed aging characteristics were unaffected. These data demonstrate the presence of a redox cycle within the plant cell cycle and that the redox state of the nuclei is an important factor in cell cycle progression. Controlled oxidation is a key feature of the early stages of the plant cell cycle. However, sustained mild oxidation restricts nuclear functions and impairs progression through the cell cycle leading to fewer cells in the root apical meristem. Antioxid. Redox Signal. 27, 1505-1519.

Nuclear localization signal-dependent and -independent movements of Drosophila melanogaster dUTPase isoforms during nuclear cleavage

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muha, Villo; Zagyva, Imre; Venkei, Zsolt

2009-04-03

Two dUTPase isoforms (23 kDa and 21 kDa) are present in the fruitfly with the sole difference of an N-terminal extension. In Drosophila embryo, both isoforms are detected inside the nucleus. Here, we investigated the function of the N-terminal segment using eYFP-dUTPase constructs. In Schneider 2 cells, only the 23 kDa construct showed nuclear localization arguing that it may contain a nuclear localization signal (NLS). Sequence comparisons identified a lysine-rich nonapeptide with similarity to the human c-myc NLS. In Drosophila embryos during nuclear cleavages, the 23 kDa isoform showed the expected localization shifts. Contrariwise, although the 21 kDa isoform wasmore » excluded from the nuclei during interphase, it was shifted to the nucleus during prophase and forthcoming mitotic steps. The observed dynamic localization character showed strict timing to the nuclear cleavage phases and explained how both isoforms can be present within the nuclear microenvironment, although at different stages of cell cycle.« less

Nuclear morphology for the detection of alterations in bronchial cells from lung cancer: an attempt to improve sensitivity and specificity.

PubMed

Fafin-Lefevre, Mélanie; Morlais, Fabrice; Guittet, Lydia; Clin, Bénédicte; Launoy, Guy; Galateau-Sallé, Françoise; Plancoulaine, Benoît; Herlin, Paulette; Letourneux, Marc

2011-08-01

To identify which morphologic or densitometric parameters are modified in cell nuclei from bronchopulmonary cancer based on 18 parameters involving shape, intensity, chromatin, texture, and DNA content and develop a bronchopulmonary cancer screening method relying on analysis of sputum sample cell nuclei. A total of 25 sputum samples from controls and 22 bronchial aspiration samples from patients presenting with bronchopulmonary cancer who were professionally exposed to cancer were used. After Feulgen staining, 18 morphologic and DNA content parameters were measured on cell nuclei, via image cytom- etry. A method was developed for analyzing distribution quantiles, compared with simply interpreting mean values, to characterize morphologic modifications in cell nuclei. Distribution analysis of parameters enabled us to distinguish 13 of 18 parameters that demonstrated significant differences between controls and cancer cases. These parameters, used alone, enabled us to distinguish two population types, with both sensitivity and specificity > 70%. Three parameters offered 100% sensitivity and specificity. When mean values offered high sensitivity and specificity, comparable or higher sensitivity and specificity values were observed for at least one of the corresponding quantiles. Analysis of modification in morphologic parameters via distribution analysis proved promising for screening bronchopulmonary cancer from sputum.

Using simulated fluorescence cell micrographs for the evaluation of cell image segmentation algorithms.

PubMed

Wiesmann, Veit; Bergler, Matthias; Palmisano, Ralf; Prinzen, Martin; Franz, Daniela; Wittenberg, Thomas

2017-03-18

Manual assessment and evaluation of fluorescent micrograph cell experiments is time-consuming and tedious. Automated segmentation pipelines can ensure efficient and reproducible evaluation and analysis with constant high quality for all images of an experiment. Such cell segmentation approaches are usually validated and rated in comparison to manually annotated micrographs. Nevertheless, manual annotations are prone to errors and display inter- and intra-observer variability which influence the validation results of automated cell segmentation pipelines. We present a new approach to simulate fluorescent cell micrographs that provides an objective ground truth for the validation of cell segmentation methods. The cell simulation was evaluated twofold: (1) An expert observer study shows that the proposed approach generates realistic fluorescent cell micrograph simulations. (2) An automated segmentation pipeline on the simulated fluorescent cell micrographs reproduces segmentation performances of that pipeline on real fluorescent cell micrographs. The proposed simulation approach produces realistic fluorescent cell micrographs with corresponding ground truth. The simulated data is suited to evaluate image segmentation pipelines more efficiently and reproducibly than it is possible on manually annotated real micrographs.

Fluorescent Magnesium Nanocomplex in Protein Scaffold for Cell Nuclei Imaging Application

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandya, Alok; Tripathi, Apritam; Purohit, Rahul

2015-10-27

Here in, we report a facile strategy for the synthesis of water-soluble ultra-fine blue emitting fluorescent Magnesium nanoparticles-protein complex (MgNC). This MgNC is demonstrated to exhibit excellent photo stability and biocompatibility. It was also observed that MgNC stain cell nuclei with high specifcity.

Release of specific proteins from nuclei of HL-60 and MOLT-4 cells by antitumor drugs having affinity to nucleic acids.

PubMed

Lassota, P; Melamed, M R; Darzynkiewicz, Z

The binding sites for mitoxantrone (MIT), Ametantrone (AMT), doxorubicin (DOX), actinomycin D (AMD) and ethidium bromide (EB) in nuclei from exponentially growing and differentiating human promyelocytic HL-60 and lymphocytic leukemic MOLT-4 cells were studied by gel electrophoresis of proteins selectively released during titration of these nuclei with the drugs. Each drug at different drug: DNA binding ratios resulted in a characteristic pattern of protein elution and/or retention. For example, in nuclei from exponentially growing HL-60 cells, MIT affected 44 nuclear proteins that were different from those affected by EB; of these 29 were progressively released at increasing MIT:DNA ratios, 11 were transiently released (i.e. only at a low MIT:DNA ratio) and 4 entrapped. Patterns of proteins displaced from nuclei of exponentially growing HL-60 cells differed from those of cells undergoing myeloid differentiation as well as from those of exponentially growing MOLT-4 cells. The first effects were seen at a binding density of approximately one drug molecule per 10-50 base pairs of DNA. The observed selective displacement of proteins may reflect drug-altered affinity of the binding sites for those proteins, for example due to a change of nucleic acid or protein conformation upon binding the ligand. The data show that the binding site(s) for each of the ligands studied is different and the differences correlate with variability in chemical structure between the ligands. The nature of the drug-affected proteins may provide clues regarding antitumor or cytotoxic mechanisms of drug action.

Total Absorption Study of Beta Decays Relevant for Nuclear Applications and Nuclear Structure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Algora, A.; Valencia, E.; Tain, J. L.

2014-06-01

We present an overview of our activities related to the study of the beta decay of neutron rich nuclei relevant for nuclear applications. Recent results of the study of the beta decay of Br using a new segmented total absorption spectrometer are presented. Our measurements were performed at the IGISOL facility using trap-assisted total absorption spectroscopy.

Flavanol binding of nuclei from tree species.

PubMed

Feucht, W; Treutter, D; Polster, J

2004-01-01

Light microscopy was used to examine the nuclei of five tree species with respect to the presence of flavanols. Flavanols develop a blue colouration in the presence of a special p-dimethylaminocinnamaldehyde (DMACA) reagent that enables those nuclei loaded with flavanols to be recognized. Staining of the nuclei was most pronounced in both Tsuga canadensis and Taxus baccata, variable in Metasequoia glyptostroboides, faint in Coffea arabica and minimal in Prunus avium. HPLC analysis showed that the five species contained substantial amounts of different flavanols such as catechin, epicatechin and proanthocyanidins. Quantitatively, total flavanols were quite different among the species. The nuclei themselves, as studied in Tsuga seed wings, were found to contain mainly catechin, much lower amounts of epicatechin and traces of proanthocyanidins. Blue-coloured nuclei located centrally in small cells were often found to maximally occupy up to 90% of a cell's radius, and the surrounding small rim of cytoplasm was visibly free of flavanols. A survey of 34 gymnosperm and angiosperm species indicated that the first group has much higher nuclear binding capacities for flavanols than the second group.

Distributed Cerebellar Motor Learning: A Spike-Timing-Dependent Plasticity Model

PubMed Central

Luque, Niceto R.; Garrido, Jesús A.; Naveros, Francisco; Carrillo, Richard R.; D'Angelo, Egidio; Ros, Eduardo

2016-01-01

Deep cerebellar nuclei neurons receive both inhibitory (GABAergic) synaptic currents from Purkinje cells (within the cerebellar cortex) and excitatory (glutamatergic) synaptic currents from mossy fibers. Those two deep cerebellar nucleus inputs are thought to be also adaptive, embedding interesting properties in the framework of accurate movements. We show that distributed spike-timing-dependent plasticity mechanisms (STDP) located at different cerebellar sites (parallel fibers to Purkinje cells, mossy fibers to deep cerebellar nucleus cells, and Purkinje cells to deep cerebellar nucleus cells) in close-loop simulations provide an explanation for the complex learning properties of the cerebellum in motor learning. Concretely, we propose a new mechanistic cerebellar spiking model. In this new model, deep cerebellar nuclei embed a dual functionality: deep cerebellar nuclei acting as a gain adaptation mechanism and as a facilitator for the slow memory consolidation at mossy fibers to deep cerebellar nucleus synapses. Equipping the cerebellum with excitatory (e-STDP) and inhibitory (i-STDP) mechanisms at deep cerebellar nuclei afferents allows the accommodation of synaptic memories that were formed at parallel fibers to Purkinje cells synapses and then transferred to mossy fibers to deep cerebellar nucleus synapses. These adaptive mechanisms also contribute to modulate the deep-cerebellar-nucleus-output firing rate (output gain modulation toward optimizing its working range). PMID:26973504

Organisation of the endosperm and endosperm-placenta syncytia in bladderworts (Utricularia, Lentibulariaceae) with emphasis on the microtubule arrangement.

PubMed

Płachno, Bartosz J; Swiątek, Piotr; Sas-Nowosielska, Hanna; Kozieradzka-Kiszkurno, Małgorzata

2013-08-01

Multinucleate cells play an important role in higher plants, especially during reproduction; however, the configurations of their cytoskeletons, which are formed as a result of mitosis without cytokinesis, have mainly been studied in coenocytes. Previous authors have proposed that in spite of their developmental origin (cell fusion or mitosis without cytokinesis), in multinucleate plant cells, radiating microtubules determine the regular spacing of individual nuclei. However, with the exception of specific syncytia induced by parasitic nematodes, there is no information about the microtubular cytoskeleton in plant heterokaryotic syncytia, i.e. when the nuclei of fused cells come from different cell pools. In this paper, we describe the arrangement of microtubules in the endosperm and special endosperm-placenta syncytia in two Utricularia species. These syncytia arise from different progenitor cells, i.e. cells of the maternal sporophytic nutritive tissue and the micropylar endosperm haustorium (both maternal and paternal genetic material). The development of the endosperm in the two species studied was very similar. We describe microtubule configurations in the three functional endosperm domains: the micropylar syncytium, the endosperm proper and the chalazal haustorium. In contrast to plant syncytia that are induced by parasitic nematodes, the syncytia of Utricularia had an extensive microtubular network. Within each syncytium, two giant nuclei, coming from endosperm cells, were surrounded by a three-dimensional cage of microtubules, which formed a huge cytoplasmic domain. At the periphery of the syncytium, where new protoplasts of the nutritive cells join the syncytium, the microtubules formed a network which surrounded small nuclei from nutritive tissue cells and were also distributed through the cytoplasm. Thus, in the Utricularia syncytium, there were different sized cytoplasmic domains, whose architecture depended on the source and size of the nuclei. The endosperm proper was isolated from maternal (ovule) tissues by a cuticle layer, so the syncytium and chalazal haustorium were the only way for nutrients to be transported from the maternal tissue towards the developing embryo.

Involvement of microtubules and 10-nm filaments in the movement and positioning of nuclei in syncytia

PubMed Central

1979-01-01

Previous studies (Holmes, K.V., and P.W. Choppin. J. Exp. Med. 124:501- 520; J. Cell Biol. 39:526-543) showed that infection of baby hamster kidney (BHK21-F) cells with the parainfluenza virus SV5 causes extensive cell fusion, that nuclei migrate in the syncytial cytoplasm and align in tightly-packed rows, and that microtubules are involved in nuclear movement and alignment. The role of microtubules, 10-nm filaments, and actin-containing microfilaments in this process has been investigated by immunofluorescence microscopy using specific antisera, time-lapse cinematography, and electron microscopy. During cell fusion, micro tubules and 10-nm filaments from many cells form large bundles which are localized between rows of nuclei. No organized bundles of actin fibers were detected in these areas, although actin fibers were observed in regions away from the aligned nuclei. Although colchicine disrupts microtubules and inhibits nuclear movement, cytochalasin B (CB; 20-50 microgram/ml) does not inhibit cell fusion or nuclear movement. However, CB alters the shape of the syncytium, resulting in long filamentous processes extending from a central region. When these processes from neighboring cells make contact, fusion occurs, and nuclei migrate through the channels which are formed. Electron and immunofluorescence microscopy reveal bundles of microtubules and 10-nm filaments in parallel arrays within these processes, but no bundles of microfilaments were detected. The effect of CB on the structural integrity of microfilaments at this high concentration (20 microgram/ml) was demonstrated by the disappearance of filaments interacting with heavy meromyosin. Cycloheximide (20 microgram/ml) inhibits protein synthesis but does not affect cell fusion, the formation of microtubules and 10-nm filament bundles, or nuclear migration and alignment; thus, continued protein synthesis is not required. The association of microtubules and 10-nm filaments with nuclear migration and alignment suggests that microtubules and 10-nm filaments are two components in a system which serves both cytoskeletal and force-generating functions in intracellular movement and position of nuclei. PMID:227913

Neuroanatomical organization of the cholinergic system in the central nervous system of a basal actinopterygian fish, the senegal bichir Polypterus senegalus.

PubMed

López, Jesús M; Perlado, Jorge; Morona, Ruth; Northcutt, R Glenn; González, Agustín

2013-01-01

Polypterid bony fishes are believed to be basal to other living ray-finned fishes, and their brain organization is therefore critical in providing information as to primitive neural characters that existed in the earliest ray-finned fishes. The cholinergic system has been characterized in more advanced ray-finned fishes, but not in polypterids. In order to establish which cholinergic neural centers characterized the earliest ray-finned fishes, the distribution of choline acetyltransferase (ChAT) is described in Polypterus and compared with the distribution of this molecule in other ray-finned fishes. Cell groups immunoreactive for ChAT were observed in the hypothalamus, the habenula, the optic tectum, the isthmus, the cranial motor nuclei, and the spinal motor column. Cholinergic fibers were observed in both the telencephalic pallium and the subpallium, in the thalamus and pretectum, in the optic tectum and torus semicircularis, in the mesencephalic tegmentum, in the cerebellar crest, in the solitary nucleus, and in the dorsal column nuclei. Comparison of the data within a segmental neuromeric context indicates that the cholinergic system in polypterid fishes is generally similar to that in other ray-finned fishes, but cholinergic-positive neurons in the pallium and subpallium, and in the thalamus and cerebellum, of teleosts appear to have evolved following the separation of polypterids and other ray-finned fishes. Copyright © 2012 Wiley Periodicals, Inc.

NeuN+ Neuronal Nuclei in Non-Human Primate Prefrontal Cortex and Subcortical White Matter After Clozapine Exposure

PubMed Central

Halene, Tobias B.; Kozlenkov, Alexey; Jiang, Yan; Mitchell, Amanda; Javidfar, Behnam; Dincer, Aslihan; Park, Royce; Wiseman, Jennifer; Croxson, Paula; Giannaris, Eustathia Lela; Hof, Patrick R.; Roussos, Panos; Dracheva, Stella; Hemby, Scott E.; Akbarian, Schahram

2016-01-01

Increased neuronal densities in subcortical white matter have been reported for some cases with schizophrenia. The underlying cellular and molecular mechanisms remain unresolved. We exposed 26 young adult macaque monkeys for 6 months to either clozapine, haloperidol or placebo and measured by structural MRI frontal gray and white matter volumes before and after treatment, followed by observer-independent, flow-cytometry-based quantification of neuronal and non-neuronal nuclei and molecular fingerprinting of cell-type specific transcripts. After clozapine exposure, the proportion of nuclei expressing the neuronal marker NeuN increased by approximately 50% in subcortical white matter, in conjunction with a more subtle and non-significant increase in overlying gray matter. Numbers and proportions of nuclei expressing the oligodendrocyte lineage marker, OLIG2, and cell-type specific RNA expression patterns, were maintained after antipsychotic drug exposure. Frontal lobe gray and white matter volumes remained indistinguishable between antipsychotic-drug-exposed and control groups. Chronic clozapine exposure increases the proportion of NeuN+ nuclei in frontal subcortical white matter, without alterations in frontal lobe volumes or cell type-specific gene expression. Further exploration of neurochemical plasticity in non-human primate brain exposed to antipsychotic drugs is warranted. PMID:26776227

Bigger Brains or Bigger Nuclei? Regulating the Size of Auditory Structures in Birds

PubMed Central

Kubke, M. Fabiana; Massoglia, Dino P.; Carr, Catherine E.

2012-01-01

Increases in the size of the neuronal structures that mediate specific behaviors are believed to be related to enhanced computational performance. It is not clear, however, what developmental and evolutionary mechanisms mediate these changes, nor whether an increase in the size of a given neuronal population is a general mechanism to achieve enhanced computational ability. We addressed the issue of size by analyzing the variation in the relative number of cells of auditory structures in auditory specialists and generalists. We show that bird species with different auditory specializations exhibit variation in the relative size of their hindbrain auditory nuclei. In the barn owl, an auditory specialist, the hind-brain auditory nuclei involved in the computation of sound location show hyperplasia. This hyperplasia was also found in songbirds, but not in non-auditory specialists. The hyperplasia of auditory nuclei was also not seen in birds with large body weight suggesting that the total number of cells is selected for in auditory specialists. In barn owls, differences observed in the relative size of the auditory nuclei might be attributed to modifications in neurogenesis and cell death. Thus, hyperplasia of circuits used for auditory computation accompanies auditory specialization in different orders of birds. PMID:14726625

Internuclear Genetic Transfer in Vegetative Dikaryons of SCHIZOPHYLLUM COMMUNE: I. Di-Mon Mating Analysis

PubMed Central

Leonard, Thomas J.; Dick, Stanley; Gaber, Richard F.

1978-01-01

A series of hemi-compatible dikaryon x monokaryon (di-mon) matings was designed to determine whether there was any genetic interaction between the dikaryotic nuclei. One of the nuclei in each dikaryon was known to carry a recessive gene (mnd) that promoted the development of an abnormal growth form, mound. Dikaryons containing both mnd + and mnd nuclei produced mosaic colonies that consisted of three distinct kinds of hyphae: mound, fruiting body, and vegetative (devoid of any multihyphal structure). When dikaryotic hyphae from each of these morphological regions were used in di-mon matings, the genetic and developmental characteristics of the selected nuclear types were examined in the resulting derived dikaryons. The results showed that fruiting-body and vegetative cells contained the expected mnd and mnd+ nuclei. Dikaryotic mound hyphae, however, contained only mnd nuclei. In a manner as yet unresolved, but clearly dependent on the presence of the mnd allele, the mnd + allele of a wild nucleus was altered to, or acquired, the mnd allele. A number of hypotheses were considered to explain the genetic event(s) that generates [mnd + mnd*] dikaryotic cells from [mnd+ + mnd] cells, but none was found to be entirely satisfactory. PMID:17248788

Deconvolution of Energy Spectra in the ATIC Experiment

NASA Technical Reports Server (NTRS)

Batkov, K. E.; Panov, A. D.; Adams, J. H.; Ahn, H. S.; Bashindzhagyan, G. L.; Chang, J.; Christl, M.; Fazley, A. R.; Ganel, O.; Gunasigha, R. M.;

Reproducibility of myelin content-based human habenula segmentation at 3 Tesla.

PubMed

Kim, Joo-Won; Naidich, Thomas P; Joseph, Joshmi; Nair, Divya; Glasser, Matthew F; O'halloran, Rafael; Doucet, Gaelle E; Lee, Won Hee; Krinsky, Hannah; Paulino, Alejandro; Glahn, David C; Anticevic, Alan; Frangou, Sophia; Xu, Junqian

2018-03-26

In vivo morphological study of the human habenula, a pair of small epithalamic nuclei adjacent to the dorsomedial thalamus, has recently gained significant interest for its role in reward and aversion processing. However, segmenting the habenula from in vivo magnetic resonance imaging (MRI) is challenging due to the habenula's small size and low anatomical contrast. Although manual and semi-automated habenula segmentation methods have been reported, the test-retest reproducibility of the segmented habenula volume and the consistency of the boundaries of habenula segmentation have not been investigated. In this study, we evaluated the intra- and inter-site reproducibility of in vivo human habenula segmentation from 3T MRI (0.7-0.8 mm isotropic resolution) using our previously proposed semi-automated myelin contrast-based method and its fully-automated version, as well as a previously published manual geometry-based method. The habenula segmentation using our semi-automated method showed consistent boundary definition (high Dice coefficient, low mean distance, and moderate Hausdorff distance) and reproducible volume measurement (low coefficient of variation). Furthermore, the habenula boundary in our semi-automated segmentation from 3T MRI agreed well with that in the manual segmentation from 7T MRI (0.5 mm isotropic resolution) of the same subjects. Overall, our proposed semi-automated habenula segmentation showed reliable and reproducible habenula localization, while its fully-automated version offers an efficient way for large sample analysis. © 2018 Wiley Periodicals, Inc.

FogBank: a single cell segmentation across multiple cell lines and image modalities.

PubMed

Chalfoun, Joe; Majurski, Michael; Dima, Alden; Stuelten, Christina; Peskin, Adele; Brady, Mary

2014-12-30

Many cell lines currently used in medical research, such as cancer cells or stem cells, grow in confluent sheets or colonies. The biology of individual cells provide valuable information, thus the separation of touching cells in these microscopy images is critical for counting, identification and measurement of individual cells. Over-segmentation of single cells continues to be a major problem for methods based on morphological watershed due to the high level of noise in microscopy cell images. There is a need for a new segmentation method that is robust over a wide variety of biological images and can accurately separate individual cells even in challenging datasets such as confluent sheets or colonies. We present a new automated segmentation method called FogBank that accurately separates cells when confluent and touching each other. This technique is successfully applied to phase contrast, bright field, fluorescence microscopy and binary images. The method is based on morphological watershed principles with two new features to improve accuracy and minimize over-segmentation. First, FogBank uses histogram binning to quantize pixel intensities which minimizes the image noise that causes over-segmentation. Second, FogBank uses a geodesic distance mask derived from raw images to detect the shapes of individual cells, in contrast to the more linear cell edges that other watershed-like algorithms produce. We evaluated the segmentation accuracy against manually segmented datasets using two metrics. FogBank achieved segmentation accuracy on the order of 0.75 (1 being a perfect match). We compared our method with other available segmentation techniques in term of achieved performance over the reference data sets. FogBank outperformed all related algorithms. The accuracy has also been visually verified on data sets with 14 cell lines across 3 imaging modalities leading to 876 segmentation evaluation images. FogBank produces single cell segmentation from confluent cell sheets with high accuracy. It can be applied to microscopy images of multiple cell lines and a variety of imaging modalities. The code for the segmentation method is available as open-source and includes a Graphical User Interface for user friendly execution.

Cell Pleomorphism and Cytoskeleton Disorganization in Human Liver Cancer.

PubMed

Cheng, Chiung-Chi; Lai, Yen-Chang Clark; Lai, Yih-Shyong; Chao, Wei-Ting; Tseng, Yu-Hui; Hsu, Yung-Hsiang; Chen, You-Yin; Liu, Yi-Hsiang

Nucleoskeleton maintains the framework of a cell nucleus that is required for a variety of nuclear functions. However, the nature of nucleoskeleton structure has not been yet clearly elucidated due to microscopy visualization limitations. Plectin, a nuclear pore-permeable component of cytoskeleton, exhibits a role of cross-linking between cytoplasmic intermediate filaments and nuclear lamins. Presumably, plectin is also a part of nucleoskeleton. Previously, we demonstrated that pleomorphism of hepatoma cells is the consequence of cytoskeletal changes mediated by plectin deficiency. In this study, we applied a variety of technologies to detect the cytoskeletons in liver cells. The images of confocal microscopy did not show the existence of plectin, intermediate filaments, microfilaments and microtubules in hepatic nuclei. However, in the isolated nuclear preparation, immunohistochemical staining revealed positive results for plectin and cytoskeletal proteins that may contribute to the contamination derived from cytoplasmic residues. Therefore, confocal microscopy provides a simple and effective technology to observe the framework of nucleoskeleton. Accordingly, we verified that cytoskeletons are not found in hepatic cell nuclei. Furthermore, the siRNA-mediated knockdown of plectin in liver cells leads to collapsed cytoskeleton, cell transformation and pleomorphic nuclei. Plectin and cytoskeletons were not detected in the nuclei of liver cells compared to the results of confocal microscopy. Despite the absence of nuclear plectin and cytoskeletal filaments, the evidence provided support that nuclear pleomorphism of cancer cells is correlated with the cytoplasmic disorganization of cytoskeleton. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Vertical uniformity of cells and nuclei in epithelial monolayers.

PubMed

Neelam, Srujana; Hayes, Peter Robert; Zhang, Qiao; Dickinson, Richard B; Lele, Tanmay P

2016-01-22

Morphological variability in cytoskeletal organization, organelle position and cell boundaries is a common feature of cultured cells. Remarkable uniformity and reproducibility in structure can be accomplished by providing cells with defined geometric cues. Cells in tissues can also self-organize in the absence of directing extracellular cues; however the mechanical principles for such self-organization are not understood. We report that unlike horizontal shapes, the vertical shapes of the cell and nucleus in the z-dimension are uniform in cells in cultured monolayers compared to isolated cells. Apical surfaces of cells and their nuclei in monolayers were flat and heights were uniform. In contrast, isolated cells, or cells with disrupted cell-cell adhesions had nuclei with curved apical surfaces and variable heights. Isolated cells cultured within micron-sized square wells displayed flat cell and nuclear shapes similar to cells in monolayers. Local disruption of nuclear-cytoskeletal linkages resulted in spatial variation in vertical uniformity. These results suggest that competition between cell-cell pulling forces that expand and shorten the vertical cell cross-section, thereby widening and flattening the nucleus, and the resistance of the nucleus to further flattening results in uniform cell and nuclear cross-sections. Our results reveal the mechanical principles of self-organized vertical uniformity in cell monolayers.

Segmentation of clustered cells in negative phase contrast images with integrated light intensity and cell shape information.

PubMed

Wang, Y; Wang, C; Zhang, Z

2018-05-01

Automated cell segmentation plays a key role in characterisations of cell behaviours for both biology research and clinical practices. Currently, the segmentation of clustered cells still remains as a challenge and is the main reason for false segmentation. In this study, the emphasis was put on the segmentation of clustered cells in negative phase contrast images. A new method was proposed to combine both light intensity and cell shape information through the construction of grey-weighted distance transform (GWDT) within preliminarily segmented areas. With the constructed GWDT, the clustered cells can be detected and then separated with a modified region skeleton-based method. Moreover, a contour expansion operation was applied to get optimised detection of cell boundaries. In this paper, the working principle and detailed procedure of the proposed method are described, followed by the evaluation of the method on clustered cell segmentation. Results show that the proposed method achieves an improved performance in clustered cell segmentation compared with other methods, with 85.8% and 97.16% accuracy rate for clustered cells and all cells, respectively. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

[Calcium distribution in the central cell of lettuce (Lactuca sativa L.) before and after pollination].

PubMed

Qiu, Yi Lan; Liu, Ru Shi; Ye, Lv; Tian, Hui

2008-02-01

Potassium antimonite precipitation was used to locate calcium in the central cell of lettuce (Lactuca sativa L.) before and after pollination. At 3d before anthesis, two polar nuclei of central cell separately located at two polarity of the cell, and few calcium precipitates (ppts) appeared in the polar nuclei and cytoplasm, but some ppts in its small vacuoles. At 2d before anthesis, two polar nuclei moved toward the middle of the cell and fused to form a secondary nucleus, and the ppts evidently increased in the nucleus and cytoplasm. At 1d before anthesis, secondary nucleus again moved toward micropylar end and located near the egg to prepare for fertilization. Calcium precipitates were mainly accumulated in the secondary nucleus. After pollination and before fertilization, the distribution of calcium ppts was similar to that before pollination. At 4h after pollination, the central cell was fertilized, and calcium ppts evidently increased in the cell and numerous were accumulated in its nucleus and cytoplasm. At 6h after pollination, the primary endosperm nucleus completed its first division and formed two dissociate endosperm nuclei, and still many calcium precipitates appeared in the nucleus and cytoplasm. With endosperm development, calcium ppts decreased in the endosperm cell. At 1d after emasculated and without pollination, the secondary nucleus of the cell still bordered on the egg and some calcium ppts appeared in the secondary nucleus. The results indicated that the temporal and spatial changes of calcium in the central cell may play an important physiological role during the development of the central cell and endosperm.

Production of neutron-rich nuclei approaching r-process by gamma-induced fission of 238U at ELI-NP

NASA Astrophysics Data System (ADS)

Mei, Bo; Balabanski, Dimiter; Constantin, Paul; Anh Le, Tuan; Viet Cuong, Phan

2018-05-01

The investigation of neutron-rich exotic nuclei is crucial not only for nuclear physics but also for nuclear astrophysics. Experimentally, only few neutron-rich nuclei near the stability have been studied, however, most neutron-rich nuclei have not been measured due to their small production cross sections as well as short half-lives. At ELI-NP, gamma beams with high intensities will open new opportunities to investigate very neutron-rich fragments produced by photofission of 238U targets in a gas cell. Based on some simulations, a novel gas cell has been designed to produce, stop and extract 238U photofission fragments. The extraction time and efficiency of photofission fragments have been optimized by using SIMION simulations. According to these simulations, a high extraction efficiency and a short extraction time can be achieved for 238U photofission fragments in the gas cell, which will allow one to measure very neutron-rich fragments with short half-lives by using the IGISOL facility proposed at ELI-NP.

Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons

PubMed Central

Krishnaswami, Suguna Rani; Grindberg, Rashel V; Novotny, Mark; Venepally, Pratap; Lacar, Benjamin; Bhutani, Kunal; Linker, Sara B; Pham, Son; Erwin, Jennifer A; Miller, Jeremy A; Hodge, Rebecca; McCarthy, James K; Kelder, Martin; McCorrison, Jamison; Aevermann, Brian D; Fuertes, Francisco Diez; Scheuermann, Richard H; Lee, Jun; Lein, Ed S; Schork, Nicholas; McConnell, Michael J; Gage, Fred H; Lasken, Roger S

2016-01-01

A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. We isolate nuclei at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at −80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing. PMID:26890679

Rhodamine B induces long nucleoplasmic bridges and other nuclear anomalies in Allium cepa root tip cells.

PubMed

Tan, Dehong; Bai, Bing; Jiang, Donghua; Shi, Lin; Cheng, Shunchang; Tao, Dongbing; Ji, Shujuan

2014-03-01

The cytogenetic toxicity of rhodamine B on root tip cells of Allium cepa was investigated. A. cepa were cultured in water (negative control), 10 ppm methyl methanesulfonate (positive control), and three concentrations of rhodamine B (200, 100, and 50 ppm) for 7 days. Rhodamine B inhibited mitotic activity; increased nuclear anomalies, including micronuclei, nuclear buds, and bridged nuclei; and induced oxidative stress in A. cepa root tissues. Furthermore, a substantial amount of long nucleoplasmic bridges were entangled together, and some nuclei were simultaneously linked to several other nuclei and to nuclear buds with nucleoplasmic bridges in rhodamine B-treated cells. In conclusion, rhodamine B induced cytogenetic effects in A. cepa root tip cells, which suggests that the A. cepa root is an ideal model system for detecting cellular interactions.

Adult granulosa cell tumor of the ovary: fine-needle-aspiration cytology of 10 cases and review of literature.

PubMed

Ali, Sarfraz; Gattuso, Paolo; Howard, Allison; Mosunjac, Marina B; Siddiqui, Momin T

2008-05-01

Adult granulosa cell tumor (GCT) of the ovary is mostly diagnosed in postmenopausal women. They typically secrete estrogen, which stimulates the endometrium to proliferate and cause abnormal bleeding. This study reviews the cytologic features of adult GCT of the ovary diagnosed by fine-needle aspiration (FNA). We reviewed slides from ten cases diagnosed by CT guided FNA from 1995 to 2007 at our institutions. Smears were stained with Diff-Quik and Papanicolaou stains. Patient's history and histologic diagnosis were also available and reviewed for all cases. The patients ranged in age from 39 to 83 yr. All 10 cases were hypercellular with both large and small overlapping cell clusters and individual cells. The cytologic features identified included: naked nuclei (10/10 cases), Call-Exner bodies (7/10 cases), blood vessels with prominent perivascular tumor cell growth (4/10 cases), spindle-shaped hyperchromatic stromal cells within cellular clusters (6/10 cases), mixed inflammation (3/10 cases), tumor cell necrosis (1/10 cases), and prominent metachromatic stroma seen in association with blood vessels (1/10 cases). Moderate to scant delicate cytoplasm was also seen (10/10 cases). Small, punctuate cytoplasmic vacuoles were also noted (7/10 cases) and were occasionally prominent (3/10 cases). In general nuclear to cytoplasmic ratios were high although lower than those typically seen in a lymphoma or small-cell carcinoma. Nuclei were generally centrally located although eccentrically located nuclei were consistently seen in a minority of cells. Nuclei were monotonous in size showing slightly convoluted (occasional rentiform and fetiform nuclei) to polygonal outlines. Prominent, central nucleoli were also seen (4/10 cases). Nuclear grooves were also seen (9/10 cases). No atypical mitotic activity was identified in any of the 10 cases (0/10 cases). In summary, the above cytologic features can also help in the cytologic diagnosis of adult GCTs.

[The frequency of sex chromatine occurring in cell nuclei of internal organs determined by the smear method (author's transl)].

PubMed

Michailow, R

1975-09-05

The frequency of sex chromatine occurring in cell nuclei of twelve organs from 25 male and female corpses was determined using the smear method. It was found to be about 60% in the case of female, and about 6% in the case of male corpses.

Immunological and biochemical evidence for nuclear localization of annexin in peas

NASA Technical Reports Server (NTRS)

Clark, G. B.; Dauwalder, M.; Roux, S. J.

1998-01-01

Immunofluorescent localization of annexins using an anti-pea annexin polyclonal antibody (anti-p35) in pea (Pisum sativum) leaf and stem epidermal peels showed staining of the nuclei and the cell periphery. Nuclear staining was also seen in cell teases prepared from pea plumules. The amount of nuclear stain was reduced both by fixation time and by dehydration and organic solvent treatment. Observation with confocal microscopy demonstrated that the anti-p35 stain was diffusely distributed throughout the nuclear structure. Immunoblots of purified nuclei, nuclear envelope matrix, nucleolar, and chromatin fractions showed a cross-reactive protein band of 35 kDa. These data are the first to show annexins localized in plant cell nuclei where they may play a role in nuclear function.

Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer).

PubMed

Chelomina, Galina N; Rozhkovan, Konstantin V; Voronova, Anastasia N; Burundukova, Olga L; Muzarok, Tamara I; Zhuravlev, Yuri N

2016-04-01

Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440-640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine.

Variation in the number of nucleoli and incomplete homogenization of 18S ribosomal DNA sequences in leaf cells of the cultivated Oriental ginseng (Panax ginseng Meyer)

PubMed Central

Chelomina, Galina N.; Rozhkovan, Konstantin V.; Voronova, Anastasia N.; Burundukova, Olga L.; Muzarok, Tamara I.; Zhuravlev, Yuri N.

2015-01-01

Background Wild ginseng, Panax ginseng Meyer, is an endangered species of medicinal plants. In the present study, we analyzed variations within the ribosomal DNA (rDNA) cluster to gain insight into the genetic diversity of the Oriental ginseng, P. ginseng, at artificial plant cultivation. Methods The roots of wild P. ginseng plants were sampled from a nonprotected natural population of the Russian Far East. The slides were prepared from leaf tissues using the squash technique for cytogenetic analysis. The 18S rDNA sequences were cloned and sequenced. The distribution of nucleotide diversity, recombination events, and interspecific phylogenies for the total 18S rDNA sequence data set was also examined. Results In mesophyll cells, mononucleolar nuclei were estimated to be dominant (75.7%), while the remaining nuclei contained two to four nucleoli. Among the analyzed 18S rDNA clones, 20% were identical to the 18S rDNA sequence of P. ginseng from Japan, and other clones differed in one to six substitutions. The nucleotide polymorphism was more expressed at the positions 440–640 bp, and distributed in variable regions, expansion segments, and conservative elements of core structure. The phylogenetic analysis confirmed conspecificity of ginseng plants cultivated in different regions, with two fixed mutations between P. ginseng and other species. Conclusion This study identified the evidences of the intragenomic nucleotide polymorphism in the 18S rDNA sequences of P. ginseng. These data suggest that, in cultivated plants, the observed genome instability may influence the synthesis of biologically active compounds, which are widely used in traditional medicine. PMID:27158239

Canine Circovirus 1 (CaCV-1) and Canine Parvovirus 2 (CPV-2): Recurrent Dual Infections in a Papillon Breeding Colony.

PubMed

Thaiwong, T; Wise, A G; Maes, R K; Mullaney, T; Kiupel, M

2016-11-01

Recurrent outbreaks of sudden death and bloody diarrhea were reported in March 2013 and February 2014 in a breeding colony of Papillon dogs. During the first outbreak, 1 adult dog and 2 eight-month-old puppies died. During the second outbreak, 2 ten-week-old puppies died. One puppy from the first outbreak and 2 puppies from the second outbreak were examined at necropsy. Histologically, all 3 puppies had severe segmental crypt necrosis of the small intestine and marked lymphoid follicle depletion in the spleen and Peyer's patches. Real-time (RT) polymerase chain reaction (PCR) demonstrated abundant canine parvovirus (CPV-2) DNA (Ct<15) in the affected small intestine, and immunohistochemistry detected large amounts of CPV-2 antigen in intestinal crypt epithelium and Kupffer cells but few positive macrophages in lymphoid organs. All puppies had marked sinusoidal histiocytosis and multifocal granulomatous inflammation in mesenteric lymph nodes and spleen, prompting additional RT-PCR testing for canine circovirus 1 (CaCV-1). Very high levels of CaCV-1 DNA (Ct<13) were detected in small intestine, lymph nodes, and spleen. In situ hybridization for CaCV-1 detected rare positive nuclei of regenerating crypt epithelium but abundant amounts of CaCV-1 nucleic acid in the cytoplasm and nuclei of histiocytes in all lymphoid tissues, including granulomatous inflammatory foci and hepatic Kupffer cells. Significant levels of CaCV-1 DNA were detected in blood and serum (Ct as low as 13) but not feces from 3 surviving dogs at 2 months or 1 year after the outbreak, respectively. We hypothesize that CPV-2 infection predisposed dogs to CaCV-1 infection and ultimately resulted in more severe clinical disease. © The Author(s) 2016.

A cytochemical and radioautographic study of the ultrastructural organization of puff-like fibrillar structures in plant interphase nuclei (Allium porrum).

PubMed

Lafontaine, J G; Luck, B T; Dontigny, D

1979-10-01

Loose, fibrillar, spherical structures have been observed during recent years in interphase nuclei of both animal and plant cells. These nuclear formations have been referred to as karyosomes, fibrillar bodies, micropuffs and centromeres. In order to gain further information on the nature of these structures, a cytochemical and radioautographic investigation was undertaken using plant meristematic cells (Allium porrum). For that purpose roots were fixed with either formaldehyde or glutaraldehyde in order to carry out cytochemical tests for DNA, RNA and proteins. Certain of the preparations were also first digested with DNase, RNase or proteinase K and then stained according to different procedures. Other specimens were labelled with thymidine for high-resolution radioautographic observations. Staining with diaminobenzidine (DAB) revealed that these nuclear puff-like formations consisted partly of a loose fibrillar meshwork containing nucleic acids. Part of this fine fibrillar reticulum persisted whether the preparations were digested with DNase or RNase before staining with DAB, thus indicating that these nuclear structures contained both DNA and RNA. The fact that these formations incorporate thymidine furnished additional support for the view that they correspond to specific chromosome segments. Staining with ethanolic phosphotungstic acid or digestion of specimens with proteinase K showed that these loose fibrillar structures also consisted of proteins. Judging from their ultrastructure, their association with the chromatin reticulum as well as from their cytochemical characteristics, these nuclear formations most likely correspond to centromeres. In view of the presence of DNA within these structures, it is possible to distinguish them from other equally spherical nuclear formations, observed in certain plant species, that have generally been referred to as karyosomes or micronucleoli and that appear to consist of ribonucleoproteins.

Analysis of Nuclear RNA Interference (RNAi) in Human Cells by Subcellular Fractionation and Argonaute Loading

PubMed Central

Gagnon, Keith T.; Li, Liande; Janowski, Bethany A.; Corey, David R.

2014-01-01

RNA interference (RNAi) is well known for its ability to regulate gene expression in the cytoplasm of mammalian cells. In mammalian cell nuclei, however, the impact of RNAi has remained more controversial. A key technical hurdle has been a lack of optimized protocols for the isolation and analysis of cell nuclei. Here we describe a simplified protocol for nuclei isolation from cultured cells that incorporates a method for obtaining nucleoplasmic and chromatin fractions and removing cytoplasmic contamination. Cell fractions can then be used to detect the presence and activity of RNAi factors in the nucleus. We present a protocol for investigating an early step in RNAi, Argonaute protein loading with small RNAs, which is enabled by our improved extract preparations. These protocols facilitate characterization of nuclear RNAi and can be applied to the analysis of other nuclear proteins and pathways. From cellular fractionation to analysis of Argonaute loading results, this protocol takes 4–6 d to complete. PMID:25079428

Impact of physical confinement on nuclei geometry and cell division dynamics in 3D spheroids.

PubMed

Desmaison, Annaïck; Guillaume, Ludivine; Triclin, Sarah; Weiss, Pierre; Ducommun, Bernard; Lobjois, Valérie

2018-06-08

Multicellular tumour spheroids are used as a culture model to reproduce the 3D architecture, proliferation gradient and cell interactions of a tumour micro-domain. However, their 3D characterization at the cell scale remains challenging due to size and cell density issues. In this study, we developed a methodology based on 3D light sheet fluorescence microscopy (LSFM) image analysis and convex hull calculation that allows characterizing the 3D shape and orientation of cell nuclei relative to the spheroid surface. By using this technique and optically cleared spheroids, we found that in freely growing spheroids, nuclei display an elongated shape and are preferentially oriented parallel to the spheroid surface. This geometry is lost when spheroids are grown in conditions of physical confinement. Live 3D LSFM analysis of cell division revealed that confined growth also altered the preferential cell division axis orientation parallel to the spheroid surface and induced prometaphase delay. These results provide key information and parameters that help understanding the impact of physical confinement on cell proliferation within tumour micro-domains.

BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models

PubMed Central

Bilgin, Cemal Cagatay; Fontenay, Gerald; Cheng, Qingsu; Chang, Hang; Han, Ju; Parvin, Bahram

2016-01-01

BioSig3D is a computational platform for high-content screening of three-dimensional (3D) cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i) morphogenesis of a panel of human mammary epithelial cell lines (HMEC), and (ii) heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation. PMID:26978075

Multi-nucleate retinal pigment epithelium cells of the human macula exhibit a characteristic and highly specific distribution.

PubMed

Starnes, Austin C; Huisingh, Carrie; McGwin, Gerald; Sloan, Kenneth R; Ablonczy, Zsolt; Smith, R Theodore; Curcio, Christine A; Ach, Thomas

2016-01-01

The human retinal pigment epithelium (RPE) is reportedly 3% bi-nucleated. The importance to human vision of multi-nucleated (MN)-RPE cells could be clarified with more data about their distribution in central retina. Nineteen human RPE-flatmounts (9 ≤ 51 years, 10 > 80 years) were imaged at 12 locations: 3 eccentricities (fovea, perifovea, near periphery) in 4 quadrants (superior, inferior, temporal, nasal). Image stacks of lipofuscin-attributable autofluorescence and phalloidin labeled F-actin cytoskeleton were obtained using a confocal fluorescence microscope. Nuclei were devoid of autofluorescence and were marked using morphometric software. Cell areas were approximated by Voronoi regions. Mean number of nuclei per cell among eccentricity/quadrant groups and by age were compared using Poisson and binominal regression models. A total of 11,403 RPE cells at 200 locations were analyzed: 94.66% mono-, 5.31% bi-, 0.02% tri-nucleate, and 0.01% with 5 nuclei. Age had no effect on number of nuclei. There were significant regional differences: highest frequencies of MN-cells were found at the perifovea (9.9%) and near periphery (6.8%). The fovea lacked MN-cells almost entirely. The nasal quadrant had significantly more MN-cells compared to other quadrants, at all eccentricities. This study demonstrates MN-RPE cells in human macula. MN-cells may arise due to endoreplication, cell fusion, or incomplete cell division. The topography of MN-RPE cells follows the topography of photoreceptors; with near-absence at the fovea (cones only) and high frequency at perifovea (highest rod density). This distribution might reflect specific requirements of retinal metabolism or other mechanisms addressable in further studies.

Multiplication free neural network for cancer stem cell detection in H-and-E stained liver images

NASA Astrophysics Data System (ADS)

Badawi, Diaa; Akhan, Ece; Mallah, Ma'en; Üner, Ayşegül; ćetin-Atalay, Rengül; ćetin, A. Enis

2017-05-01

Markers such as CD13 and CD133 have been used to identify Cancer Stem Cells (CSC) in various tissue images. It is highly likely that CSC nuclei appear as brown in CD13 stained liver tissue images. We observe that there is a high correlation between the ratio of brown to blue colored nuclei in CD13 images and the ratio between the dark blue to blue colored nuclei in H&E stained liver images. Therefore, we recommend that a pathologist observing many dark blue nuclei in an H&E stained tissue image may also order CD13 staining to estimate the CSC ratio. In this paper, we describe a computer vision method based on a neural network estimating the ratio of dark blue to blue colored nuclei in an H&E stained liver tissue image. The neural network structure is based on a multiplication free operator using only additions and sign operations. Experimental results are presented.

Evolution of TUNEL-labeling in the rat lens after in vivo exposure to just above threshold dose UVB.

PubMed

Kronschläger, Martin; Yu, Zhaohua; Talebizadeh, Nooshin; Meyer, Linda M; Hallböök, Finn; Söderberg, Per G

2013-08-01

To quantitatively analyse the evolution of TUNEL-labeling, after in vivo exposure to UVB. Altogether, 16 Sprague Dawley rats were unilaterally exposed in vivo for 15 min to close to threshold dose, 5 kJ/m(2), of ultraviolet radiation in the 300 nm wavelength region. Animals were sacrificed in groups of 4 at 1, 5, 24 and 120 h after exposure. For each animal, both eye globes were removed and frozen. The frozen eye was cryo-sectioned in 10 µm thick midsagittal sections. From each globe, three midsagittal sections with at least five sections interval in between were mounted on a microscope slide. Sections were TUNEL-labeled and counter stained with DAPI. For quantification of apoptosis, a fluorescence microscope was used. In sections with a continuous epithelial cell surface, the number of lens epithelial cell nuclei and the number of TUNEL-positive epithelial cell nuclei was counted. The total number of TUNEL-positive epithelial cell nuclei for all three sections of one lens in relation to the total number of epithelial cell nuclei for all three sections of the same lens was compared between exposed and contralateral not exposed lens for each animal. The relative difference of the fraction of TUNEL-positive nuclei between exposed and contralateral not exposed lens increased gradually, peaked in the time interval 5-120 h after exposure, and then declined. Close to threshold dose of UVB induces TUNEL-labeling that peaks in the time window 5-120 h after exposure to UVB.

Over-expression of GFP-FEZ1 causes generation of multi-lobulated nuclei mediated by microtubules in HEK293 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lanza, Daniel C.F.; Trindade, Daniel M.; Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP

2008-06-10

FEZ1 (Fasciculation and elongation protein zeta 1) is an ortholog of the Caenorhabditis elegans protein UNC-76, involved in neuronal development and axon outgrowth, in that worm. Mammalian FEZ1 has already been reported to cooperate with PKC-zeta in the differentiation and polarization of PC12 neuronal cells. Furthermore, FEZ1 is associated with kinesin 1 and JIP1 to form a cargo-complex responsible for microtubule based transport of mitochondria along axons. FEZ1 can also be classified as a hub protein, since it was reported to interact with over 40 different proteins in yeast two-hybrid screens, including at least nine nuclear proteins. Here, we transientlymore » over-expressed GFP-FEZ1full in human HEK293 and HeLa cells in order to study the sub-cellular localization of GFP-FEZ1. We observed that over 40% of transiently transfected cells at 3 days post-transfection develop multi-lobulated nuclei, which are also called flower-like nuclei. We further demonstrated that GFP-FEZ1 localizes either to the cytoplasm or the nuclear fraction, and that the appearance of the flower-like nuclei depends on intact microtubule function. Finally, we show that FEZ1 co-localizes with both, {alpha}- and especially with {gamma}-tubulin, which localizes as a centrosome like structure at the center of the multiple lobules. In summary, our data suggest that FEZ1 has an important centrosomal function and supply new mechanistic insights to the formation of flower-like nuclei, which are a phenotypical hallmark of human leukemia cells.« less

Uptake and metabolism of (/sup 3/H)testosterone in the brain, pituitary gland and genital tract of the male cynomolgus monkey

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bonsall, R.W.; Rees, H.D.; Micheal, R.P.

1986-03-01

To study the mechanism by which testosterone restores the sexual potency of castrated cynomolgus monkeys, two males (body weights 5.2 and 5.3 kg) were castrated and, 3 days later, injected with 3 mCi (/sup 3/H)testosterone ((/sup 3/H)T) as an intravenous bolus. After 30 min, males were killed and brains and samples of other tissues were rapidly removed and placed on ice. Samples were dissected from the right halves of the brain and homogenized. Purified cell nuclei were prepared and ether extracts were analyzed by reverse-phase HPCL. Generally, unchanged (/sup 3/H)T was the major form of radioactivity in brain and pituitarymore » gland, but in cell nuclei from hypothalamus, preoptic area and amygdala, a large proportion (34 - 61%) was in the form of (/sup 3/H)estradiol ((/sup 4/H)E/sub 2/). Little or no (/sup 3/H)dihydrotestosterone ((/sup 3/H)DHT) was detected in cell nuclei from any brain region or from pituitary gland. However, (/sup 3/H)DHT was the major form (61 - 95%) of radioactivity in cell nuclei from glans penis, prostrate and seminal vesicles. In autoradiograms of the left halves of the same brains, the percentage of cells that accumulated radioactivity in their nuclei was high in specific regions of the hypothalamus, preoptic areas and amygdala. The authors conclude that the peripheral actions of T are mediated via DHT, but its central actions are dependent on unchanged T or on E/sub 2/ formed locally by aromatization.« less

Exploiting the dynamics of S-phase tracers in developing brain: interkinetic nuclear migration for cells entering versus leaving the S-phase

NASA Technical Reports Server (NTRS)

Hayes, N. L.; Nowakowski, R. S.

2000-01-01

Two S-phase markers for in vivo studies of cell proliferation in the developing central nervous system, tritiated thymidine ((3)H-TdR) and bromodeoxyuridine (BUdR), were compared using double-labeling techniques in the developing mouse cortex at embryonic day 14 (E14). The labeling efficiencies and detectability of the two tracers were approximately equivalent, and there was no evidence of significant tracer interactions that depend on order of administration. For both tracers, the loading time needed to label an S-phase cell to detectability is estimated at <0.2 h shortly after the injection of the label, but, as the concentration of the label falls, it increases to approximately 0.65 h after about 30 min. Thereafter, cells that enter the S-phase continue to become detectably labeled for approximately 5-6 h. The approximate equivalence of these two tracers was exploited to observe directly the numbers and positions of nuclei entering (labeled with the second tracer only) and leaving (labeled with the first tracer only) the S-phase. As expected, the numbers of nuclei entering and leaving the S-phase both increased as the interval between the two injections lengthened. Also, nuclei leaving the S-phase rapidly move towards the ventricular surface during G2, but, unexpectedly, the distribution of the entering nuclei does not differ significantly from the distribution of the nuclei in the S-phase. This indicates that: (1) the extent and rate of abventricular nuclear movement during G1 is variable, such that not all nuclei traverse the entire width of the ventricular zone, and (2) interkinetic nuclear movements are minimal during S-phase. Copyright 2000 S. Karger AG, Basel.

Survey statistics of automated segmentations applied to optical imaging of mammalian cells.

PubMed

Bajcsy, Peter; Cardone, Antonio; Chalfoun, Joe; Halter, Michael; Juba, Derek; Kociolek, Marcin; Majurski, Michael; Peskin, Adele; Simon, Carl; Simon, Mylene; Vandecreme, Antoine; Brady, Mary

2015-10-15

The goal of this survey paper is to overview cellular measurements using optical microscopy imaging followed by automated image segmentation. The cellular measurements of primary interest are taken from mammalian cells and their components. They are denoted as two- or three-dimensional (2D or 3D) image objects of biological interest. In our applications, such cellular measurements are important for understanding cell phenomena, such as cell counts, cell-scaffold interactions, cell colony growth rates, or cell pluripotency stability, as well as for establishing quality metrics for stem cell therapies. In this context, this survey paper is focused on automated segmentation as a software-based measurement leading to quantitative cellular measurements. We define the scope of this survey and a classification schema first. Next, all found and manually filteredpublications are classified according to the main categories: (1) objects of interests (or objects to be segmented), (2) imaging modalities, (3) digital data axes, (4) segmentation algorithms, (5) segmentation evaluations, (6) computational hardware platforms used for segmentation acceleration, and (7) object (cellular) measurements. Finally, all classified papers are converted programmatically into a set of hyperlinked web pages with occurrence and co-occurrence statistics of assigned categories. The survey paper presents to a reader: (a) the state-of-the-art overview of published papers about automated segmentation applied to optical microscopy imaging of mammalian cells, (b) a classification of segmentation aspects in the context of cell optical imaging, (c) histogram and co-occurrence summary statistics about cellular measurements, segmentations, segmented objects, segmentation evaluations, and the use of computational platforms for accelerating segmentation execution, and (d) open research problems to pursue. The novel contributions of this survey paper are: (1) a new type of classification of cellular measurements and automated segmentation, (2) statistics about the published literature, and (3) a web hyperlinked interface to classification statistics of the surveyed papers at https://isg.nist.gov/deepzoomweb/resources/survey/index.html.

Segmented all-electron Gaussian basis sets of double and triple zeta qualities for Fr, Ra, and Ac

NASA Astrophysics Data System (ADS)

Campos, C. T.; de Oliveira, A. Z.; Ferreira, I. B.; Jorge, F. E.; Martins, L. S. C.

2017-05-01

Segmented all-electron basis sets of valence double and triple zeta qualities plus polarization functions for the elements Fr, Ra, and Ac are generated using non-relativistic and Douglas-Kroll-Hess (DKH) Hamiltonians. The sets are augmented with diffuse functions with the purpose to describe appropriately the electrons far from the nuclei. At the DKH-B3LYP level, first atomic ionization energies and bond lengths, dissociation energies, and polarizabilities of a sample of diatomics are calculated. Comparison with theoretical and experimental data available in the literature is carried out. It is verified that despite the small sizes of the basis sets, they are yet reliable.

Neutron Resonance Spin Determination Using Multi-Segmented Detector DANCE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baramsai, B.; Mitchell, G. E.; Chyzh, A.

2011-06-01

A sensitive method to determine the spin of neutron resonances is introduced based on the statistical pattern recognition technique. The new method was used to assign the spins of s-wave resonances in {sup 155}Gd. The experimental neutron capture data for these nuclei were measured with the DANCE (Detector for Advanced Neutron Capture Experiment) calorimeter at the Los Alamos Neutron Science Center. The highly segmented calorimeter provided detailed multiplicity distributions of the capture {gamma}-rays. Using this information, the spins of the neutron capture resonances were determined. With these new spin assignments, level spacings are determined separately for s-wave resonances with J{supmore » {pi}} = 1{sup -} and 2{sup -}.« less

A method for estimating the accuracy of measurements of optical characteristics of the nuclei of blood cells in the diagnosis of acute leukemia

NASA Astrophysics Data System (ADS)

Polyakov, E. V.; Nikitaev, V. G.

2017-01-01

The work is devoted to investigation of the random component of the measurement error of the nuclei structure characteristics, which are used in the method of structural elements to measure the differences of blood cells of different types. This method is realized in information-measuring system of the analysis of micropreparations of blood cells in the diagnosis of acute leukemia and its variants.

A nuclear F-actin scaffold stabilizes ribonucleoprotein droplets against gravity in large cells.

PubMed

Feric, Marina; Brangwynne, Clifford P

2013-10-01

The size of a typical eukaryotic cell is of the order of ∼10 μm. However, some cell types grow to very large sizes, including oocytes (immature eggs) of organisms from humans to starfish. For example, oocytes of the frog Xenopus laevis grow to a diameter ≥1 mm. They have a correspondingly large nucleus (germinal vesicle) of ∼450 μm in diameter, which is similar to smaller somatic nuclei, but contains a significantly higher concentration of actin. The form and structure of this nuclear actin remain controversial, and its potential mechanical role within these large nuclei is unknown. Here, we use a microrheology and quantitative imaging approach to show that germinal vesicles contain an elastic F-actin scaffold that mechanically stabilizes these large nuclei against gravitational forces, which are usually considered negligible within cells. We find that on actin disruption, ribonucleoprotein droplets, including nucleoli and histone locus bodies, undergo gravitational sedimentation and fusion. We develop a model that reveals how gravity becomes an increasingly potent force as cells and their nuclei grow larger than ∼10 μm, explaining the requirement for a stabilizing nuclear F-actin scaffold in large Xenopus oocytes. All life forms are subject to gravity, and our results may have broad implications for cell growth and size control.

A nuclear F-actin scaffold stabilizes RNP droplets against gravity in large cells

PubMed Central

Feric, Marina; Brangwynne, Clifford P.

2013-01-01

The size of a typical eukaryotic cell is on the order of ≈10 μm. However, some cell types grow to very large sizes, including oocytes (immature eggs) of organisms from humans to starfish. For example, oocytes of the frog X. laevis grow to a diameter ≥1 mm. They contain a correspondingly large nucleus (germinal vesicle, GV) of ≈450 μm in diameter, which is similar to smaller somatic nuclei, but contains a significantly higher concentration of actin. The form and structure of this nuclear actin remain controversial, and its potential mechanical role within these large nuclei is unknown. Here, we use a microrheology and quantitative imaging approach to show that GVs contain an elastic F-actin scaffold that mechanically stabilizes these large nuclei against gravitational forces, which are usually considered negligible within cells. We find that upon actin disruption, RNA/protein droplets, including nucleoli and histone locus bodies (HLBs), undergo gravitational sedimentation and fusion. We develop a model that reveals how gravity becomes an increasingly potent force as cells and their nuclei grow larger than ≈10 μm, explaining the requirement for a stabilizing nuclear F-actin scaffold in large X. laevis ooctyes. All life forms are subject to gravity, and our results may have broad implications for cell growth and size control. PMID:23995731

DNA replication after mutagenic treatment in Hordeum vulgare.

PubMed

Kwasniewska, Jolanta; Kus, Arita; Swoboda, Monika; Braszewska-Zalewska, Agnieszka

2016-12-01

The temporal and spatial properties of DNA replication in plants related to DNA damage and mutagenesis is poorly understood. Experiments were carried out to explore the relationships between DNA replication, chromatin structure and DNA damage in nuclei from barley root tips. We quantitavely analysed the topological organisation of replication foci using pulse EdU labelling during the S phase and its relationship with the DNA damage induced by mutagenic treatment with maleic hydrazide (MH), nitroso-N-methyl-urea (MNU) and gamma ray. Treatment with mutagens did not change the characteristic S-phase patterns in the nuclei; however, the frequencies of the S-phase-labelled cells after treatment differed from those observed in the control cells. The analyses of DNA replication in barley nuclei were extended to the micronuclei induced by mutagens. Replication in the chromatin of the micronuclei was rare. The results of simultanous TUNEL reaction to identify cells with DNA strand breaks and the labelling of the S-phase cells with EdU revealed the possibility of DNA replication occurring in damaged nuclei. For the first time, the intensity of EdU fluorescence to study the rate of DNA replication was analysed. Copyright © 2016 Elsevier B.V. All rights reserved.

Three-Dimensional, Live-Cell Imaging of Chromatin Dynamics in Plant Nuclei Using Chromatin Tagging Systems.

PubMed

Hirakawa, Takeshi; Matsunaga, Sachihiro

2016-01-01

In plants, chromatin dynamics spatiotemporally change in response to various environmental stimuli. However, little is known about chromatin dynamics in the nuclei of plants. Here, we introduce a three-dimensional, live-cell imaging method that can monitor chromatin dynamics in nuclei via a chromatin tagging system that can visualize specific genomic loci in living plant cells. The chromatin tagging system is based on a bacterial operator/repressor system in which the repressor is fused to fluorescent proteins. A recent refinement of promoters for the system solved the problem of gene silencing and abnormal pairing frequencies between operators. Using this system, we can detect the spatiotemporal dynamics of two homologous loci as two fluorescent signals within a nucleus and monitor the distance between homologous loci. These live-cell imaging methods will provide new insights into genome organization, development processes, and subnuclear responses to environmental stimuli in plants.

Structural connectivity of the developing human amygdala.

PubMed

Saygin, Zeynep M; Osher, David E; Koldewyn, Kami; Martin, Rebecca E; Finn, Amy; Saxe, Rebecca; Gabrieli, John D E; Sheridan, Margaret

2015-01-01

A large corpus of research suggests that there are changes in the manner and degree to which the amygdala supports cognitive and emotional function across development. One possible basis for these developmental differences could be the maturation of amygdalar connections with the rest of the brain. Recent functional connectivity studies support this conclusion, but the structural connectivity of the developing amygdala and its different nuclei remains largely unstudied. We examined age related changes in the DWI connectivity fingerprints of the amygdala to the rest of the brain in 166 individuals of ages 5-30. We also developed a model to predict age based on individual-subject amygdala connectivity, and identified the connections that were most predictive of age. Finally, we segmented the amygdala into its four main nucleus groups, and examined the developmental changes in connectivity for each nucleus. We observed that with age, amygdalar connectivity becomes increasingly sparse and localized. Age related changes were largely localized to the subregions of the amygdala that are implicated in social inference and contextual memory (the basal and lateral nuclei). The central nucleus' connectivity also showed differences with age but these differences affected fewer target regions than the basal and lateral nuclei. The medial nucleus did not exhibit any age related changes. These findings demonstrate increasing specificity in the connectivity patterns of amygdalar nuclei across age.

Study of HeLa cells clone survival after X-ray irradiation in the presence of cisplatin

NASA Astrophysics Data System (ADS)

Baulin, A. A.; Sukhikh, E. S.; Vasilyev, S. A.; Sukhikh, L. G.; Sheino, I. N.

2017-09-01

Radiation therapy in the presence of heavy elements nuclei (Z > 53) is widely developed these days. The presence of such nuclei in cancer cells results in the local increase of energy release from primary photon beam thus increasing relative biological efficiency. In this paper we present the preliminary results of the cell survival study while irradiating cells by X-Ray photon beam in the presence of cisplatin (Pt, Z = 78). The preliminary results show the decrease of the cell survival in the presence of both radiation and cisplatin.

Nuclear transfer of synchronized African wild cat somatic cells into enucleated domestic cat oocytes

USGS Publications Warehouse

Gomez, M.C.; Jenkins, J.A.; Giraldo, A.; Harris, R.F.; King, A.; Dresser, B.L.; Pope, C.E.

2003-01-01

The African wild cat is one of the smallest wild cats and its future is threatened by hybridization with domestic cats. Nuclear transfer, a valuable tool for retaining genetic variability, offers the possibility of species continuation rather than extinction. The aim of this study was to investigate the ability of somatic cell nuclei of the African wild cat (AWC) to dedifferentiate within domestic cat (DSH) cytoplasts and to support early development after nuclear transplantation. In experiment 1, distributions of AWC and DSH fibroblasts in each cell-cycle phase were assessed by flow cytometry using cells cultured to confluency and disaggregated with pronase, trypsin, or mechanical separation. Trypsin (89.0%) and pronase (93.0%) yielded higher proportions of AWC nuclei in the G0/G1 phase than mechanical separation (82.0%). In contrast, mechanical separation yielded higher percentages of DSH nuclei in the G0/G1 phase (86.6%) than pronase (79.7%) or trypsin (74.2%) treatments. In both species, pronase induced less DNA damage than trypsin. In experiment 2, the effects of serum starvation, culture to confluency, and exposure to roscovitine on the distribution of AWC and DSH fibroblasts in various phases of the cell cycle were determined. Flow cytometry analyses revealed that the dynamics of the cell cycle varied as culture conditions were modified. Specifically, a higher percentage of AWC and DSH nuclei were in the G0/G1 phase after cells were serum starved (83% vs. 96%) than were present in cycling cells (50% vs. 64%), after contact inhibition (61% vs. 88%), or after roscovitine (56% vs. 84%) treatment, respectively. In experiment 3, we evaluated the effects of cell synchronization and oocyte maturation (in vivo vs. in vitro) on the reconstruction and development of AWC-DSH- and DSH-DSH-cloned embryos. The method of cell synchronization did not affect the fusion and cleavage rate because only a slightly higher percentage of fused couplets cleaved when donor nuclei were synchronized by serum starvation (83.0%) than after roscovitine (80.0%) or contact-inhibition (80.0%). The fusion efficiency of in vivo and in vitro matured oocytes used as recipient cytoplasts of AWC donor nuclei (86.6% vs. 85.2%) was similar to the rates obtained with DSH donor nuclei, 83.7% vs. 73.0%, respectively. The only significant effect of source of donor nucleus (AWC vs. DSH) was on the rate of blastocyst formation in vitro. A higher percentage of the embryos derived from AWC nuclei developed to the blastocyst stage than did embryos produced from DSH nuclei, 24.2% vs. 3.3%, respectively (P < 0.05). In experiment 4, the effect of calcium in the fusion medium on induction of oocyte activation and development of AWC-DSH-cloned embryos was determined. The presence of calcium in the fusion medium induced a high incidence of cleavage of DSH oocytes (54.3%), while oocyte cleavage frequency was much lower in the absence of calcium (16.6%). The presence or absence of calcium in the fusion medium did not affect the fusion, cleavage, and blastocyst development of AWC-DSH-cloned embryos. In experiment 5, AWC-DSH-cloned embryos were transferred to the uteri of 11 synchronized domestic cat recipients on Day 6 or 7 after oocyte aspiration. Recipients were assessed by ultrasonography on Day 21 postovulation, but no pregnancies were observed. In the present study, after NT, AWC donor nuclei were able to dedifferentiate in DSH cytoplasts and support high rates of blastocyst development in vitro. Incomplete reprogramming of the differentiated nucleus may be a major constraint to the in vivo developmental potential of the embryos.

Anatomical evidence for brainstem circuits mediating feeding motor programs in the leopard frog, Rana pipiens.

PubMed

Anderson, C W

2001-09-01

Using injections of small molecular weight fluorescein dextran amines, combined with activity-dependent uptake of sulforhodamine 101 (SR101), brainstem circuits presumed to be involved in feeding motor output were investigated. As has been shown previously in other studies, projections to the cerebellar nuclei were identified from the cerebellar cortex, the trigeminal motor nucleus, and the vestibular nuclei. Results presented here suggest an additional pathway from the hypoglossal motor nuclei to the cerebellar nucleus as well as an afferent projection from the peripheral hypoglossal nerve to the Purkinje cell layer of the cerebellar cortex. Injections in the cerebellar cortex combined with retrograde labeling of the peripheral hypoglossal nerve demonstrate anatomical convergence at the level of the medial reticular formation. This suggests a possible integrative region for afferent feedback from the hypoglossal nerve and information through the Purkinje cell layer of the cerebellar cortex. The activity-dependent uptake of SR101 additionally suggests a reciprocal, polysynaptic pathway between this same area of the medial reticular formation and the trigeminal motor nuclei. The trigeminal motor neurons innervate the m adductor mandibulae, the primary mouth-closing muscle. The SR101 uptake clearly labeled the ventrolateral hypoglossal nuclei, the medial reticular formation, and the Purkinje cell layer of the cerebellar cortex. Unlike retrograde labeling of the peripheral hypoglossal nerve, stimulating the hypoglossal nerve while SR101 was bath-applied labeled trigeminal motor neurons. This, combined with the dextran labeling, suggests a reciprocal connection between the trigeminal motor nuclei and the cerebellar nuclei, as well as the medulla. Taken together, these data are important for understanding the neurophysiological pathways used to coordinate the proper timing of an extremely rapid, goal-directed movement and may prove useful for elucidating some of the first principles of sensorimotor integration.

Spatial distribution of centromeres and telomeres at interphase varies among Brachypodium species

PubMed Central

Idziak, Dominika; Robaszkiewicz, Ewa; Hasterok, Robert

2015-01-01

In this study the 3-D distribution of centromeres and telomeres was analysed in the interphase nuclei of three Brachypodium species, i.e. B. distachyon (2n=10), B. stacei (2n=20) and B. hybridum (2n=30), which is presumably a hybrid between the first two species. Using fluorescence in situ hybridization (FISH) with centromeric and telomeric DNA probes, it was observed that the majority of B. distachyon nuclei in the root tip cells displayed the Rabl configuration while both B. stacei and B. hybridum mostly lacked the centromere–telomere polarization. In addition, differentiated leaf cells of B. distachyon did not display the Rabl pattern. In order to analyse the possible connection between the occurrence of the Rabl pattern and the phase of cell cycle or DNA content, FISH was combined with digital image cytometry. The results revealed that the frequency of nuclei with the Rabl configuration in the root tip nuclei was positively correlated with an increase in DNA content, which resulted from DNA replication. Also, the analysis of the influence of the nuclear shape on the nuclear architecture indicated that an increasing elongation of the nuclei negatively affected the occurrence of the Rabl pattern. Some possible explanations of these phenomena are discussed. PMID:26208647

Replication labeling patterns and chromosome territories typical of mammalian nuclei are conserved in the early metazoan Hydra.

PubMed

Alexandrova, Olga; Solovei, Irina; Cremer, Thomas; David, Charles N

2003-12-01

To investigate the evolutionary conservation of higher order nuclear architecture previously described for mammalian cells we have analyzed the nuclear architecture of the simple polyp Hydra. These diploblastic organisms have large nuclei (8-10 microm) containing about 3x10(9) bp of DNA organized in 15 chromosome pairs. They belong to the earliest metazoan phylum and are separated from mammals by at least 600 million years. Single and double pulse labeling with halogenated nucleotides (bromodeoxyuridine, iododeoxyuridine and chlorodeoxyuridine) revealed striking similarities to the known sequence of replication labeling patterns in mammalian nuclei. These patterns reflect a persistent nuclear arrangement of early, mid-, and late replicating chromatin foci that could be identified during all stages of interphase over at least 5-10 cell generations. Segregation of labeled chromatids led after several cell divisions to nuclei with single or a few labeled chromosome territories. In such nuclei distinct clusters of labeled chromatin foci were separated by extended nuclear areas with non-labeled chromatin, which is typical of a territorial arrangement of interphase chromosomes. Our results indicate the conservation of fundamental features of higher order chromatin arrangements throughout the evolution of metazoan animals and suggest the existence of conserved mechanism(s) controlling this architecture.

Immunohistochemical detection of tumor suppressor gene p53 protein in feline injection site-associated sarcomas.

PubMed

Nambiar, P R; Jackson, M L; Ellis, J A; Chelack, B J; Kidney, B A; Haines, D M

2001-03-01

Sarcomas associated with injection sites are a rare but important problem in cats. Immunohistochemical detection of p53 protein may correlate to mutation of the p53 tumor suppressor gene, a gene known to be important in oncogenesis. The expression of nuclear p53 protein in 40 feline injection site-assocated sarcomas was examined by immunohistochemical staining. In 42.5% (17/40), tumor cell nuclei were stained darkly; in 20% (8/40), tumor cell nuclei were stained palely; and in 37.5% (15/40), tumor cell nuclei were unstained. Immunohistochemical detection of p53 protein in a proportion of injection site-associated sarcomas suggests that mutation of the p53 gene may play a role in the pathogenesis of these tumors.

Variations in cell morphology in the canine cruciate ligament complex.

PubMed

Smith, K D; Vaughan-Thomas, A; Spiller, D G; Clegg, P D; Innes, J F; Comerford, E J

2012-08-01

Cell morphology may reflect the mechanical environment of tissues and influence tissue physiology and response to injury. Normal cruciate ligaments (CLs) from disease-free stifle joints were harvested from dog breeds with a high (Labrador retriever) and low (Greyhound) risk of cranial cruciate ligament (CCL) rupture. Antibodies against the cytoskeletal components vimentin and alpha tubulin were used to analyse cell morphology; nuclei were stained with 4',6-diamidino-2-phenylindole, and images were collected using conventional and confocal microscopy. Both cranial and caudal CLs contained cells of heterogenous morphologies. Cells were arranged between collagen bundles and frequently had cytoplasmic processes. Some of these processes were long (type A cells), others were shorter, thicker and more branched (type B cells), and some had no processes (type C cells). Processes were frequently shown to contact other cells, extending longitudinally and transversely through the CLs. Cells with longer processes had fusiform nuclei, and those with no processes had rounded nuclei and were more frequent in the mid-substance of both CLs. Cells with long processes were more commonly noted in the CLs of the Greyhound. As contact between cells may facilitate direct communication, variances in cell morphology between breeds at a differing risk of CCL rupture may reflect differences in CL physiology. Copyright © 2012 Elsevier Ltd. All rights reserved.

DNA content of hepatocyte and erythrocyte nuclei of the spined loach (Cobitis taenia L.) and its polyploid forms.

PubMed

Juchno, Dorota; Lackowska, Bozena; Boron, Alicja; Kilarski, Wincenty

2010-09-01

We analyzed the DNA content of hepatocyte and erythrocyte nuclei of the spined loach Cobitis taenia (diploid) and its allopolyploid forms. Twenty triploid females and one tetraploid were used. At least 20,000 hepatocyte and erythrocyte nuclei were acquired and analyzed by flow cytometry. C. taenia erythrocyte nuclei contain 3.15 +/- 0.21 pg of DNA and the hepatocyte nuclei 4.45 +/- 0.46 pg of DNA. Triploid Cobitis have 5.08 +/- 0.41 pg of DNA in erythrocyte nuclei and 6.11 +/- 0.40 pg of DNA in hepatocyte nuclei, whereas the tetraploid erythrocyte and hepatocyte nuclei contained 6.60 and 7.40 pg of DNA, respectively. In general, the DNA contents correlate positively with the ploidy level of the fish investigated. The DNA content variation in the hepatocyte and erythrocyte nuclei may be due to differences in extent of chromatin condensation, which is more pronounced in the erythrocyte than hepatocyte nuclei, or to the several orders of ploidy that occur in the parenchymal liver cells.

A new fluorescent imaging procedure in vivo for evaluation of the retinal microcirculation in rats.

PubMed

Kimura, H; Kiryu, J; Nishiwaki, H; Ogura, Y

1995-03-01

We investigated a new method for in vivo evaluation of the retinal microcirculation in rats using a cell-permeant fluorescent dye, acridine orange (AO), which stains cell nuclei and cytoplasm, and a scanning laser ophthalmoscope (SLO). AO, which binds and interacts with DNA and RNA, and thus stains cell nuclei and cytoplasm, was administered intravenously to rats. Fluorescein angiography was performed after administration of the AO, and fundus images were recorded on S-VHS videotape by means of an SLO. Argon laser was used as an exciter of the dye. The retinal vessels were stained with the dye, rendering the retinal microvasculature clearly visible. Cell nuclei and vessel walls were observed as greater fluorescence and lesser fluorescence, respectively. Leukocytes were also observed as highly fluorescent dots moving through the vessels. The results suggest that SLO visualization of AO uptake by cells may be a useful procedure for the evaluation of retinal microcirculation in vivo in rats.

[Morphological studies of rat adrenal glands after space flight on "Kosmos-1667"].

PubMed

Prodan, N G; Bara'nska, V

1989-01-01

Histological and histomorphometric examinations of rat adrenals after a 7-day flight revealed the following changes: blood congestion in the cortex and medulla, progressive delipoidization of the cortex, slight enlargement of the nuclear volume of glomerular and fascicular zones, vacuolization of the cytoplasm of medulla cells, reduction of the area of noradrenocyte islets and cell nuclei of the medulla; the adrenal weight remained however unchanged. It is concluded that an early period of adaptation to microgravity was accompanied by a weak stress-reaction. Upon return to Earth the rats developed an acute gravitational stress. From the morphological point of view the stress manifested as: increased volume of nuclei in fascicular cells, decreased content of lipids in them, and greater vacuolization of the cytoplasm of medulla cells. The lack of medulla hypertrophy, reduction of the area of noradrenocyte islets and nuclei of medulla cells suggest that 7-day exposure to microgravity did not exert of stimulating effect on the sympathetic system of rats.

Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis

PubMed Central

1993-01-01

We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn2+, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis. PMID:8408207

Segmentation and Quantitative Analysis of Apoptosis of Chinese Hamster Ovary Cells from Fluorescence Microscopy Images.

PubMed

Du, Yuncheng; Budman, Hector M; Duever, Thomas A

2017-06-01

Accurate and fast quantitative analysis of living cells from fluorescence microscopy images is useful for evaluating experimental outcomes and cell culture protocols. An algorithm is developed in this work to automatically segment and distinguish apoptotic cells from normal cells. The algorithm involves three steps consisting of two segmentation steps and a classification step. The segmentation steps are: (i) a coarse segmentation, combining a range filter with a marching square method, is used as a prefiltering step to provide the approximate positions of cells within a two-dimensional matrix used to store cells' images and the count of the number of cells for a given image; and (ii) a fine segmentation step using the Active Contours Without Edges method is applied to the boundaries of cells identified in the coarse segmentation step. Although this basic two-step approach provides accurate edges when the cells in a given image are sparsely distributed, the occurrence of clusters of cells in high cell density samples requires further processing. Hence, a novel algorithm for clusters is developed to identify the edges of cells within clusters and to approximate their morphological features. Based on the segmentation results, a support vector machine classifier that uses three morphological features: the mean value of pixel intensities in the cellular regions, the variance of pixel intensities in the vicinity of cell boundaries, and the lengths of the boundaries, is developed for distinguishing apoptotic cells from normal cells. The algorithm is shown to be efficient in terms of computational time, quantitative analysis, and differentiation accuracy, as compared with the use of the active contours method without the proposed preliminary coarse segmentation step.

Java Web Start based software for automated quantitative nuclear analysis of prostate cancer and benign prostate hyperplasia.

PubMed

Singh, Swaroop S; Kim, Desok; Mohler, James L

2005-05-11

Androgen acts via androgen receptor (AR) and accurate measurement of the levels of AR protein expression is critical for prostate research. The expression of AR in paired specimens of benign prostate and prostate cancer from 20 African and 20 Caucasian Americans was compared to demonstrate an application of this system. A set of 200 immunopositive and 200 immunonegative nuclei were collected from the images using a macro developed in Image Pro Plus. Linear Discriminant and Logistic Regression analyses were performed on the data to generate classification coefficients. Classification coefficients render the automated image analysis software independent of the type of immunostaining or image acquisition system used. The image analysis software performs local segmentation and uses nuclear shape and size to detect prostatic epithelial nuclei. AR expression is described by (a) percentage of immunopositive nuclei; (b) percentage of immunopositive nuclear area; and (c) intensity of AR expression among immunopositive nuclei or areas. The percent positive nuclei and percent nuclear area were similar by race in both benign prostate hyperplasia and prostate cancer. In prostate cancer epithelial nuclei, African Americans exhibited 38% higher levels of AR immunostaining than Caucasian Americans (two sided Student's t-tests; P < 0.05). Intensity of AR immunostaining was similar between races in benign prostate. The differences measured in the intensity of AR expression in prostate cancer were consistent with previous studies. Classification coefficients are required due to non-standardized immunostaining and image collection methods across medical institutions and research laboratories and helps customize the software for the specimen under study. The availability of a free, automated system creates new opportunities for testing, evaluation and use of this image analysis system by many research groups who study nuclear protein expression.

Smart markers for watershed-based cell segmentation.

PubMed

Koyuncu, Can Fahrettin; Arslan, Salim; Durmaz, Irem; Cetin-Atalay, Rengul; Gunduz-Demir, Cigdem

2012-01-01

Automated cell imaging systems facilitate fast and reliable analysis of biological events at the cellular level. In these systems, the first step is usually cell segmentation that greatly affects the success of the subsequent system steps. On the other hand, similar to other image segmentation problems, cell segmentation is an ill-posed problem that typically necessitates the use of domain-specific knowledge to obtain successful segmentations even by human subjects. The approaches that can incorporate this knowledge into their segmentation algorithms have potential to greatly improve segmentation results. In this work, we propose a new approach for the effective segmentation of live cells from phase contrast microscopy. This approach introduces a new set of "smart markers" for a marker-controlled watershed algorithm, for which the identification of its markers is critical. The proposed approach relies on using domain-specific knowledge, in the form of visual characteristics of the cells, to define the markers. We evaluate our approach on a total of 1,954 cells. The experimental results demonstrate that this approach, which uses the proposed definition of smart markers, is quite effective in identifying better markers compared to its counterparts. This will, in turn, be effective in improving the segmentation performance of a marker-controlled watershed algorithm.

Permeabilization of the nuclear envelope following nanosecond pulsed electric field exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, Gary L., E-mail: gary.l.thompson.3@gmail.com; Roth, Caleb C.; Kuipers, Marjorie A.

2016-01-29

Permeabilization of cell membranes occurs upon exposure to a threshold absorbed dose (AD) of nanosecond pulsed electric fields (nsPEF). The ultimate, physiological bioeffect of this exposure depends on the type of cultured cell and environment, indicating that cell-specific pathways and structures are stimulated. Here we investigate 10 and 600 ns duration PEF effects on Chinese hamster ovary (CHO) cell nuclei, where our hypothesis is that pulse disruption of the nuclear envelope membrane leads to observed cell death and decreased viability 24 h post-exposure. To observe short-term responses to nsPEF exposure, CHO cells have been stably transfected with two fluorescently-labeled proteins known tomore » be sequestered for cellular chromosomal function within the nucleus – histone-2b (H2B) and proliferating cell nuclear antigen (PCNA). H2B remains associated with chromatin after nsPEF exposure, whereas PCNA leaks out of nuclei permeabilized by a threshold AD of 10 and 600 ns PEF. A downturn in 24 h viability, measured by MTT assay, is observed at the number of pulses required to induce permeabilization of the nucleus. - Highlights: • The ability of nsPEF to damage nuclear structures within cells is investigated. • Leakage of proliferating nuclear antigen from nuclei is induced by nsPEF. • High doses of nsPEF disrupt cortical lamin and cause chromatin decompaction. • Histone H2B remains attached to chromatin following nsPEF exposure. • DNA does not leak out of nsPEF-permeabilized nuclei.« less

Comparison of segmentation algorithms for fluorescence microscopy images of cells.

PubMed

Dima, Alden A; Elliott, John T; Filliben, James J; Halter, Michael; Peskin, Adele; Bernal, Javier; Kociolek, Marcin; Brady, Mary C; Tang, Hai C; Plant, Anne L

2011-07-01

The analysis of fluorescence microscopy of cells often requires the determination of cell edges. This is typically done using segmentation techniques that separate the cell objects in an image from the surrounding background. This study compares segmentation results from nine different segmentation techniques applied to two different cell lines and five different sets of imaging conditions. Significant variability in the results of segmentation was observed that was due solely to differences in imaging conditions or applications of different algorithms. We quantified and compared the results with a novel bivariate similarity index metric that evaluates the degree of underestimating or overestimating a cell object. The results show that commonly used threshold-based segmentation techniques are less accurate than k-means clustering with multiple clusters. Segmentation accuracy varies with imaging conditions that determine the sharpness of cell edges and with geometric features of a cell. Based on this observation, we propose a method that quantifies cell edge character to provide an estimate of how accurately an algorithm will perform. The results of this study will assist the development of criteria for evaluating interlaboratory comparability. Published 2011 Wiley-Liss, Inc.

Cytoarchitectonic study of the brain of a dwarf snakehead, Channa gachua (Ham.). I. The telencephalon.

PubMed

Baile, Vidya V; Patle, Pratap J

2011-12-01

Cytoarchitectonic pattern of the telencephalon of a dwarf snakehead, Channa gachua, is studied by serial transverse sections of the brain (Kluver and Barrera staining). On the anteriormost extremity of the telencephalon, olfactory bulbs terminate that are sessile. The olfactory bulbs comprise four concentric layers, which from outside toward the center are olfactory nerve layer, a glomerular layer, mitral cell layer, and internal cell layer. Large terminal nerve ganglion cells are prominently visible in the dorsomedial position where the bulbs terminate on the telencephalon. In all, 24 nuclei are identified in the telencephalon on ventral and dorsal areas and are named according to their position. Ventral telencephalon exhibits 11 nuclei. On the dorsal telencephalon, there are 13 nuclei. These again are named according to their position on dorsal, ventral, median, lateral, or posterior part. This study reported for the first time in this fish will be useful in tracing the neuronal system of Channa gachua and subsequent studies of the functional aspects of these nuclei in the regulation of reproductive cycle of this species.

Incidence and variation of interpretably bone (os incae) in northeastern Thailand.

PubMed

Thanapaisal, Chaiwit; Duangthongpon, Pichayen; Kitkuandee, Amnat; Chaiciwamongkol, Kowit; Morthong, Vilaiwan

2013-09-01

The squamous segment of occipital bone consists of cartilaginous and membranous origin. The cartilaginous part develops to supra-occipital bone. The membranous part has three primary ossification centers on each side. The first pair ossification center lies above the cartilaginous part between the superior nuchal line and the highest nuchal line and fuse with the cartilaginous part to form a supra-occipital segment of occipital bone. The second and third pairs have two nuclei each forming lateral and medial plates. All of these ossification centers fuse to form squamous segments of occipital bone. The fusion failure between ossification centers of second and third pair nuclei with each other or supra-occipital segment causes separated bone(s) called interparietal bone(s) or os incae. The interparietal bone should be differentiated from Wormian (intrasutural) bone. The incidence from various studies ranges from 0.37% to 9.50% of the population. To study the incidence and variation of interparietal bone in Northeastern Thailand as compared with other studies. A total of 400 Thai native skulls (276 male and 124 female) from the collection of Anatomical Museum of the Faculty of Medicine Khon Kaen University aged from 16 to 93 years old were examined by naked eye and photographed. Wormian bone was excluded by shape and site. The statistical method used was percentage of relative frequency. The incidence of interparietal bone in Northeastern Thailand is 7.25% (29 from 400). Males have a two times higher incidence rate than females, (8.33% versus 4.84%). Eleven patterns of interparietal bone were found. Fusion failure of a third pair ossification center is more common than second pair Knowledge of interparietal bone is useful for neurosurgeons and radiologists to avoid missed diagnosis of skull fracture. Presented interparietal bone may cause difficulty in surgery of occipital and parietal bone. Forensic scientist can use interparietal bone for personal identification.

Nuclear transition moment measurements of neutron rich nuclei

NASA Astrophysics Data System (ADS)

Starosta, Krzysztof

2009-10-01

The Recoil Distance Method (RDM) and related Doppler Shift Attenuation Method (DSAM) are well-established tools for lifetime measurements following nuclear reactions near the Coulomb barrier. Recently, the RDM was implemented at National Superconducting Cyclotron Laboratory (NSCL) at Michigan State University using NSCL/K"oln plunger device and a unique combination of the state-of-the-art instruments available there. Doppler-shift lifetime measurements following Coulomb excitation, knock-out, and fragmentation at intermediate energies of ˜100 MeV/u hold the promise of providing lifetime information for excited states in a wide range of unstable nuclei. So far, the method was used to investigate the collectivity of the neutron-rich ^16,18,20C, ^62,64,66Fe, ^70,72Ni, ^110,114Pd isotopes and also of the neutron-deficient N=Z ^64Ge. A significant fraction of these experiments was performed using NSCL's Segmented Germanium Array instrumented with the Digital Data Acquisition System which enables gamma-ray tracking. The impact of GRETINA and gamma-ray tracking on RDM and DSAM studies of neutron-rich nuclei will be discussed.

[Cytologic diagnosis of adenoid cystic carcinoma of salivary glands and distinction from basal cell adenoma].

PubMed

Bai, Y P; Zhang, Y; Tian, C; Xing, L; Liu, H G

2018-04-08

Objective: To describe the cytologic features of adenoid cystic carcinoma (ADCC) of salivary glands, and to identify distinguishing cytologic features of ADCC and basal cell adenoma (BCA). Methods: A retrospective review of cytology smears of 30 cases of ADCC and 12 cases of BCA of salivary glands were performed. All cases were collected from Beijing Tongren Hospital, Capital Medical University from January 2010 to January 2017. Except for 2 aspirate smears of ADCC, all were touch imprint smears. All cases had further histological confirmation. Results: Neoplastic ductal cells of ADCC were arranged in three-dimensional clusters, sheets and singles. Hyaline globules were found in most cases (20/30, 66.7%). The nuclei were round to oval, showing varying degrees of nuclear atypia. These included (1) the nuclei were hyperchromatic, demonstrating coarse or slightly coarse, irregularly distributed chromatin; (2) the nuclei were slightly large and vary in size; (3) appearance of the nuclei had a different degree of irregularity (often mild). Nucleoli were common seen (21/30, 70.0%), and were prominent in some cases. Mitosis and necrosis were rare. Cytologically, BCA showed cell arrangements and nuclear features overlapped with those of ADCC. The cytologic difference between these two tumors included: (1) the tumor cells presented rarely in singles; (2) hyaline globules were very uncommon (1/12) in BCA; (3) nuclei of BCA were hypochromatic or slightly hyperchromatic, homogeneous and uniform in appearance and size, overall without nuclear atypia and they were smaller and slender then those of ADCC and (4) individual cells of BCA showed relatively abundant cytoplasm. Conclusions: The cytologic features of ADCC and BCA both overlap and different from each other. Most cases can be diagnosed by cytologic examination. The presence of hyaline globules is an important diagnostic clue of ADCC, although not pathognomonic. Nuclear atypia of neoplastic ductal cells is an essential cytological feature in the diagnosis of ADCC, and is the most reliable point for differential diagnosis of ADCC and BCA.

Assessment of cell death mechanisms triggered by 177Lu-anti-CD20 in lymphoma cells.

PubMed

Azorín-Vega, E; Rojas-Calderón, E; Martínez-Ventura, B; Ramos-Bernal, J; Serrano-Espinoza, L; Jiménez-Mancilla, N; Ordaz-Rosado, D; Ferro-Flores, G

2018-08-01

The aim of this research was to evaluate the cell cycle redistribution and activation of early and late apoptotic pathways in lymphoma cells after treatment with 177 Lu-anti-CD20. Experimental and computer models were used to calculate the radiation absorbed dose to cancer cell nuclei. The computer model (Monte Carlo, PENELOPE) consisted of twenty spheres representing cells with an inner sphere (cell nucleus) embedded in culture media. Radiation emissions of the radiopharmaceutical located in cell membranes and in culture media were considered for nuclei dose calculations. Flow cytometric analyses demonstrated that doses as low as 4.8Gy are enough to induce cell cycle arrest and activate late apoptotic pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

AUTOMATED CELL SEGMENTATION WITH 3D FLUORESCENCE MICROSCOPY IMAGES.

PubMed

Kong, Jun; Wang, Fusheng; Teodoro, George; Liang, Yanhui; Zhu, Yangyang; Tucker-Burden, Carol; Brat, Daniel J

2015-04-01

A large number of cell-oriented cancer investigations require an effective and reliable cell segmentation method on three dimensional (3D) fluorescence microscopic images for quantitative analysis of cell biological properties. In this paper, we present a fully automated cell segmentation method that can detect cells from 3D fluorescence microscopic images. Enlightened by fluorescence imaging techniques, we regulated the image gradient field by gradient vector flow (GVF) with interpolated and smoothed data volume, and grouped voxels based on gradient modes identified by tracking GVF field. Adaptive thresholding was then applied to voxels associated with the same gradient mode where voxel intensities were enhanced by a multiscale cell filter. We applied the method to a large volume of 3D fluorescence imaging data of human brain tumor cells with (1) small cell false detection and missing rates for individual cells; and (2) trivial over and under segmentation incidences for clustered cells. Additionally, the concordance of cell morphometry structure between automated and manual segmentation was encouraging. These results suggest a promising 3D cell segmentation method applicable to cancer studies.

Flow of essential elements in subcellular fractions during oxidative stress.

PubMed

Lago, Larissa; Nunes, Emilene A; Vigato, Aryane A; Souza, Vanessa C O; Barbosa, Fernando; Sato, João R; Batista, Bruno L; Cerchiaro, Giselle

2017-02-01

Essential trace elements are commonly found in altered concentrations in the brains of patients with neurodegenerative diseases. Many studies in trace metal determination and quantification are conducted in tissue, cell culture or whole brain. In the present investigation, we determined by ICP-MS Fe, Cu, Zn, Ca, Se, Co, Cr, Mg, and Mn in organelles (mitochondria, nuclei) and whole motor neuron cell cultured in vitro. We performed experiments using two ways to access oxidative stress: cell treatments with H 2 O 2 or Aβ-42 peptide in its oligomeric form. Both treatments caused accumulation of markers of oxidative stress, such as oxidized proteins and lipids, and alteration in DNA. Regarding trace elements, cells treated with H 2 O 2 showed higher levels of Zn and lower levels of Ca in nuclei when compared to control cells with no oxidative treatments. On the other hand, cells treated with Aβ-42 peptide in its oligomeric form showed higher levels of Mg, Ca, Fe and Zn in nuclei when compared to control cells. These differences showed that metal flux in cell organelles during an intrinsic external oxidative condition (H 2 O 2 treatment) are different from an intrinsic external neurodegenerative treatment.

Alterations in the nuclear proteome of HIV-1 infected T-cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

DeBoer, Jason; Jagadish, Teena; Haverland, Nicole A.

Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified fourmore » clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.« less

Introduced T cell receptor variable region gene segments recombine in pre-B cells: evidence that B and T cells use a common recombinase.

PubMed

Yancopoulos, G D; Blackwell, T K; Suh, H; Hood, L; Alt, F W

1986-01-31

We have recently proposed that a common recombinase performs all of the many variable region gene assembly events in B and T cells, and that the specificity of these joining events is mediated by regulating the "accessibility" of the involved gene segments. To test this possibility, we have introduced "accessible" T cell receptor (TCR) variable region gene segments into a pre-B cell line capable of recombining endogenous and transfected immunoglobulin (Ig) variable region gene segments. Although the corresponding "inaccessible" endogenous TCR gene segments do not rearrange in this line or in B cells in general, the introduced TCR gene segments join very frequently and, in fact, closely resemble introduced Ig gene segments in their recombination characteristics. These observations suggest a new role for conventional Ig transcriptional enhancers--recombinational enhancement. Our studies provide insight into additional aspects of the joining mechanism such as N region insertion, aberrant joining, and recombination-recognition sequence requirements for joining.

Using white-light spectroscopy for size determination of tissue phantoms

NASA Astrophysics Data System (ADS)

Vitol, Elina A.; Kurzweg, Timothy P.; Nabet, Bahram

2005-09-01

Along with breast and cervical cancer, esophageal adenocarcinoma is one of the most common types of cancers. The characteristic features of pre-cancerous tissues are the increase in cell proliferation rate and cell nuclei enlargement, which both take place in the epithelium of human body surfaces. However, in the early stages of cancer these changes are very small and difficult to detect, even for expert pathologists. The aim of our research is to develop an optical probe for in vivo detection of nuclear size changes using white light scattering from cell nuclei. The probe will be employed through an endoscope and will be used for the medical examination of the esophagus. The proposed method of examination will be noninvasive, cheap, and specific, compared to a biopsy. Before the construction of this probe, we have developed theory to determine the nuclei size from the reflection data. In this first stage of our research, we compare experimental and theoretical scattered light intensities. Our theoretical model includes the values of scatterer size from which we can extract the nuclei size value. We first performed the study of polystyrene microspheres, acting as a tissue phantom. Spectral and angular distributions of scattered white light from tissue phantoms were studied. Experimental results show significant differences between the spectra of microspheres of different sizes and demonstrate almost linear relation between the number of spectral oscillations and the size of microspheres. Best results were achieved when the scattered light spectrum was collected at 30° to the normal of the sample surface. We present these research results in this paper. In ongoing work, normal and cancerous mammalian cell studies are being performed in order to determine cell nuclei size correlation with the size of microspheres through the light scattering spectrum observation.

Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin

PubMed Central

Diaz Perez, Silvia V.; Kim, Rachel; Li, Ziwei; Marquez, Victor E.; Patel, Sanjeet; Plath, Kathrin; Clark, Amander T.

2012-01-01

Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1–UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state. PMID:22058289

Defining external factors that determine neuronal survival, apoptosis and necrosis during excitotoxic injury using a high content screening imaging platform.

PubMed

Anilkumar, Ujval; Weisova, Petronela; Schmid, Jasmin; Bernas, Tytus; Huber, Heinrich J; Düssmann, Heiko; Connolly, Niamh M C; Prehn, Jochen H M

2017-01-01

Cell death induced by excessive glutamate receptor overactivation, excitotoxicity, has been implicated in several acute and chronic neurological disorders. While numerous studies have demonstrated the contribution of biochemically and genetically activated cell death pathways in excitotoxic injury, the factors mediating passive, excitotoxic necrosis are less thoroughly investigated. To address this question, we developed a high content screening (HCS) based assay to collect high volumes of quantitative cellular imaging data and elucidated the effects of intrinsic and external factors on excitotoxic necrosis and apoptosis. The analysis workflow consisted of robust nuclei segmentation, tracking and a classification algorithm, which enabled automated analysis of large amounts of data to identify and quantify viable, apoptotic and necrotic neuronal populations. We show that mouse cerebellar granule neurons plated at low or high density underwent significantly increased necrosis compared to neurons seeded at medium density. Increased extracellular Ca2+ sensitized neurons to glutamate-induced excitotoxicity, but surprisingly potentiated cell death mainly through apoptosis. We also demonstrate that inhibition of various cell death signaling pathways (including inhibition of calpain, PARP and AMPK activation) primarily reduced excitotoxic apoptosis. Excitotoxic necrosis instead increased with low extracellular glucose availability. Our study is the first of its kind to establish and implement a HCS based assay to investigate the contribution of external and intrinsic factors to excitotoxic apoptosis and necrosis.

Defining external factors that determine neuronal survival, apoptosis and necrosis during excitotoxic injury using a high content screening imaging platform

PubMed Central

Weisova, Petronela; Schmid, Jasmin; Bernas, Tytus; Huber, Heinrich J.; Düssmann, Heiko; Connolly, Niamh M. C.; Prehn, Jochen H. M.

2017-01-01

Cell death induced by excessive glutamate receptor overactivation, excitotoxicity, has been implicated in several acute and chronic neurological disorders. While numerous studies have demonstrated the contribution of biochemically and genetically activated cell death pathways in excitotoxic injury, the factors mediating passive, excitotoxic necrosis are less thoroughly investigated. To address this question, we developed a high content screening (HCS) based assay to collect high volumes of quantitative cellular imaging data and elucidated the effects of intrinsic and external factors on excitotoxic necrosis and apoptosis. The analysis workflow consisted of robust nuclei segmentation, tracking and a classification algorithm, which enabled automated analysis of large amounts of data to identify and quantify viable, apoptotic and necrotic neuronal populations. We show that mouse cerebellar granule neurons plated at low or high density underwent significantly increased necrosis compared to neurons seeded at medium density. Increased extracellular Ca2+ sensitized neurons to glutamate-induced excitotoxicity, but surprisingly potentiated cell death mainly through apoptosis. We also demonstrate that inhibition of various cell death signaling pathways (including inhibition of calpain, PARP and AMPK activation) primarily reduced excitotoxic apoptosis. Excitotoxic necrosis instead increased with low extracellular glucose availability. Our study is the first of its kind to establish and implement a HCS based assay to investigate the contribution of external and intrinsic factors to excitotoxic apoptosis and necrosis. PMID:29145487

Low levels of citrin (SLC25A13) expression in adult mouse brain restricted to neuronal clusters.

PubMed

Contreras, Laura; Urbieta, Almudena; Kobayashi, Keiko; Saheki, Takeyori; Satrústegui, Jorgina

2010-04-01

The mitochondrial aspartate-glutamate carriers (AGC) aralar (SLC25A12) and citrin (SLC25A13) are components of the malate aspartate shuttle (MAS), a major intracellular pathway to transfer reducing equivalents from NADH to the mitochondrial matrix. Aralar is the main AGC isoform present in the adult brain, and it is expressed mainly in neurons. To search for the other AGC isoform, citrin, in brain glial cells, we used a citrin knockout mouse in which the lacZ gene was inserted into the citrin locus as reporter gene. In agreement with the low citrin levels known to be present in the adult mouse brain, beta-galactosidase expression was very low. Surprisingly, unlike the case with astroglial cultures that express citrin, no beta-galactosidase was found in brain glial cells. It was confined to neuronal cells within discrete neuronal clusters. Double-immunolabelling experiments showed that beta-galactosidase colocalized not with glial cell markers but with the pan-neuronal marker NeuN. The deep cerebellar nuclei and a few midbrain nuclei (reticular tegmental pontine nuclei; magnocellular red nuclei) were the regions where beta-galactosidase expression was highest, and it was up-regulated in fasted mice, as was also the case for liver beta-galactosidase. The results support the notion that glial cells have much lower AGC levels and MAS activity than neurons. (c) 2009 Wiley-Liss, Inc.

The complex pericentriolar material 1 protein allows differentiation between myonuclei and nuclei of satellite cells of the skeletal muscle.

PubMed

Brunn, Anna

2018-05-27

The original article by Winje et al., entitled "Specific labelling of myonuclei by an antibody against pericentriolar material 1 (PCM1) on skeletal muscle tissue sections" 1 , sheds new light on the issue of heterogeneity of skeletal muscle and, thus, the problem to reliably distinguish between myonuclei versus nuclei of satellite cells of the skeletal muscle which are intimately associated. At the light microscopical level this differentiation is particularly difficult since only nuclei inside the muscle fiber are defined as true myonuclei. This is a major problem in analyses that use tissue homogenates, while in situ immunohistochemical studies using appropriate antibodies usually allow differentiation of cell populations. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Nonserotonergic projection neurons in the midbrain raphe nuclei contain the vesicular glutamate transporter VGLUT3.

PubMed

Jackson, Jesse; Bland, Brian H; Antle, Michael C

2009-01-01

The brainstem raphe nuclei are typically assigned a role in serotonergic brain function. However, numerous studies have reported that a large proportion of raphe projection cells are nonserotonergic. The identity of these projection cells is unknown. Recent studies have reported that the vesicular glutamate transporter VGLUT3 is found in both serotonergic and nonserotonergic neurons in both the median raphe (MR) and dorsal raphe (DR) nuclei. We injected the retrograde tracer cholera toxin subunit B into either the dorsal hippocampus or the medial septum (MS) and used triple labeled immunofluorescence to determine if nonserotonergic raphe cells projecting to these structures contained VGLUT3. Consistent with previous studies, only about half of retrogradely labeled MR neurons projecting to the hippocampus contained serotonin, whereas a majority of the retrogradely labeled nonserotonergic cells contained VGLUT3. Similar patterns were observed for MR cells projecting to the MS. About half of retrogradely labeled nonserotonergic neurons in the DR contained VGLUT3. Additionally, a large number of retrogradely labeled cells in the caudal linear and interpeduncular nuclei projecting to the MS were found to contain VGLUT3. These data suggest the enigmatic nonserotonergic projection from the MR to forebrain regions may be glutamatergic. In addition, these results demonstrate a dissociation between glutamatergic and serotonergic MR afferent inputs to the MS and hippocampus suggesting divergent and/or complementary roles of these pathways in modulating cellular activity within the septohippocampal network.

[A morphometric analysis of the nuclei and nucleoli in tumor cells in lymphogranulomatosis, diffuse large B-cell lymphoma and anaplastic large cell lymphoma].

PubMed

Gorgidze, L A; Vorob'ev, I A

2009-01-01

To make a comparative morphometric analysis of the nuclei and nucleoli of tumor cells in lymphogranulomatosis (LGM), diffuse large B-cell lymphoma (DLBCL) and anaplastic large cell lymphoma (ALCL) for differential diagnosis of these lymphomas. Biopsy material (lymph node biopsies) was frozen in hexane, fixed and stained, then microscopic pictures were made. Mean area of tumor cell nuclei in LGM was 97.25 +/- 68.77 mcm2, in DLBCL and ALCL--55.89 +/- 20.13 mcm2 and 70.31 +/- 34.64 mcm2, respectively. The area differences were significant (p < 0.001). Hodgkin's and Berezovsky-Rid-Sternberg cell bucleoli area was the largest (11.44 +/- 7.83 mcm2). The nucleoli of the former are larger than those of the latter. Mean area of the nucleoli in DLBCL was 3.05 +/- 1.58, in ALCL--5.53 +/- 4.94 mcm2. The differences are significant (p < 0.001). Nucleoli in Hodgkin 's cells are significantly larger than those in the tumor cells in ALCL and DLBCL and the nucleoli with the area more than 12 mcm2 can be used in differential diagnosis between LGM and DLBCL but not between LGM and ALCL.

Relationship between DNA ploidy level and tumor sociology behavior in 12 nervous cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kiss, R.; Camby, I.; Salmon, I.

1995-06-01

Cell population sociology was studied in two medulloblastomas and 10 astrocytic human tumor cell lines by means of the characterization of the structure of neoplastic cell colonies growing on histological slides. This was carried out via digital cell image analysis of Feulgen-stained nuclei, to which the Delaunay triangulation and Voronoi paving mathematical techniques were applied. Such assessments were compared to the DNA ploidy level (assessed by means of DNA histogram typing). The results show that the cell colony architecture characteristics differed markedly according to whether the cell lines were euploid (diploid or tetraploid) or aneuploid (hyperdiploid, triploid, hypertriploid, or polymorphic).more » In fact, the cell colonies from the euploid cell nuclei populations were larger and more dense than those from the aneuploid ones. Furthermore, for an identical period of culture, the cell lines from high-grade malignant astrocytic tumors (glioblastomas) exhibited cell colonies that were larger and more dense than those in cell lines from low-grade astrocytic tumors (astrocytomas). In each of these two groups, the diploid cell nuclei populations exhibited cell colonies larger and more dense than the nondiploid colonies. The present methodology is now being applied in vivo to histological sections of surgically removed human brain tumors in order to distinguish between high-risk clinical subgroups and medium-risk subgroups in clearly circumscribed histopathological groups. 38 refs., 5 figs., 2 tabs.« less

A spectral k-means approach to bright-field cell image segmentation.

PubMed

Bradbury, Laura; Wan, Justin W L

2010-01-01

Automatic segmentation of bright-field cell images is important to cell biologists, but difficult to complete due to the complex nature of the cells in bright-field images (poor contrast, broken halo, missing boundaries). Standard approaches such as level set segmentation and active contours work well for fluorescent images where cells appear as round shape, but become less effective when optical artifacts such as halo exist in bright-field images. In this paper, we present a robust segmentation method which combines the spectral and k-means clustering techniques to locate cells in bright-field images. This approach models an image as a matrix graph and segment different regions of the image by computing the appropriate eigenvectors of the matrix graph and using the k-means algorithm. We illustrate the effectiveness of the method by segmentation results of C2C12 (muscle) cells in bright-field images.

Central Topography of Cranial Motor Nuclei Controlled by Differential Cadherin Expression

PubMed Central

Astick, Marc; Tubby, Kristina; Mubarak, Waleed M.; Guthrie, Sarah; Price, Stephen R.

2014-01-01

Summary Neuronal nuclei are prominent, evolutionarily conserved features of vertebrate central nervous system (CNS) organization [1]. Nuclei are clusters of soma of functionally related neurons and are located in highly stereotyped positions. Establishment of this CNS topography is critical to neural circuit assembly. However, little is known of either the cellular or molecular mechanisms that drive nucleus formation during development, a process termed nucleogenesis [2–5]. Brainstem motor neurons, which contribute axons to distinct cranial nerves and whose functions are essential to vertebrate survival, are organized exclusively as nuclei. Cranial motor nuclei are composed of two main classes, termed branchiomotor/visceromotor and somatomotor [6]. Each of these classes innervates evolutionarily distinct structures, for example, the branchial arches and eyes, respectively. Additionally, each class is generated by distinct progenitor cell populations and is defined by differential transcription factor expression [7, 8]; for example, Hb9 distinguishes somatomotor from branchiomotor neurons. We characterized the time course of cranial motornucleogenesis, finding that despite differences in cellular origin, segregation of branchiomotor and somatomotor nuclei occurs actively, passing through a phase of each being intermingled. We also found that differential expression of cadherin cell adhesion family members uniquely defines each motor nucleus. We show that cadherin expression is critical to nucleogenesis as its perturbation degrades nucleus topography predictably. PMID:25308074

High frame-rate resolution of cell division during Candida albicans filamentation

PubMed Central

Thomson, Darren D.; Berman, Judith; Brand, Alexandra C.

2016-01-01

The commensal yeast, Candida albicans, is an opportunistic pathogen in humans and forms filaments called hyphae and pseudohyphae, in which cell division requires precise temporal and spatial control to produce mononuclear cell compartments. High-frame-rate live-cell imaging (1 frame/min) revealed that nuclear division did not occur across the septal plane. We detected the presence of nucleolar fragments that may be extrachromosomal molecules carrying the ribosomal RNA genes. Cells occasionally maintained multiple nucleoli, suggesting either polyploidy, multiple nuclei and/or aneuploidy of ChrR., while the migration pattern of sister nuclei differed between unbranched and branched hyphae. The presented movie challenges and extends previous concepts of C. albicans cell division. PMID:26854071

α-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected Heliothis zea Larvae and Spodoptera frugiperda Cells

PubMed Central

Grula, Marjori A.; Buller, Patricia L.; Weaver, Robert F.

1981-01-01

[3H]RNA was synthesized in nuclei isolated at various times postinfection from the fat bodies of Heliothis zea larvae infected with H. zea nuclear polyhedrosis virus and from cultured Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus. To detect virus-specific RNA synthesis, the [3H]RNA was hybridized to denatured viral DNA immobilized on nitrocellulose filters. Nuclear polyhedrosis virus-specific RNA synthesis in the infected nuclei isolated from H. zea larval fat bodies and S. frugiperda cells was only inhibited 20 to 25% by concentrations of α-amanitin sufficient to inhibit the host RNA polymerase II. In addition, a productive nuclear polyhedrosis virus infection was obtained in S. frugiperda cells grown in the presence of an α-amanitin concentration that inhibited 90% of the cellular RNA polymerase II activity. The cellular RNA polymerase II enzyme remained sensitive to α-amanitin during infection, and there was no evidence that a virus-coded, α-amanitin-resistant enzyme was synthesized after the onset of infection. The data suggest that the bulk of nuclear polyhedrosis virus-specific RNA synthesis in isolated nuclei is transcribed by an enzyme other than the host RNA polymerase II. PMID:16789208

Spatiotemporal expression of Ezh2 in the developing mouse cochlear sensory epithelium.

PubMed

Chen, Yan; Li, Wenyan; Li, Wen; Chai, Renjie; Li, Huawei

2016-09-01

The enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) is a histone-lysine Nmethyltransferase enzyme that participates in DNA methylation. Ezh2 has also been reported to play crucial roles in stem cell proliferation and differentiation. However, the detailed expression profile of Ezh2 during mouse cochlear development has not been investigated. Here, we examined the spatiotemporal expression of Ezh2 in the cochlea during embryonic and postnatal development. Ezh2 expression began to be observed in the whole otocyst nuclei at embryonic day 9.5 (E9.5). At E12.5, Ezh2 was expressed in the nuclei of the cochlear prosensory epithelium. At E13.5 and E15.5, Ezh2 was expressed from the apical to the basal turns in the nuclei of the differentiating cochlear epithelium. At postnatal day (P) 0 and 7, the Ezh2 expression was located in the nuclei of the cochlear epithelium in all three turns and could be clearly seen in outer and inner hair cells, supporting cells, the stria vascularis, and spiral ganglion cells. Ezh2 continued to be expressed in the cochlear epithelium of adult mice. Our results provide the basic Ezh2 expression pattern and might be useful for further investigating the detailed role of Ezh2 during cochlear development.

Nuclear Physics in a biological context

NASA Astrophysics Data System (ADS)

Discher, Dennis

2012-02-01

A solid tissue can be soft like fat or brain, stiff like striated muscle and heart, or rigid like bone -- and of course every cell has a nucleus that contributes in some way small or large to tissue mechanics. Indeed, nuclei generally exhibit rheology and plasticity that reflects both the chromatin and the nuclear envelope proteins called lamins, all of which change in differentiation. Profiling of tissue nuclei shows that the nuclear intermediate filament protein Lamin-A/C varies over 30-fold between adult tissues and scales strongly with micro-elasticity of tissue, while other nuclear envelope components such as Lamin-B exhibit small variations. Lamin-A/C has been implicated in aging syndromes that affect muscle and fat but not brain, and we find nuclei in brain-derived cells are indeed dominated by Lamin-B and are much softer than nuclei derived from muscle cells with predominantly Lamin-A/C. In vitro, matrix elasticity can affect expression of nuclear envelope components in adult stem cells, and major changes in Lamin-A/C are indeed shown to direct lineage with lower levels favoring soft tissue and higher levels promoting rigid tissue lineage. Further molecular studies provide evidence that the nucleus transduces physical stress. References: (1) J.D. Pajerowski, K.N. Dahl, F.L. Zhong, P.J. Sammak, and D.E. Discher. Physical plasticity of the nucleus in stem cell differentiation. PNAS 104: 15619-15624 (2007). (2) A. Buxboim, I. Ivanova, and D.E. Discher. Matrix Elasticity, Cytoskeletal Forces, and Physics of the Nucleus: how deeply do cells `feel' outside and in? Journal of Cell Science 123: 297-308 (2010).

Spatial distribution of centromeres and telomeres at interphase varies among Brachypodium species.

PubMed

Idziak, Dominika; Robaszkiewicz, Ewa; Hasterok, Robert

2015-11-01

In this study the 3-D distribution of centromeres and telomeres was analysed in the interphase nuclei of three Brachypodium species, i.e. B. distachyon (2n=10), B. stacei (2n=20) and B. hybridum (2n=30), which is presumably a hybrid between the first two species. Using fluorescence in situ hybridization (FISH) with centromeric and telomeric DNA probes, it was observed that the majority of B. distachyon nuclei in the root tip cells displayed the Rabl configuration while both B. stacei and B. hybridum mostly lacked the centromere-telomere polarization. In addition, differentiated leaf cells of B. distachyon did not display the Rabl pattern. In order to analyse the possible connection between the occurrence of the Rabl pattern and the phase of cell cycle or DNA content, FISH was combined with digital image cytometry. The results revealed that the frequency of nuclei with the Rabl configuration in the root tip nuclei was positively correlated with an increase in DNA content, which resulted from DNA replication. Also, the analysis of the influence of the nuclear shape on the nuclear architecture indicated that an increasing elongation of the nuclei negatively affected the occurrence of the Rabl pattern. Some possible explanations of these phenomena are discussed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Diversity of vestibular nuclei neurons targeted by cerebellar nodulus inhibition

PubMed Central

Meng, Hui; Blázquez, Pablo M; Dickman, J David; Angelaki, Dora E

2014-01-01

Abstract A functional role of the cerebellar nodulus and ventral uvula (lobules X and IXc,d of the vermis) for vestibular processing has been strongly suggested by direct reciprocal connections with the vestibular nuclei, as well as direct vestibular afferent inputs as mossy fibres. Here we have explored the types of neurons in the macaque vestibular nuclei targeted by nodulus/ventral uvula inhibition using orthodromic identification from the caudal vermis. We found that all nodulus-target neurons are tuned to vestibular stimuli, and most are insensitive to eye movements. Such non-eye-movement neurons are thought to project to vestibulo-spinal and/or thalamo-cortical pathways. Less than 20% of nodulus-target neurons were sensitive to eye movements, suggesting that the caudal vermis can also directly influence vestibulo-ocular pathways. In general, response properties of nodulus-target neurons were diverse, spanning the whole continuum previously described in the vestibular nuclei. Most nodulus-target cells responded to both rotation and translation stimuli and only a few were selectively tuned to translation motion only. Other neurons were sensitive to net linear acceleration, similar to otolith afferents. These results demonstrate that, unlike the flocculus and ventral paraflocculus which target a particular cell group, nodulus/ventral uvula inhibition targets a large diversity of cell types in the vestibular nuclei, consistent with a broad functional significance contributing to vestibulo-ocular, vestibulo-thalamic and vestibulo-spinal pathways. PMID:24127616

Tracking cells in Life Cell Imaging videos using topological alignments.

PubMed

Mosig, Axel; Jäger, Stefan; Wang, Chaofeng; Nath, Sumit; Ersoy, Ilker; Palaniappan, Kannap-pan; Chen, Su-Shing

2009-07-16

With the increasing availability of live cell imaging technology, tracking cells and other moving objects in live cell videos has become a major challenge for bioimage informatics. An inherent problem for most cell tracking algorithms is over- or under-segmentation of cells - many algorithms tend to recognize one cell as several cells or vice versa. We propose to approach this problem through so-called topological alignments, which we apply to address the problem of linking segmentations of two consecutive frames in the video sequence. Starting from the output of a conventional segmentation procedure, we align pairs of consecutive frames through assigning sets of segments in one frame to sets of segments in the next frame. We achieve this through finding maximum weighted solutions to a generalized "bipartite matching" between two hierarchies of segments, where we derive weights from relative overlap scores of convex hulls of sets of segments. For solving the matching task, we rely on an integer linear program. Practical experiments demonstrate that the matching task can be solved efficiently in practice, and that our method is both effective and useful for tracking cells in data sets derived from a so-called Large Scale Digital Cell Analysis System (LSDCAS). The source code of the implementation is available for download from http://www.picb.ac.cn/patterns/Software/topaln.

The study of muscle remodeling in Drosophila metamorphosis using in vivo microscopy and bioimage informatics

PubMed Central

2012-01-01

Background Metamorphosis in insects transforms the larval into an adult body plan and comprises the destruction and remodeling of larval and the generation of adult tissues. The remodeling of larval into adult muscles promises to be a genetic model for human atrophy since it is associated with dramatic alteration in cell size. Furthermore, muscle development is amenable to 3D in vivo microscopy at high cellular resolution. However, multi-dimensional image acquisition leads to sizeable amounts of data that demand novel approaches in image processing and analysis. Results To handle, visualize and quantify time-lapse datasets recorded in multiple locations, we designed a workflow comprising three major modules. First, the previously introduced TLM-converter concatenates stacks of single time-points. The second module, TLM-2D-Explorer, creates maximum intensity projections for rapid inspection and allows the temporal alignment of multiple datasets. The transition between prepupal and pupal stage serves as reference point to compare datasets of different genotypes or treatments. We demonstrate how the temporal alignment can reveal novel insights into the east gene which is involved in muscle remodeling. The third module, TLM-3D-Segmenter, performs semi-automated segmentation of selected muscle fibers over multiple frames. 3D image segmentation consists of 3 stages. First, the user places a seed into a muscle of a key frame and performs surface detection based on level-set evolution. Second, the surface is propagated to subsequent frames. Third, automated segmentation detects nuclei inside the muscle fiber. The detected surfaces can be used to visualize and quantify the dynamics of cellular remodeling. To estimate the accuracy of our segmentation method, we performed a comparison with a manually created ground truth. Key and predicted frames achieved a performance of 84% and 80%, respectively. Conclusions We describe an analysis pipeline for the efficient handling and analysis of time-series microscopy data that enhances productivity and facilitates the phenotypic characterization of genetic perturbations. Our methodology can easily be scaled up for genome-wide genetic screens using readily available resources for RNAi based gene silencing in Drosophila and other animal models. PMID:23282138

[Spatial arrangement of macro-, midi- and microchromosomes in transcriptionally active nuclei of growing oocytes in birds of the order galliformes].

PubMed

Maslova, A V; Krasikova, A V

2011-01-01

Three-dimensional genome organization in the cell nucleus reflects its functional state and is one of the regulatory levels of gene expression. Thus, a detailed exploration of the interrelations between the genome spatial organization and functioning is essential. In this work, three-dimensional genome organization in growing oocytes of Galliform birds, with giant transcriptionally active nucleus characterized by nearly absolute lack of structural constraints on chromosome decondensation, is analyzed in detail. Radial distribution of three groups of chromosomes with different size and gene density in the nuclei of chicken and Japanese quail oocytes was analyzed using confocal laser scanning microscopy followed by the 3D-reconstruction. The chromosome position relative to the nuclear center was estimated by analyzing its localization in the certain radial zones of the nucleus and direct distance measurements from the centre of the nucleus to the terminal regions and the center of gravity of the chromosome. It has been shown that, in the transcriptionally active nuclei of avian oocytes, chromosomes are located at a significant distance from the nuclear envelope and the gene-rich microchromosomes have no preferential location close to the center of the nucleus and are localized mainly at the periphery of the region occupied by the whole chromosome set. Therefore the radial distribution of lampbrush chromosomes in the oocyte nucleus does not obey the regularity of the spatial arrangement of chromosomes in the interphase nucleus according to which the gene-rich chromosome territories are located at the nuclear center and the gene-poor ones are at the nuclear periphery. With the help of visualization of 3D-preserved lampbrush chromosomes in the intact nucleus, we have confirmed the presence of the repulsion forces between the lateral loops of lampbrush half-bivalents and the lack of interactions between the heterochromatic segments of different bivalents at the lampbrush stage of oogenesis.

Meckelin 3 Is Necessary for Photoreceptor Outer Segment Development in Rat Meckel Syndrome

PubMed Central

Tiwari, Sarika; Hudson, Scott; Gattone, Vincent H.; Miller, Caroline; Chernoff, Ellen A. G.; Belecky-Adams, Teri L.

2013-01-01

Ciliopathies lead to multiorgan pathologies that include renal cysts, deafness, obesity and retinal degeneration. Retinal photoreceptors have connecting cilia joining the inner and outer segment that are responsible for transport of molecules to develop and maintain the outer segment process. The present study evaluated meckelin (MKS3) expression during outer segment genesis and determined the consequences of mutant meckelin on photoreceptor development and survival in Wistar polycystic kidney disease Wpk/Wpk rat using immunohistochemistry, analysis of cell death and electron microscopy. MKS3 was ubiquitously expressed throughout the retina at postnatal day 10 (P10) and P21. However, in the mature retina, MKS3 expression was restricted to photoreceptors and the retinal ganglion cell layer. At P10, both the wild type and homozygous Wpk mutant retina had all retinal cell types. In contrast, by P21, cells expressing rod- and cone-specific markers were fewer in number and expression of opsins appeared to be abnormally localized to the cell body. Cell death analyses were consistent with the disappearance of photoreceptor-specific markers and showed that the cells were undergoing caspase-dependent cell death. By electron microscopy, P10 photoreceptors showed rudimentary outer segments with an axoneme, but did not develop outer segment discs that were clearly present in the wild type counterpart. At p21 the mutant outer segments appeared much the same as the P10 mutant outer segments with only a short axoneme, while the wild-type controls had developed outer segments with many well-organized discs. We conclude that MKS3 is not important for formation of connecting cilium and rudimentary outer segments, but is critical for the maturation of outer segment processes. PMID:23516626

Functional Characterization of G12, a Gene Required for Mitotic Progression during Gastrulation in Zebrafish

NASA Technical Reports Server (NTRS)

Reinsch, Sigrid; Conway, Gregory; Dalton, Bonnie P. (Technical Monitor)

2002-01-01

In a differential RNA display screen we have isolated a zebrafish gene, G12, for which homologs can only be found in DNA databases for vertebrates, but not invertebrates. This suggests that this is a gene required specifically in vertebrates. G12 expression is upregulated at mid-blastula transition (MBT). Morpholino inactivation of this gene by injection into 1-cell embryos results in mitotic defects and apoptosis shortly after MBT. Nuclei in morpholino treated embryos also display segregation defects. We have characterized the localization of this gene as a GFP fusion in live and fixed embryos. Overexpression of G12-GFP is non-toxic. Animals retain GFP expression for at least 7 days with no developmental defects, Interestingly in these animals G12-GFP is never detectable in blood cells though blood is present. In the deep cells of early embryos, G 12GFP is localized to nuclei and cytoskeletal elements in interphase and to the centrosome and spindle apparatus during mitosis. In the EVL, G12-GFP shows additional localization to the cell periphery, especially in mitosis. In the yolk syncytium, G12-GFP again localizes to nuclei and strongly to cytoplasmic microtubules of migrating nuclei at the YSL margin. Morpholinc, injection specifically into the YSL after cellularization blocks epiboly and nuclei of the YSL show mitotic defects while deep cells show no mitotic defects and continue to divide. Rescue experiments in which morpholino and G12-GFP RNA are co-injected indicate partial rescue by the G12-GFP. The rescue is cell autonomous; that is, regions of the embryo with higher G12-GFP expression show fewer mitotic defects. Spot 14, the human bomolog of G12, has been shown to be amplified in aggressive breast tumors. This finding, along with our functional and morphological data suggest that G12 and spot 14 are vertebrate-specific and may function either as mitotic checkpoints or as structural components of the spindle apparatus.

Distinct development of the trigeminal sensory nuclei in platypus and echidna.

PubMed

Ashwell, Ken W S; Hardman, Craig D

2012-01-01

Both lineages of the modern monotremes have been reported to be capable of electroreception using the trigeminal pathways and it has been argued that electroreception arose in an aquatic platypus-like ancestor of both modern monotreme groups. On the other hand, the trigeminal sensory nuclear complex of the platypus is highly modified for processing tactile and electrosensory information from the bill, whereas the trigeminal sensory nuclear complex of the short-beaked echidna (Tachyglossus aculeatus) is not particularly specialized. If the common ancestor for both platypus and echidna were an electroreceptively and trigeminally specialized aquatic feeder, one would expect the early stages of development of the trigeminal sensory nuclei in both species to show evidence of structural specialization from the outset. To determine whether this is the case, we examined the development of the trigeminal sensory nuclei in the platypus and short-beaked echidna using the Hill and Hubrecht embryological collections. We found that the highly specialized features of the platypus trigeminal sensory nuclei (i.e. the large size of the principal nucleus and oral part of the spinal trigeminal nuclear complex, and the presence of a dorsolateral parvicellular segment in the principal nucleus) appear around the time of hatching in the platypus, but are never seen at any stage in the echidna. Our findings support the proposition that the modern echidna and platypus are derived from a common ancestor with only minimal trigeminal specialization and that the peculiar anatomy of the trigeminal sensory nuclei in the modern platypus emerged in the ornithorhynchids after divergence from the tachyglossids. Copyright © 2012 S. Karger AG, Basel.

Stage and cell-specific expression and intracellular localization of the small heat shock protein Hsp27 during oogenesis and spermatogenesis in the Mediterranean fruit fly, Ceratitis capitata.

PubMed

Economou, Katerina; Kotsiliti, Elena; Mintzas, Anastassios C

2017-01-01

The cell-specific expression and intracellular distribution of the small heat protein Hsp27 was investigated in the ovaries and testes of the Mediterranean fruit fly, Ceratitis capitata (medfly), under both normal and heat shock conditions. For this study, a gfp-hsp27 strain was used to detect the chimeric protein by confocal microscopy. In unstressed ovaries, the protein was expressed throughout egg development in a stage and cell-specific pattern. In germarium, the protein was detected in the cytoplasm of the somatic cells in both unstressed and heat-shocked ovaries. In the early stages of oogenesis of unstressed ovaries, the protein was mainly located in the perinuclear region of the germ cells and in the cytoplasm of the follicle cells, while in later stages (9-10) it was distributed in the cytoplasm of the germ cells. In late stages (12-14), the protein changed localization pattern and was exclusively associated with the nuclei of the somatic cells. In heat shocked ovaries, the protein was mainly located in the nuclei of the somatic cells throughout egg chamber's development. In unstressed testes, the chimeric protein was detected in the nuclei of primary spermatocytes and in the filamentous structures of spermatid bundles, called actin cones. Interestingly, after a heat shock, the protein presented the same cell-specific localization pattern as in unstressed testes. Furthermore, the protein was also detected in the nuclei of the epithelial cells of the deferent duct, the accessory glands and the ejaculatory bulb. Our data suggest that medfly Hsp27 may have cell-specific functions, especially in the nucleus. Moreover, the association of this protein to actin cones during spermatid individualization, suggests a possible role of the protein in the formation and stabilization of actin cones. Copyright © 2016 Elsevier Ltd. All rights reserved.

A comparative study on the blood and milk cell counts of healthy, subclinical, and clinical mastitis Karan Fries cows

PubMed Central

Alhussien, Mohanned; Kaur, Mandheer; Manjari, Pasumarti; Kimothi, Shiv Prasad; Mohanty, Ashok K.; Dang, Ajay K.

2015-01-01

Aim: The present study was aimed to study the use of cell counts as an early indicator of mammary health. Materials and Methods: Milk and blood cell counts were estimated from 8 healthy, 8 subclinical (SCM), and 8 clinically mastitis (CM) groups of Karan Fries (KF) cows. Results: Total leucocyte counts and neutrophil percent in blood and milk somatic cells and milk neutrophil percent of healthy cows increased significantly (p<0.05) in SCM cows and CM cows. Viability of blood and milk neutrophils was more in healthy cows, but decreased significantly (p<0.05) in SCM and CM cows. Significant (p<0.05) decrease were also observed in both the blood and milk lymphocytes and monocytes of SCM and CM cows. Phagocytic activity (PA) of blood neutrophils also decreased significantly (p<0.05) in SCM cows. There was no difference between the PA of SCM and CM cows. Milk neutrophil percent was more in the SCM and clinically infected milk than in the blood of these cows. About 96-97% of the neutrophils had segmented nucleus in both healthy and subclinical milk, whereas, 2-3% were having band shaped or immature nuclei. There was a significant decrease in the segmented neutrophils, whereas, band neutrophils increase significantly to about 5% in the infected milk of mastitic cows. Viability of the milk neutrophils decreased more in case of subclinical and clinical milk as compared to that of blood. PA was found to be highest in the milk of healthy group of cows, but decreased significantly (p<0.05) in subclinically infected cows. However, there was no difference between the PA of milk neutrophils of SCM and CM cows. PA of milk was also found to be significantly lower in the milk of healthy cows when compared to that of blood neutrophils. Conclusion: This study indicated that percent neutrophils and their type in conjunction with milk somatic cell counts can be used as a more reliable indicator of mammary health in cows. PMID:27047156

ACME: Automated Cell Morphology Extractor for Comprehensive Reconstruction of Cell Membranes

PubMed Central

Mosaliganti, Kishore R.; Noche, Ramil R.; Xiong, Fengzhu; Swinburne, Ian A.; Megason, Sean G.

2012-01-01

The quantification of cell shape, cell migration, and cell rearrangements is important for addressing classical questions in developmental biology such as patterning and tissue morphogenesis. Time-lapse microscopic imaging of transgenic embryos expressing fluorescent reporters is the method of choice for tracking morphogenetic changes and establishing cell lineages and fate maps in vivo. However, the manual steps involved in curating thousands of putative cell segmentations have been a major bottleneck in the application of these technologies especially for cell membranes. Segmentation of cell membranes while more difficult than nuclear segmentation is necessary for quantifying the relations between changes in cell morphology and morphogenesis. We present a novel and fully automated method to first reconstruct membrane signals and then segment out cells from 3D membrane images even in dense tissues. The approach has three stages: 1) detection of local membrane planes, 2) voting to fill structural gaps, and 3) region segmentation. We demonstrate the superior performance of the algorithms quantitatively on time-lapse confocal and two-photon images of zebrafish neuroectoderm and paraxial mesoderm by comparing its results with those derived from human inspection. We also compared with synthetic microscopic images generated by simulating the process of imaging with fluorescent reporters under varying conditions of noise. Both the over-segmentation and under-segmentation percentages of our method are around 5%. The volume overlap of individual cells, compared to expert manual segmentation, is consistently over 84%. By using our software (ACME) to study somite formation, we were able to segment touching cells with high accuracy and reliably quantify changes in morphogenetic parameters such as cell shape and size, and the arrangement of epithelial and mesenchymal cells. Our software has been developed and tested on Windows, Mac, and Linux platforms and is available publicly under an open source BSD license (https://github.com/krm15/ACME). PMID:23236265

Structural Connectivity of the Developing Human Amygdala

PubMed Central

Saygin, Zeynep M.; Osher, David E.; Koldewyn, Kami; Martin, Rebecca E.; Finn, Amy; Saxe, Rebecca; Gabrieli, John D.E.; Sheridan, Margaret

2015-01-01

A large corpus of research suggests that there are changes in the manner and degree to which the amygdala supports cognitive and emotional function across development. One possible basis for these developmental differences could be the maturation of amygdalar connections with the rest of the brain. Recent functional connectivity studies support this conclusion, but the structural connectivity of the developing amygdala and its different nuclei remains largely unstudied. We examined age related changes in the DWI connectivity fingerprints of the amygdala to the rest of the brain in 166 individuals of ages 5-30. We also developed a model to predict age based on individual-subject amygdala connectivity, and identified the connections that were most predictive of age. Finally, we segmented the amygdala into its four main nucleus groups, and examined the developmental changes in connectivity for each nucleus. We observed that with age, amygdalar connectivity becomes increasingly sparse and localized. Age related changes were largely localized to the subregions of the amygdala that are implicated in social inference and contextual memory (the basal and lateral nuclei). The central nucleus’ connectivity also showed differences with age but these differences affected fewer target regions than the basal and lateral nuclei. The medial nucleus did not exhibit any age related changes. These findings demonstrate increasing specificity in the connectivity patterns of amygdalar nuclei across age. PMID:25875758

What is a segment?

PubMed

Hannibal, Roberta L; Patel, Nipam H

2013-12-17

Animals have been described as segmented for more than 2,000 years, yet a precise definition of segmentation remains elusive. Here we give the history of the definition of segmentation, followed by a discussion on current controversies in defining a segment. While there is a general consensus that segmentation involves the repetition of units along the anterior-posterior (a-p) axis, long-running debates exist over whether a segment can be composed of only one tissue layer, whether the most anterior region of the arthropod head is considered segmented, and whether and how the vertebrate head is segmented. Additionally, we discuss whether a segment can be composed of a single cell in a column of cells, or a single row of cells within a grid of cells. We suggest that 'segmentation' be used in its more general sense, the repetition of units with a-p polarity along the a-p axis, to prevent artificial classification of animals. We further suggest that this general definition be combined with an exact description of what is being studied, as well as a clearly stated hypothesis concerning the specific nature of the potential homology of structures. These suggestions should facilitate dialogue among scientists who study vastly differing segmental structures.

An Algorithm to Automate Yeast Segmentation and Tracking

PubMed Central

Doncic, Andreas; Eser, Umut; Atay, Oguzhan; Skotheim, Jan M.

2013-01-01

Our understanding of dynamic cellular processes has been greatly enhanced by rapid advances in quantitative fluorescence microscopy. Imaging single cells has emphasized the prevalence of phenomena that can be difficult to infer from population measurements, such as all-or-none cellular decisions, cell-to-cell variability, and oscillations. Examination of these phenomena requires segmenting and tracking individual cells over long periods of time. However, accurate segmentation and tracking of cells is difficult and is often the rate-limiting step in an experimental pipeline. Here, we present an algorithm that accomplishes fully automated segmentation and tracking of budding yeast cells within growing colonies. The algorithm incorporates prior information of yeast-specific traits, such as immobility and growth rate, to segment an image using a set of threshold values rather than one specific optimized threshold. Results from the entire set of thresholds are then used to perform a robust final segmentation. PMID:23520484

Rapid auxin-induced nitric oxide accumulation and subsequent tyrosine nitration of proteins during adventitious root formation in sunflower hypocotyls

PubMed Central

Yadav, Sunita; David, Anisha; Baluška, František; Bhatla, Satish C.

2013-01-01

Using NO specific probe (MNIP-Cu), rapid nitric oxide (NO) accumulation as a response to auxin (IAA) treatment has been observed in the protoplasts from the hypocotyls of sunflower seedlings (Helianthus annuus L.). Incubation of protoplasts in presence of NPA (auxin efflux blocker) and PTIO (NO scavenger) leads to significant reduction in NO accumulation, indicating that NO signals represent an early signaling event during auxin-induced response. A surge in NO production has also been demonstrated in whole hypocotyl explants showing adventitious root (AR) development. Evidence of tyrosine nitration of cytosolic proteins as a consequence of NO accumulation has been provided by western blot analysis and immunolocalization in the sections of AR producing hypocotyl segments. Most abundant anti-nitrotyrosine labeling is evident in proteins ranging from 25–80 kDa. Tyrosine nitration of a particular protein (25 kDa) is completely absent in presence of NPA (which suppresses AR formation). Similar lack of tyrosine nitration of this protein is also evident in other conditions which do not allow AR differentiation. Immunofluorescent localization experiments have revealed that non-inductive treatments (such as PTIO) for AR develpoment from hypocotyl segments coincide with symplastic and apoplastic localization of tyrosine nitrated proteins in the xylem elements, in contrast with negligible (and mainly apoplastic) nitration of proteins in the interfascicular cells and phloem elements. Application of NPA does not affect tyrosine nitration of proteins even in the presence of an external source of NO (SNP). Tyrosine nitrated proteins are abundant around the nuclei in the actively dividing cells of the root primordium. Thus, NO-modulated rapid response to IAA treatment through differential distribution of tyrosine nitrated proteins is evident as an inherent aspect of the AR development. PMID:23299324

Granulysin Produced by Uterine Natural Killer Cells Induces Apoptosis of Extravillous Trophoblasts in Spontaneous Abortion

PubMed Central

Nakashima, Akitoshi; Shiozaki, Arihiro; Myojo, Subaru; Ito, Mika; Tatematsu, Mikiko; Sakai, Masatoshi; Takamori, Yasushi; Ogawa, Kazuyuki; Nagata, Kinya; Saito, Shigeru

2008-01-01

Immune changes are known to occur in recurrent spontaneous abortion, but it is unclear whether either maternal natural killer (NK) cells or T cells attack fetus-derived trophoblasts. To clarify the immunological causes of spontaneous abortion, we examined the relationship between cytotoxic granule proteins in decidual lymphocytes, such as granulysin, granzyme B, and perforin, and the induction of apoptosis in extravillous trophoblasts (EVTs). The number of granulysin-positive CD56bright NK cells increased significantly in the decidua basalis during spontaneous abortion compared with normal pregnancy; however, granzyme B- and perforin-positive cells did not change. Interestingly, the expression of granulysin was also detected in the nuclei of EVTs in spontaneous abortion samples. When IL-2-stimulated CD56bright NK cells were cocultured with EVT cells (HTR-8/SV40neo), granulysin was found initially in the cytoplasm and then accumulated in the nuclei of the HTR-8/SV40neo cells. Furthermore, transfected cells expressing a GFP-granulysin fusion protein induced apoptosis in HTR-8/SV40neo cells independently of caspases. Our results suggest that granulysin-positive uterine NK cells attack EVTs; subsequently, the uNK-derived granulysin actively accumulates in the nuclei of EVTs, causing the death of EVTs due to apoptosis. These data support a new apoptosis pathway for trophoblasts via uNK-derived granulysin, suggesting that granulysin is involved in spontaneous abortion. PMID:18688023

Role of gelsolin interaction with actin in regulation and creation of actin nuclei in chemotactic peptide activated polymorphonuclear neutrophils.

PubMed Central

Deaton, J D; Guerrero, T; Howard, T H

1992-01-01

In vitro Ca++ activates gelsolin to sever F-actin and form a gelsolin-actin (GA) complex at the+end of F-actin that is not dissociated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) but is separated by EGTA+PIP/PIP2. The gelsolin blocks the+end on the actin filament, but the-end of the filament can still initiate actin polymerization. In thrombin activated platelets, evidence suggests that severing of F-actin by gelsolin increases GA complex, creates one-end actin nucleus and one cryptic+end actin nucleus per cut, and then dissociates to yield free+ends to nucleate rapid actin assembly. We examined the role of F-actin severing in creation and regulation of nuclei and polymerization in polymorphonuclear neutrophils (PMNs). At 2-s intervals after formyl peptide (FMLP) activation of endotoxin free (ETF) PMNs, change in GA complex was correlated with change in+end actin nuclei,-end actin nuclei, and F-actin content. GA complex was quantitated by electrophoretograms of proteins absorbed by antigelsolin from cells lysed in 10 mM EGTA,+end actin nuclei as cytochalasin (CD) sensitive and-end actin nuclei as CD insensitive increases in G-pyrenyl actin polymerization rates induced by the same PMNs, and F-actin content by NBDphallacidin binding to fixed cells. Thirty three percent of gelsolin was in GA complex in basal ETF PMNs; from 2-6 s, GA complexes dissociate (low = 15% at 10 s) and sequentially+end nuclei and F-actin content and then-end nuclei increase to a maximum at 10 s. At > s GA complex increase toward basal and + end nuclei and F-actin content returned toward basal. These kinetic data show gelsolin regulates availability of + end nuclei and actin polymerization in FMLP. However, absence of an initial increase in GA complex or - end nucleating activity shows FMLP activation does not cause gelsolin to sever F- or to bind G-actin to create cryptic + end nuclei in PMNs; the results suggest the + nucleus formation is gelsolin independent. PMID:1337290

Role of gelsolin interaction with actin in regulation and creation of actin nuclei in chemotactic peptide activated polymorphonuclear neutrophils.

PubMed

Deaton, J D; Guerrero, T; Howard, T H

1992-12-01

In vitro Ca++ activates gelsolin to sever F-actin and form a gelsolin-actin (GA) complex at the+end of F-actin that is not dissociated by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) but is separated by EGTA+PIP/PIP2. The gelsolin blocks the+end on the actin filament, but the-end of the filament can still initiate actin polymerization. In thrombin activated platelets, evidence suggests that severing of F-actin by gelsolin increases GA complex, creates one-end actin nucleus and one cryptic+end actin nucleus per cut, and then dissociates to yield free+ends to nucleate rapid actin assembly. We examined the role of F-actin severing in creation and regulation of nuclei and polymerization in polymorphonuclear neutrophils (PMNs). At 2-s intervals after formyl peptide (FMLP) activation of endotoxin free (ETF) PMNs, change in GA complex was correlated with change in+end actin nuclei,-end actin nuclei, and F-actin content. GA complex was quantitated by electrophoretograms of proteins absorbed by antigelsolin from cells lysed in 10 mM EGTA,+end actin nuclei as cytochalasin (CD) sensitive and-end actin nuclei as CD insensitive increases in G-pyrenyl actin polymerization rates induced by the same PMNs, and F-actin content by NBDphallacidin binding to fixed cells. Thirty three percent of gelsolin was in GA complex in basal ETF PMNs; from 2-6 s, GA complexes dissociate (low = 15% at 10 s) and sequentially+end nuclei and F-actin content and then-end nuclei increase to a maximum at 10 s. At > s GA complex increase toward basal and + end nuclei and F-actin content returned toward basal. These kinetic data show gelsolin regulates availability of + end nuclei and actin polymerization in FMLP. However, absence of an initial increase in GA complex or - end nucleating activity shows FMLP activation does not cause gelsolin to sever F- or to bind G-actin to create cryptic + end nuclei in PMNs; the results suggest the + nucleus formation is gelsolin independent.

Fos-defined activity in rat brainstem following centripetal acceleration.

PubMed

Kaufman, G D; Anderson, J H; Beitz, A J

1992-11-01

To identify rat brainstem nuclei involved in the initial, short-term response to a change in gravito-inertial force, adult Long-Evans rats were rotated in the horizontal plane for 90 min in complete darkness after they were eccentrically positioned off the axis of rotation (off-axis) causing a centripetal acceleration of 2 g. Neural activation was defined by the brainstem distribution of the c-fos primary response gene protein, Fos, using immunohistochemistry. The Fos labeling in off-axis animals was compared with that of control animals who were rotated on the axis of rotation (on-axis) with no centripetal acceleration, or who were restrained but not rotated. In the off-axis animals there was a significant labeling of neurons: in the inferior, medial, and y-group subnuclei of the vestibular complex; in subnuclei of the inferior olive, especially the dorsomedial cell column; in midbrain nuclei, including the interstitial nucleus of Cajal, nucleus of Darkschewitsch, Edinger-Westphal nucleus, and dorsolateral periaqueductal gray; in autonomic centers including the solitary nucleus, area postrema, and locus coeruleus; and in reticular nuclei including the lateral reticular nucleus and the lateral parabrachial nucleus. Also, there was greater Fos expression in the dorsomedial cell column, the principal inferior olive subnuclei, inferior vestibular nucleus, the dorsolateral central gray, and the locus coeruleus in animals who had their heads restrained compared to animals whose heads were not restrained. As one control, the vestibular neuroepithelium was destroyed by injecting sodium arsanilate into the middle ear, bilaterally. This resulted in a complete lack of Fos labeling in the vestibular nuclei and the inferior olive, and a significant reduction in labeling in other nuclei in the off-axis condition, indicating that these nuclei have a significant labyrinth-sensitive component to their Fos labeling. The data indicate that several novel brainstem regions, including the dorsomedial cell column of the inferior olive and the periaqueductal gray, as well as more traditional brainstem nuclei including vestibular and oculomotor related nuclei, respond to otolith activation during a sustained centripetal acceleration.

Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function

PubMed Central

Zhang, Lixia; Kendrick, Christina; Jülich, Dörthe; Holley, Scott A.

2010-01-01

Summary Cell division, differentiation and morphogenesis are coordinated during embryonic development and frequently in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and utilize the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is substantially driven by cell migration. We find that the segmentation clock, which regulates somitogenesis, functions normally in the absence of cell cycle progression and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1−/−. The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle, somites appear to hyper-epithelialize as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for normal segmental arrangement of epithelial borders and internal mesenchymal cells. PMID:18480162

Effects of cerebellar nuclear inactivation on the learning of a complex forelimb movement in cats.

PubMed

Wang, J J; Shimansky, Y; Bracha, V; Bloedel, J R

1998-05-01

The purpose of this study was to determine the effects of inactivating concurrently the cerebellar interposed and dentate nuclei on the capacity of cats to acquire and retain a complex, goal-directed forelimb movement. To assess the effects on acquisition, cats were required to learn to move a vertical manipulandum bar through a two-segment template with a shape approximating an inverted "L" after the injection of muscimol (saline for the control group) in the interposed and dentate cerebellar nuclei. During training periods, they were exposed progressively to more difficult templates, which were created by decreasing the angle between the two segments of the template. After determining the most difficult template the injected animals could learn within the specified time and performance constraints, the retraining phase of the experiment was initiated in which the cats were required to execute the same sequence of templates in the absence of any injection. This stage of the experiment assessed retention and determined the extent of any relearning required to execute the task at criterion levels. Next, the animals were overtrained without any injection on the most difficult template they could perform. Finally, to determine the effects of nuclear inactivation on retention after extensive retraining, their capacity to perform the same template was determined after muscimol injection in the interposed and dentate nuclei. The findings show that during the inactivation of the dentate and interposed nuclei the animals could learn to execute the more difficult templates. However, when required to execute the most difficult template learned under muscimol on the day after injections were discontinued, the cats had to "relearn" (reacquire) the movement. Finally, when the cerebellar nuclei were inactivated after the animals learned the task in the absence of any injections during the retraining phase, retention was not blocked. The data indicate that the intermediate and lateral cerebellum are not required either for learning this type of complex voluntary movement or for retaining the capacity to perform the task once it is learned. Nevertheless, when the cerebellum becomes available for executing a task learned in the absence of this structure, reacquisition of the behavior usually is necessary. It is hypothesized that the relearning observed after acquisition during muscimol inactivation reflects the tendency of the system to incorporate the cerebellum into the interactions responsible for the learning and performance of a motor sequence that is optimal for executing the task.

Cosmic Ray Energetics And Mass (CREAM) Launch and Operations

NASA Astrophysics Data System (ADS)

Seo, Eun-Suk

We request continued NASA support for the on-going Cosmic Ray Energetics And Mass (CREAM) project. The balloon-borne CREAM instrument was flown for 161 days in six flights over Antarctica, the longest known exposure for a single balloon project. Building on the success of those balloon missions, one of the two balloon payloads was successfully transformed for exposure on the International Space Station (ISS) Japanese Experiment Module Exposed Facility (JEM EF). Following completion of its systemlevel qualification and verification, this ISS-CREAM payload was delivered to the NASA Kennedy Space Center in August 2015 to await its launch to the ISS. The ISS-CREAM mission would achieve the primary science objectives of the Advanced Cosmic-ray Composition Experiment for the Space Station (ACCESS), which was given high priority in the 2001 NRC Decadal Study Report. Its nuclei composition data between 10^12 and 10^15 eV would enable detailed study of the spectral hardening first reported by the CREAM balloon project and recently confirmed for protons and helium by the PAMELA and AMS-02 space missions using permanent magnet spectrometers. In addition, multiTeV energy electron data allow searches for local sources and the signature of darkmatter, etc. The ISS-CREAM instrument is configured with redundant and complementary particle detectors capable of precise measurements of elemental spectra for Z = 1 - 26 nuclei, as well as electrons. The four layers of its finely segmented Silicon Charge Detector provide charge measurements, and its ionization calorimeter provides energy measurements. Its segmented scintillator-based Top and Bottom Counting Detectors separate electrons from nuclei using shower profile differences. Its Boronated Scintillator Detector distinguishes electrons from nuclei by detecting thermal neutrons that are dominant in nuclei induced showers. An order of magnitude increase in data collecting power is possible by utilizing the ISS to reach the highest energies practical with direct measurements. The ISSCREAM launch is currently manifested on SpaceX-12, which is scheduled for April 2017. It is expected to accumulate a total of > 4.5 years exposure during the grant period. The study of cosmic accelerators supports the Science Mission Directorate's Goal for Astrophysics in NASA's 2010 Science Plan, "Discover how the universe works, explore how the universe began and evolved, and search for Earth-like planets." It specifically addresses the Science Question, "How do matter, energy, space and time behave under the extraordinarily diverse conditions of the cosmos?"

Cosmic Ray Energetics And Mass (CREAM) launch and operations, PSU Co-I

NASA Astrophysics Data System (ADS)

Nutter, Scott

This is the Northern Kentucky University Co-I proposal to request continued NASA support for the on-going Cosmic Ray Energetics And Mass (CREAM) project. The balloon-borne CREAM instrument was flown for 161 days in six flights over Antarctica, the longest known exposure for a single balloon project. Building on the success of those balloon missions, one of the two balloon payloads was successfully transformed for exposure on the International Space Station (ISS) Japanese Experiment Module Exposed Facility (JEM EF). Following completion of its system-level qualification and verification, this ISS-CREAM payload was delivered to the NASA Kennedy Space Center in August 2015 to await its launch to the ISS. The ISS-CREAM mission would achieve the primary science objectives of the Advanced Cosmic-ray Composition Experiment for the Space Station (ACCESS), which was given high priority in the 2001 NRC Decadal Study Report. Its nuclei composition data between 10^12 and 10^15 eV would enable detailed study of the spectral hardening first reported by the CREAM balloon project and recently confirmed for protons and helium by the PAMELA and AMS-02 space missions using permanent magnet spectrometers. In addition, multiTeV energy electron data allow searches for local sources and the signature of darkmatter, etc. The ISS-CREAM instrument is configured with redundant and complementary particle detectors capable of precise measurements of elemental spectra for Z = 1 - 26 nuclei, as well as electrons. The four layers of its finely segmented Silicon Charge Detector provide charge measurements, and its ionization calorimeter provides energy measurements. Its segmented scintillator-based Top and Bottom Counting Detectors separate electrons from nuclei using shower profile differences. Its Boronated Scintillator Detector distinguishes electrons from nuclei by detecting thermal neutrons that are dominant in nuclei induced showers. An order of magnitude increase in data collecting power is possible by utilizing the ISS to reach the highest energies practical with direct measurements. The ISSCREAM launch is currently manifested on SpaceX-12, which is scheduled for April 2017. It is expected to accumulate a total of > 4.5 years exposure during the grant period. The study of cosmic accelerators supports the Science Mission Directorate's Goal for Astrophysics in NASA's 2010 Science Plan, "Discover how the universe works, explore how the universe began and evolved, and search for Earth-like planets."It specifically addresses the Science Question, "How do matter, energy, space and time behave under the extraordinarily diverse conditions of the cosmos?"

Cell segmentation in histopathological images with deep learning algorithms by utilizing spatial relationships.

PubMed

Hatipoglu, Nuh; Bilgin, Gokhan

2017-10-01

In many computerized methods for cell detection, segmentation, and classification in digital histopathology that have recently emerged, the task of cell segmentation remains a chief problem for image processing in designing computer-aided diagnosis (CAD) systems. In research and diagnostic studies on cancer, pathologists can use CAD systems as second readers to analyze high-resolution histopathological images. Since cell detection and segmentation are critical for cancer grade assessments, cellular and extracellular structures should primarily be extracted from histopathological images. In response, we sought to identify a useful cell segmentation approach with histopathological images that uses not only prominent deep learning algorithms (i.e., convolutional neural networks, stacked autoencoders, and deep belief networks), but also spatial relationships, information of which is critical for achieving better cell segmentation results. To that end, we collected cellular and extracellular samples from histopathological images by windowing in small patches with various sizes. In experiments, the segmentation accuracies of the methods used improved as the window sizes increased due to the addition of local spatial and contextual information. Once we compared the effects of training sample size and influence of window size, results revealed that the deep learning algorithms, especially convolutional neural networks and partly stacked autoencoders, performed better than conventional methods in cell segmentation.

Respiratory herpesvirus infection in two Indian Ringneck parakeets.

PubMed

Lazic, Tatjana; Ackermann, Mark R; Drahos, Jo M; Stasko, Judith; Haynes, Joseph S

2008-03-01

A flock of Indian Ringneck parakeets (Psittacula krameri manillensis) was imported to the United States from Australia. Soon after, 1 parakeet suddenly died, and a second parakeet died after a 2-day course of illness, which consisted of anorexia, lethargy, emaciation, and dyspnea. At necropsy, the affected birds had diffuse consolidation and red discoloration of the lungs, as well as thickened, congested air sacs. The microscopic examination revealed multifocal, necrotizing bronchitis, parabronchitis, and interstitial pneumonia. The lumen of the affected airways contained numerous, large syncytial cells with up to 15 nuclei. The nuclei of these syncytial cells often contained large, eosinophilic inclusion bodies, consistent with herpesvirus. The epithelium of the trachea and air sacs was hypertrophied and contained syncytial cells with intranuclear inclusion bodies similar to the bronchi. In addition, a few intranuclear inclusion bodies were also present in the epithelial cells that line the air capillaries. On ultrastructural examination, the nuclei of degenerating epithelial cells contained clusters of viral nucleocapsid proteins and unenveloped, icosahedral, viral particles that were approximately 90 nm in diameter. In addition, some epithelial cells contained clusters of enveloped viral particles approximately 105 nm in diameter, within the cytocavitary network. These lesions are characteristic of those caused by respiratory herpesvirus of parakeets.

The cytology of a thyroid granular cell tumor.

PubMed

Chang, Shu-Mei; Wei, Chang-Kuo; Tseng, Chih-En

2009-01-01

Granular cell tumor (GCT) of the thyroid is rare. Before this report, only four cases of thyroid GCT have been reported, none of which presented a cytopathological examination. In this paper, we report the fine needle aspiration cytology and pathological analysis of a thyroid GCT from a 12-year-old girl who presented with a painless neck mass. The tumor cells were single, in syncytial clusters, or pseudofollicles, contained small round, oval, or spindle nuclei, indistinct nucleoli, and a large amount of grayish, granular fragile cytoplasm. The background contained granular debris and naked nuclei. A differential diagnosis of thyroid GCT with more frequent thyroid lesions containing cytoplasmic granules, including Hurthle cells, macrophages, follicular cells, and cells of black thyroid syndrome, was also performed.

Mapping of enkephalins and adrenocorticotropic hormone in the squirrel monkey brainstem.

PubMed

Duque-Díaz, Ewing; Díaz-Cabiale, Zaida; Narváez, José Angel; Coveñas, Rafael

2017-03-01

An immunocytochemical technique has been used to study for the first time the distribution of fibers and cell bodies containing leucine-enkephalin (leu-enk), methionine-enkephalin (met-enk) or adrenocorticotropic hormone (ACTH) in the whole brainstem of the squirrel monkey Saimiri sciureus. Cell bodies containing leu-enk or met-enk were found in the superior colliculus and the formatio reticularis tegmenti mesencephali, respectively. No immunoreactive cell bodies containing ACTH were observed. Leu-enk-immunoreactive fibers were observed in 40 brainstem nuclei/tracts/regions, fibers containing met-enk were found in 38 brainstem nuclei/tracts/regions and fibers containing ACTH were found in 26 nuclei/tracts/regions. In the latter case, the density of immunoreactive fibers was always low. A high/moderate density of leu-enk- or met-enk-immunoreactive fibers were found in 18 and 16 brainstem nuclei/tracts/regions, respectively. The distribution of immunoreactive fibers containing leu-enk or met-enk was quite similar, with both leu-enk and met-enk observed in 82.5 % of the squirrel monkey brainstem nuclei/tracts/regions. This relationship is less marked for met-enk and ACTH (60.5 %) and even lower for leu-enk and ACTH (52.5 %). In 42.5 % of the nuclei/tracts/regions of the squirrel monkey brainstem (colliculus superior, substantia grisea centralis, nucleus interpeduncularis, nucleus tractus spinalis nervi trigemini, nucleus tractus solitarii, nucleus parabrachialis, formatio reticularis, substantia nigra), we observed fibers containing all three neuropeptides. The widespread distribution reported here suggests that enkephalins and ACTH can be involved in several physiological functions. The distribution of the immunoreactive fibers reported here is quite similar to that previously reported for enkephalins and ACTH in Macaca species and humans.

Diffusion Tensor Imaging Based Thalamic Segmentation in Deep Brain Stimulation for Chronic Pain Conditions

PubMed Central

Kim, Won; Chivukula, Srinivas; Hauptman, Jason; Pouratian, Nader

2016-01-01

Background/Aims Thalamic deep brain stimulation (DBS) for the treatment of medically refractory pain has largely been abandoned on account of its inconsistent and oftentimes poor efficacy. Our aim here was to use diffusion tensor imaging (DTI)-based segmentation to assess the internal thalamic nuclei of patients who have undergone thalamic DBS for intractable pain and retrospectively correlate lead position with clinical outcome. Methods DTI-based segmentation was performed on 5 patients who underwent sensory thalamus DBS for chronic pain. Postoperative computed tomography (CT) images obtained for electrode placement were fused with preoperative MRIs that had undergone DTI-based thalamic segmentation. Sensory thalamus maps of 4 patients were analyzed for lead positioning and interpatient variability. Results Four patients who experienced significant pain relief following DBS demonstrated contact positions within the DTI-determined sensory thalamus or in its vicinity, whereas one who did not respond to stimulation did not. Only four voxels (2%) within the sensory thalamus were mutually shared among patients; 108 voxels (58%) were uniquely represented. Conclusions DTI-based segmentation of the thalamus can be used to confirm thalamic lead placement relative to the sensory thalamus, and may serve as a useful tool to guide thalamic DBS electrode implantation in the future. PMID:27537848

STUDIES ON ISOLATED NUCLEI. I. ISOLATION AND CHEMICAL CHARACTERIZATION OF A NUCLEAR FRACTION FROM GUINEA PIG LIVER.

PubMed

MAGGIO, R; SIEKEVITZ, P; PALADE, G E

1963-08-01

This article describes a method for the isolation of nuclei from guinea pig liver. It involves the homogenization of the tissue in 0.88 M sucrose-1.5 mM CaCl(2) followed by centrifugation in a discontinuous density gradient in which the upper phase is the homogenate and the lower phase is 2.2 M sucrose-0.5 mM CaCl(2). Based on DNA recovery, the isolated fraction contains 25 to 30 per cent of the nuclei of the original homogenate. Electron microscopical observations showed that approximately 88 per cent of the isolated nuclei come from liver cells (the rest from von Kupffer cells and leucocytes) and that approximately 90 per cent of the nuclei appear intact, with well preserved nucleoli, nucleoplasm, nuclear envelope, and pores. Cytoplasmic contamination is minimal and consists primarily of the nuclear envelope and its attached ribosomes. The nuclear fraction consists of approximately 22.3 per cent DNA, approximately 4.7 per cent RNA, and approximately 73 per cent protein, the DNA/RNA ratio being 4.7. Data on RNA extractibility by phosphate and salt and on the base composition of total nuclear RNA are included.

Some Pecularities of the Graviresponse in Vaucheria

NASA Technical Reports Server (NTRS)

Gavrilova, O. V.; Rudanova, E. E.; Voloshko, L. N.; Gabova, A. V.

1996-01-01

The growth and position of nuclei in the siphonaceous alga Vaucheria sessilis were investigated under conditions of hypergravity and hypogravity. Under hypergravity conditions, active sporogenesis was observed. The accumulation of nuclei in the apical and branching zone preceeded the sporogenesis. The anti-microtubular agent, colchicine inhibits sporogenesis and the response of Vaucheria to hypergravity. Under hypogravity conditions, the quantity of nuclei increased throughout the whole branch. Colchicine prevents the migration of nuclei from the apical zone to the basal part of the branch. The anti-actin agent phalloidin prevents the formation of an actin network, and phalloidin-poisoned cells lose a cluster of nuclei in the apical zone. However, the gravity dependent response is less pronounced. It is supposed that, in Vaucheria sessilis, the primary stages of the reception and translation of gravitational signals coincide with those for light signals and active division of nuclei in the growth zone is an integral part of the graviresponse.

Transplanted hematopoietic stem cells demonstrate impaired sarcoglycan expression after engraftment into cardiac and skeletal muscle.

PubMed

Lapidos, Karen A; Chen, Yiyin E; Earley, Judy U; Heydemann, Ahlke; Huber, Jill M; Chien, Marcia; Ma, Averil; McNally, Elizabeth M

2004-12-01

Pluripotent bone marrow-derived side population (BM-SP) stem cells have been shown to repopulate the hematopoietic system and to contribute to skeletal and cardiac muscle regeneration after transplantation. We tested BM-SP cells for their ability to regenerate heart and skeletal muscle using a model of cardiomyopathy and muscular dystrophy that lacks delta-sarcoglycan. The absence of delta-sarcoglycan produces microinfarcts in heart and skeletal muscle that should recruit regenerative stem cells. Additionally, sarcoglycan expression after transplantation should mark successful stem cell maturation into cardiac and skeletal muscle lineages. BM-SP cells from normal male mice were transplanted into female delta-sarcoglycan-null mice. We detected engraftment of donor-derived stem cells into skeletal muscle, with the majority of donor-derived cells incorporated within myofibers. In the heart, donor-derived nuclei were detected inside cardiomyocytes. Skeletal muscle myofibers containing donor-derived nuclei generally failed to express sarcoglycan, with only 2 sarcoglycan-positive fibers detected in the quadriceps muscle from all 14 mice analyzed. Moreover, all cardiomyocytes with donor-derived nuclei were sarcoglycan-negative. The absence of sarcoglycan expression in cardiomyocytes and skeletal myofibers after transplantation indicates impaired differentiation and/or maturation of bone marrow-derived stem cells. The inability of BM-SP cells to express this protein severely limits their utility for cardiac and skeletal muscle regeneration.

What is a segment?

PubMed Central

2013-01-01

Animals have been described as segmented for more than 2,000 years, yet a precise definition of segmentation remains elusive. Here we give the history of the definition of segmentation, followed by a discussion on current controversies in defining a segment. While there is a general consensus that segmentation involves the repetition of units along the anterior-posterior (a-p) axis, long-running debates exist over whether a segment can be composed of only one tissue layer, whether the most anterior region of the arthropod head is considered segmented, and whether and how the vertebrate head is segmented. Additionally, we discuss whether a segment can be composed of a single cell in a column of cells, or a single row of cells within a grid of cells. We suggest that ‘segmentation’ be used in its more general sense, the repetition of units with a-p polarity along the a-p axis, to prevent artificial classification of animals. We further suggest that this general definition be combined with an exact description of what is being studied, as well as a clearly stated hypothesis concerning the specific nature of the potential homology of structures. These suggestions should facilitate dialogue among scientists who study vastly differing segmental structures. PMID:24345042

A fast and efficient segmentation scheme for cell microscopic image.

PubMed

Lebrun, G; Charrier, C; Lezoray, O; Meurie, C; Cardot, H

2007-04-27

Microscopic cellular image segmentation schemes must be efficient for reliable analysis and fast to process huge quantity of images. Recent studies have focused on improving segmentation quality. Several segmentation schemes have good quality but processing time is too expensive to deal with a great number of images per day. For segmentation schemes based on pixel classification, the classifier design is crucial since it is the one which requires most of the processing time necessary to segment an image. The main contribution of this work is focused on how to reduce the complexity of decision functions produced by support vector machines (SVM) while preserving recognition rate. Vector quantization is used in order to reduce the inherent redundancy present in huge pixel databases (i.e. images with expert pixel segmentation). Hybrid color space design is also used in order to improve data set size reduction rate and recognition rate. A new decision function quality criterion is defined to select good trade-off between recognition rate and processing time of pixel decision function. The first results of this study show that fast and efficient pixel classification with SVM is possible. Moreover posterior class pixel probability estimation is easy to compute with Platt method. Then a new segmentation scheme using probabilistic pixel classification has been developed. This one has several free parameters and an automatic selection must dealt with, but criteria for evaluate segmentation quality are not well adapted for cell segmentation, especially when comparison with expert pixel segmentation must be achieved. Another important contribution in this paper is the definition of a new quality criterion for evaluation of cell segmentation. The results presented here show that the selection of free parameters of the segmentation scheme by optimisation of the new quality cell segmentation criterion produces efficient cell segmentation.

21 CFR 640.15 - Segments for testing.

Code of Federal Regulations, 2014 CFR

2014-04-01

... cells. (c) All segments accompanying a unit of Red Blood Cells shall be filled at the time the blood is... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.15 Segments for testing... provided with each unit of Whole Blood or Red Blood Cells when issued or reissued. (b) Before they are...

21 CFR 640.15 - Segments for testing.

Code of Federal Regulations, 2011 CFR

2011-04-01

... cells. (c) All segments accompanying a unit of Red Blood Cells shall be filled at the time the blood is... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.15 Segments for testing... provided with each unit of Whole Blood or Red Blood Cells when issued or reissued. (b) Before they are...

21 CFR 640.15 - Segments for testing.

Code of Federal Regulations, 2013 CFR

2013-04-01

... cells. (c) All segments accompanying a unit of Red Blood Cells shall be filled at the time the blood is... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.15 Segments for testing... provided with each unit of Whole Blood or Red Blood Cells when issued or reissued. (b) Before they are...

21 CFR 640.15 - Segments for testing.

Code of Federal Regulations, 2012 CFR

2012-04-01

... cells. (c) All segments accompanying a unit of Red Blood Cells shall be filled at the time the blood is... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.15 Segments for testing... provided with each unit of Whole Blood or Red Blood Cells when issued or reissued. (b) Before they are...

21 CFR 640.15 - Segments for testing.

Code of Federal Regulations, 2010 CFR

2010-04-01

... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.15 Segments for testing... provided with each unit of Whole Blood or Red Blood Cells when issued or reissued. (b) Before they are... cells. (c) All segments accompanying a unit of Red Blood Cells shall be filled at the time the blood is...

Sex differences and the roles of sex steroids in apoptosis of sexually dimorphic nuclei of the preoptic area in postnatal rats.

PubMed

Tsukahara, S

2009-03-01

The brain contains several sexually dimorphic nuclei that exhibit sex differences with respect to cell number. It is likely that the control of cell number by apoptotic cell death in the developing brain contributes to creating sex differences in cell number in sexually dimorphic nuclei, although the mechanisms responsible for this have not been determined completely. The milieu of sex steroids in the developing brain affects sexual differentiation in the brain. The preoptic region of rats has two sexually dimorphic nuclei. The sexually dimorphic nucleus of the preoptic area (SDN-POA) has more neurones in males, whereas the anteroventral periventricular nucleus (AVPV) has a higher cell density in females. Sex differences in apoptotic cell number arise in the SDN-POA and AVPV of rats in the early postnatal period, and an inverse correlation exists between sex differences in apoptotic cell number and the number of living cells in the mature period. The SDN-POA of postnatal male rats exhibits a higher expression of anti-apoptotic Bcl-2 and lower expression of pro-apoptotic Bax compared to that in females and, as a potential result, apoptotic cell death via caspase-3 activation more frequently occurs in the SDN-POA of females. The patterns of expression of Bcl-2 and Bax in the SDN-POA of postnatal female rats are changed to male-typical ones by treatment with oestrogen, which is normally synthesised from testicular androgen and affects the developing brain in males. In the AVPV of postnatal rats, apoptotic regulation also differs between the sexes, although Bcl-2 expression is increased and Bax expression and caspase-3 activity are decreased in females. The mechanisms of apoptosis possibly contributing to the creation of sex differences in cell number and the roles of sex steroids in apoptosis are discussed.

Isolation of the constitutive heterochromatin from mouse liver nuclei.

PubMed

Zatsepina, Olga V; Zharskaya, Oxana O; Prusov, Andrei N

2008-01-01

A method for isolation of constitutive heterochromatin (chromocenters) from nuclei of mouse liver cells is described. This method is based on the higher resistance of chromocenters to low ionic strength treatment as compared with that of nucleoli and euchromatin. The method allows separation of chromocenters that are essentially free of nucleoli and other nuclear contaminants. In contrast to nuclei and nucleoli, isolated chromocenters are characterized by a simpler protein composition and contain a smaller number of proteins (especially of high molecular weight proteins). They possess telomeric DNA and telomerase activity that suggests a tight association of chromocenters with the telomerase complex in mouse hepatocyte nuclei.

Bovine oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP).

PubMed

Tani, Tetsuya; Shimada, Hiroaki; Kato, Yoko; Tsunoda, Yukio

2007-01-01

Despite the long-held assumption that reprogramming factors are present in mammalian oocytes at the second metaphase stage, the molecular nature of these factors is not known. Here, we demonstrated that oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP). Injection of TCTP double-stranded RNA into germinal vesicle oocytes decreased the potential of nuclear-transferred (NT) oocytes, but not in vitro fertilized oocytes, to develop into blastocysts. Phosphorylated TCTP is considered to facilitate the first step of somatic cell reprogramming. After transfer of blastocysts that developed from NT oocytes fused with cumulus cells in which phosphorylated TCTP peptide was previously incorporated, the recipient pregnancy rate (47%) increased and the abortion rate (13%) decreased. Moreover, all seven cloned calves survived for at least 1 month after parturition, and had no morphologic abnormalities. The present study demonstrated that pretreatment of donor cells with phosphorylated TCTP peptide has a beneficial effect on the potential of bovine somatic cell nuclei to develop into normal cloned calves. Before widespread application of TCTP for bovine cloning, however, a large-scale embryo transfer study using different donor cell lines of various origins is necessary.

Revisiting point FRAP to quantitatively characterize anomalous diffusion in live cells.

PubMed

Daddysman, Matthew K; Fecko, Christopher J

2013-02-07

Fluorescence recovery after photobleaching (FRAP) is widely used to interrogate diffusion and binding of proteins in live cells. Herein, we apply two-photon excited FRAP with a diffraction limited bleaching and observation volume to study anomalous diffusion of unconjugated green fluorescence protein (GFP) in vitro and in cells. Experiments performed on dilute solutions of GFP reveal that reversible fluorophore bleaching can be mistakenly interpreted as anomalous diffusion. We derive a reaction-diffusion FRAP model that includes reversible photobleaching, and demonstrate that it properly accounts for these photophysics. We then apply this model to investigate the diffusion of GFP in HeLa cells and polytene cells of Drosophila larval salivary glands. GFP exhibits anomalous diffusion in the cytoplasm of both cell types and in HeLa nuclei. Polytene nuclei contain optically resolvable chromosomes, permitting FRAP experiments that focus separately on chromosomal or interchrosomal regions. We find that GFP exhibits anomalous diffusion in chromosomal regions but diffuses normally in regions devoid of chromatin. This observation indicates that obstructed transport through chromatin and not crowding by macromolecules is a source of anomalous diffusion in polytene nuclei. This behavior is likely true in other cells, so it will be important to account for this type of transport physics and for reversible photobleaching to properly interpret future FRAP experiments on DNA-binding proteins.

Defining multiple, distinct, and shared spatiotemporal patterns of DNA replication and endoreduplication from 3D image analysis of developing maize (Zea mays L.) root tip nuclei.

PubMed

Bass, Hank W; Hoffman, Gregg G; Lee, Tae-Jin; Wear, Emily E; Joseph, Stacey R; Allen, George C; Hanley-Bowdoin, Linda; Thompson, William F

2015-11-01

Spatiotemporal patterns of DNA replication have been described for yeast and many types of cultured animal cells, frequently after cell cycle arrest to aid in synchronization. However, patterns of DNA replication in nuclei from plants or naturally developing organs remain largely uncharacterized. Here we report findings from 3D quantitative analysis of DNA replication and endoreduplication in nuclei from pulse-labeled developing maize root tips. In both early and middle S phase nuclei, flow-sorted on the basis of DNA content, replicative labeling was widely distributed across euchromatic regions of the nucleoplasm. We did not observe the perinuclear or perinucleolar replicative labeling patterns characteristic of middle S phase in mammals. Instead, the early versus middle S phase patterns in maize could be distinguished cytologically by correlating two quantitative, continuous variables, replicative labeling and DAPI staining. Early S nuclei exhibited widely distributed euchromatic labeling preferentially localized to regions with weak DAPI signals. Middle S nuclei also exhibited widely distributed euchromatic labeling, but the label was preferentially localized to regions with strong DAPI signals. Highly condensed heterochromatin, including knobs, replicated during late S phase as previously reported. Similar spatiotemporal replication patterns were observed for both mitotic and endocycling maize nuclei. These results revealed that maize euchromatin exists as an intermingled mixture of two components distinguished by their condensation state and replication timing. These different patterns might reflect a previously described genome organization pattern, with "gene islands" mostly replicating during early S phase followed by most of the intergenic repetitive regions replicating during middle S phase.

Joint level-set and spatio-temporal motion detection for cell segmentation.

PubMed

Boukari, Fatima; Makrogiannis, Sokratis

2016-08-10

Cell segmentation is a critical step for quantification and monitoring of cell cycle progression, cell migration, and growth control to investigate cellular immune response, embryonic development, tumorigenesis, and drug effects on live cells in time-lapse microscopy images. In this study, we propose a joint spatio-temporal diffusion and region-based level-set optimization approach for moving cell segmentation. Moving regions are initially detected in each set of three consecutive sequence images by numerically solving a system of coupled spatio-temporal partial differential equations. In order to standardize intensities of each frame, we apply a histogram transformation approach to match the pixel intensities of each processed frame with an intensity distribution model learned from all frames of the sequence during the training stage. After the spatio-temporal diffusion stage is completed, we compute the edge map by nonparametric density estimation using Parzen kernels. This process is followed by watershed-based segmentation and moving cell detection. We use this result as an initial level-set function to evolve the cell boundaries, refine the delineation, and optimize the final segmentation result. We applied this method to several datasets of fluorescence microscopy images with varying levels of difficulty with respect to cell density, resolution, contrast, and signal-to-noise ratio. We compared the results with those produced by Chan and Vese segmentation, a temporally linked level-set technique, and nonlinear diffusion-based segmentation. We validated all segmentation techniques against reference masks provided by the international Cell Tracking Challenge consortium. The proposed approach delineated cells with an average Dice similarity coefficient of 89 % over a variety of simulated and real fluorescent image sequences. It yielded average improvements of 11 % in segmentation accuracy compared to both strictly spatial and temporally linked Chan-Vese techniques, and 4 % compared to the nonlinear spatio-temporal diffusion method. Despite the wide variation in cell shape, density, mitotic events, and image quality among the datasets, our proposed method produced promising segmentation results. These results indicate the efficiency and robustness of this method especially for mitotic events and low SNR imaging, enabling the application of subsequent quantification tasks.

Multilevel Space-Time Aggregation for Bright Field Cell Microscopy Segmentation and Tracking

PubMed Central

Inglis, Tiffany; De Sterck, Hans; Sanders, Geoffrey; Djambazian, Haig; Sladek, Robert; Sundararajan, Saravanan; Hudson, Thomas J.

2010-01-01

A multilevel aggregation method is applied to the problem of segmenting live cell bright field microscope images. The method employed is a variant of the so-called “Segmentation by Weighted Aggregation” technique, which itself is based on Algebraic Multigrid methods. The variant of the method used is described in detail, and it is explained how it is tailored to the application at hand. In particular, a new scale-invariant “saliency measure” is proposed for deciding when aggregates of pixels constitute salient segments that should not be grouped further. It is shown how segmentation based on multilevel intensity similarity alone does not lead to satisfactory results for bright field cells. However, the addition of multilevel intensity variance (as a measure of texture) to the feature vector of each aggregate leads to correct cell segmentation. Preliminary results are presented for applying the multilevel aggregation algorithm in space time to temporal sequences of microscope images, with the goal of obtaining space-time segments (“object tunnels”) that track individual cells. The advantages and drawbacks of the space-time aggregation approach for segmentation and tracking of live cells in sequences of bright field microscope images are presented, along with a discussion on how this approach may be used in the future work as a building block in a complete and robust segmentation and tracking system. PMID:20467468

Automatic Cell Segmentation Using a Shape-Classification Model in Immunohistochemically Stained Cytological Images

NASA Astrophysics Data System (ADS)

Shah, Shishir

This paper presents a segmentation method for detecting cells in immunohistochemically stained cytological images. A two-phase approach to segmentation is used where an unsupervised clustering approach coupled with cluster merging based on a fitness function is used as the first phase to obtain a first approximation of the cell locations. A joint segmentation-classification approach incorporating ellipse as a shape model is used as the second phase to detect the final cell contour. The segmentation model estimates a multivariate density function of low-level image features from training samples and uses it as a measure of how likely each image pixel is to be a cell. This estimate is constrained by the zero level set, which is obtained as a solution to an implicit representation of an ellipse. Results of segmentation are presented and compared to ground truth measurements.

Enlarged squamous cell nuclei in cervical cytologic specimens from perimenopausal women ("PM Cells") : a cause of ASC overdiagnosis.

PubMed

Cibas, Edmund S; Browne, Tara-Jane; Bassichis, Michelle H Mantel; Lee, Kenneth R

2005-07-01

We studied the appropriateness of interpreting squamous cells with enlarged, smooth, bland nuclei in perimenopausal women ("PM cells") as atypical squamous cells (ASCs). Papanicolaou smears (Paps) from 100 women (40-55 years old) with a cytologic interpretation of ASC of undetermined significance (ASCUS) and human papillomavirus (HPV) testing or a biopsy within 6 months were reviewed by 2 observers without knowledge of the biopsy diagnosis or HPV results. Cases in which both reviewers agreed that the Paps were diagnosed more properly as "negative for intraepithelial lesion or malignancy" were compared with cases of "true ASCUS," using histologic squamous intraepithelial lesion and/or a positive high-risk HPV test as a positive outcome (abnormal follow-up). Of 100 cases, 28 were reclassified as benign by both observers. In 15 of these, the original ASCUS interpretation was based on cells with bland nuclear enlargement (2-3 times the area of intermediate cell nuclei), smooth nuclear membranes, and fine chromatin. Abnormal follow-up was identified in 1 (7%) of 15 benign cases but in 30 (42%) of 72 true ASCUS cases (P = .023). PM cells are a significant cause of ASC overdiagnosis in women 40 to 55 years old. Cervical Paps with cells no more atypical than these can be interpreted safely as negative for intraepithelial lesion or malignancy.

Basal ganglia structure in Tourette's disorder and/or attention-deficit/hyperactivity disorder.

PubMed

Forde, Natalie J; Zwiers, Marcel P; Naaijen, Jilly; Akkermans, Sophie E A; Openneer, Thaira J C; Visscher, Frank; Dietrich, Andrea; Buitelaar, Jan K; Hoekstra, Pieter J

2017-04-01

Tourette's disorder and attention-deficit/hyperactivity disorder often co-occur and have both been associated with structural variation of the basal ganglia. However, findings are inconsistent and comorbidity is often neglected. T1-weighted magnetic resonance images from children (n = 141, 8 to 12 years) with Tourette's disorder and/or attention-deficit/hyperactivity disorder and controls were processed with the Oxford Centre for Functional MRI [Magnetic resonance imaging] of the Brain (FMRIB) integrated registration and segmentation tool to determine basal ganglia nuclei volume and shape. Across all participants, basal ganglia nuclei volume and shape were estimated in relation to Tourette's disorder (categorical), attention-deficit/hyperactivity disorder severity (continuous across all participants), and their interaction. The analysis revealed no differences in basal ganglia nuclei volumes or shape between children with and without Tourette's disorder, no association with attention-deficit/hyperactivity disorder severity, and no interaction between the two. We found no evidence that Tourette's disorder, attention-deficit/hyperactivity disorder severity, or a combination thereof are associated with structural variation of the basal ganglia in 8- to 12-year-old patients. © 2016 International Parkinson and Movement Disorder Society. © 2016 International Parkinson and Movement Disorder Society.

Commissioning a Rotating Target Wheel Assembly for Heavy Element Studies

NASA Astrophysics Data System (ADS)

Fields, L. D.; Bennett, M. E.; Mayorov, D. A.; Folden, C. M.

2013-10-01

The heaviest elements are produced artificially by fusing nuclei of light elements within an accelerator to form heavier nuclei. The most direct method to increase the production rate of nuclei is to increase the beam intensity, necessitating the use of a rotating target to minimize damage to the target by deposited heat. Such a target wheel was constructed for heavy element research at Texas A&M University, Cyclotron Institute, consisting of a wheel with three banana-shaped target cutouts. The target is designed to rotate at 1700 rpm, and a fiber optic cable provides a signal to trigger beam pulsing in order to avoid irradiating the spokes between target segments. Following minor mechanical modifications and construction of a dedicated electrical panel, the rotating target assembly was commissioned for a beam experiment. A 15 MeV/u beam of 20Ne was delivered from the K500 cyclotron and detected by a ruggedized silicon detector. The beam pulsing response time was characterized as a function of the rational frequency of the target wheel. Preliminary analysis suggests that the K500 is capable of pulsing at rates of up to 250 Hz, which is sufficient for planned future experiments. Funded by DOE and NSF-REU Program.

Deep learning for digital pathology image analysis: A comprehensive tutorial with selected use cases

PubMed Central

Janowczyk, Andrew; Madabhushi, Anant

2016-01-01

Background: Deep learning (DL) is a representation learning approach ideally suited for image analysis challenges in digital pathology (DP). The variety of image analysis tasks in the context of DP includes detection and counting (e.g., mitotic events), segmentation (e.g., nuclei), and tissue classification (e.g., cancerous vs. non-cancerous). Unfortunately, issues with slide preparation, variations in staining and scanning across sites, and vendor platforms, as well as biological variance, such as the presentation of different grades of disease, make these image analysis tasks particularly challenging. Traditional approaches, wherein domain-specific cues are manually identified and developed into task-specific “handcrafted” features, can require extensive tuning to accommodate these variances. However, DL takes a more domain agnostic approach combining both feature discovery and implementation to maximally discriminate between the classes of interest. While DL approaches have performed well in a few DP related image analysis tasks, such as detection and tissue classification, the currently available open source tools and tutorials do not provide guidance on challenges such as (a) selecting appropriate magnification, (b) managing errors in annotations in the training (or learning) dataset, and (c) identifying a suitable training set containing information rich exemplars. These foundational concepts, which are needed to successfully translate the DL paradigm to DP tasks, are non-trivial for (i) DL experts with minimal digital histology experience, and (ii) DP and image processing experts with minimal DL experience, to derive on their own, thus meriting a dedicated tutorial. Aims: This paper investigates these concepts through seven unique DP tasks as use cases to elucidate techniques needed to produce comparable, and in many cases, superior to results from the state-of-the-art hand-crafted feature-based classification approaches. Results: Specifically, in this tutorial on DL for DP image analysis, we show how an open source framework (Caffe), with a singular network architecture, can be used to address: (a) nuclei segmentation (F-score of 0.83 across 12,000 nuclei), (b) epithelium segmentation (F-score of 0.84 across 1735 regions), (c) tubule segmentation (F-score of 0.83 from 795 tubules), (d) lymphocyte detection (F-score of 0.90 across 3064 lymphocytes), (e) mitosis detection (F-score of 0.53 across 550 mitotic events), (f) invasive ductal carcinoma detection (F-score of 0.7648 on 50 k testing patches), and (g) lymphoma classification (classification accuracy of 0.97 across 374 images). Conclusion: This paper represents the largest comprehensive study of DL approaches in DP to date, with over 1200 DP images used during evaluation. The supplemental online material that accompanies this paper consists of step-by-step instructions for the usage of the supplied source code, trained models, and input data. PMID:27563488

Deep learning for digital pathology image analysis: A comprehensive tutorial with selected use cases.

PubMed

Janowczyk, Andrew; Madabhushi, Anant

2016-01-01

Deep learning (DL) is a representation learning approach ideally suited for image analysis challenges in digital pathology (DP). The variety of image analysis tasks in the context of DP includes detection and counting (e.g., mitotic events), segmentation (e.g., nuclei), and tissue classification (e.g., cancerous vs. non-cancerous). Unfortunately, issues with slide preparation, variations in staining and scanning across sites, and vendor platforms, as well as biological variance, such as the presentation of different grades of disease, make these image analysis tasks particularly challenging. Traditional approaches, wherein domain-specific cues are manually identified and developed into task-specific "handcrafted" features, can require extensive tuning to accommodate these variances. However, DL takes a more domain agnostic approach combining both feature discovery and implementation to maximally discriminate between the classes of interest. While DL approaches have performed well in a few DP related image analysis tasks, such as detection and tissue classification, the currently available open source tools and tutorials do not provide guidance on challenges such as (a) selecting appropriate magnification, (b) managing errors in annotations in the training (or learning) dataset, and (c) identifying a suitable training set containing information rich exemplars. These foundational concepts, which are needed to successfully translate the DL paradigm to DP tasks, are non-trivial for (i) DL experts with minimal digital histology experience, and (ii) DP and image processing experts with minimal DL experience, to derive on their own, thus meriting a dedicated tutorial. This paper investigates these concepts through seven unique DP tasks as use cases to elucidate techniques needed to produce comparable, and in many cases, superior to results from the state-of-the-art hand-crafted feature-based classification approaches. Specifically, in this tutorial on DL for DP image analysis, we show how an open source framework (Caffe), with a singular network architecture, can be used to address: (a) nuclei segmentation (F-score of 0.83 across 12,000 nuclei), (b) epithelium segmentation (F-score of 0.84 across 1735 regions), (c) tubule segmentation (F-score of 0.83 from 795 tubules), (d) lymphocyte detection (F-score of 0.90 across 3064 lymphocytes), (e) mitosis detection (F-score of 0.53 across 550 mitotic events), (f) invasive ductal carcinoma detection (F-score of 0.7648 on 50 k testing patches), and (g) lymphoma classification (classification accuracy of 0.97 across 374 images). This paper represents the largest comprehensive study of DL approaches in DP to date, with over 1200 DP images used during evaluation. The supplemental online material that accompanies this paper consists of step-by-step instructions for the usage of the supplied source code, trained models, and input data.

Establishment of segment polarity in the ectoderm of the leech Helobdella

NASA Technical Reports Server (NTRS)

Seaver, E. C.; Shankland, M.

2001-01-01

The segmented ectoderm and mesoderm of the leech arise via a stereotyped cell lineage from embryonic stem cells called teloblasts. Each teloblast gives rise to a column of primary blast cell daughters, and the blast cells generate descendant clones that serve as the segmental repeats of their particular teloblast lineage. We have examined the mechanism by which the leech primary blast cell clones acquire segment polarity - i.e. a fixed sequence of positional values ordered along the anteroposterior axis of the segmental repeat. In the O and P teloblast lineages, the earliest divisions of the primary blast cell segregate anterior and posterior cell fates along the anteroposterior axis. Using a laser microbeam, we ablated single cells from both o and p blast cell clones at stages when the clone was two to four cells in length. The developmental fate of the remaining cells was characterized with rhodamine-dextran lineage tracer. Twelve different progeny cells were ablated, and in every case the ablation eliminated the normal descendants of the ablated cell while having little or no detectable effect on the developmental fate of the remaining cells. This included experiments in which we specifically ablated those blast cell progeny that are known to express the engrailed gene, or their lineal precursors. These findings confirm and extend a previous study by showing that the establishment of segment polarity in the leech ectoderm is largely independent of cell interactions conveyed along the anteroposterior axis. Both intercellular signaling and engrailed expression play an important role in the segment polarity specification of the Drosophila embryo, and our findings suggest that there may be little or no conservation of this developmental mechanism between those two organisms.

Nicotine Affects Bone Resorption and Suppresses the Expression of Cathepsin K, MMP-9 and Vacuolar-Type H+-ATPase d2 and Actin Organization in Osteoclasts

PubMed Central

Tanaka, Hideki; Tanabe, Natsuko; Kawato, Takayuki; Nakai, Kumiko; Kariya, Taro; Matsumoto, Sakurako; Zhao, Ning; Motohashi, Masafumi; Maeno, Masao

2013-01-01

Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10−5, 10−4, or 10−3 M nicotine and/or 50 µM α-bungarotoxin (btx), an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1–5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei), and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization. PMID:23555029

Comparison of parameter-adapted segmentation methods for fluorescence micrographs.

PubMed

Held, Christian; Palmisano, Ralf; Häberle, Lothar; Hensel, Michael; Wittenberg, Thomas

2011-11-01

Interpreting images from fluorescence microscopy is often a time-consuming task with poor reproducibility. Various image processing routines that can help investigators evaluate the images are therefore useful. The critical aspect for a reliable automatic image analysis system is a robust segmentation algorithm that can perform accurate segmentation for different cell types. In this study, several image segmentation methods were therefore compared and evaluated in order to identify the most appropriate segmentation schemes that are usable with little new parameterization and robustly with different types of fluorescence-stained cells for various biological and biomedical tasks. The study investigated, compared, and enhanced four different methods for segmentation of cultured epithelial cells. The maximum-intensity linking (MIL) method, an improved MIL, a watershed method, and an improved watershed method based on morphological reconstruction were used. Three manually annotated datasets consisting of 261, 817, and 1,333 HeLa or L929 cells were used to compare the different algorithms. The comparisons and evaluations showed that the segmentation performance of methods based on the watershed transform was significantly superior to the performance of the MIL method. The results also indicate that using morphological opening by reconstruction can improve the segmentation of cells stained with a marker that exhibits the dotted surface of cells. Copyright © 2011 International Society for Advancement of Cytometry.

Cell and current collector felt arrangement for solid oxide electrochemical cell combinations

DOEpatents

Reichner, Philip

1988-01-01

A solid electrolyte electrochemical cell combination 1 is made, comprising an annular, axially elongated, inner electrode 2 containing at least one interior gas feed conduit 3; annular solid electrolyte segments 4 around and covering portions of the inner electrode; annular outer electrode segments 6 around and covering portions of the electrolyte segments; electronically conducting, non-porous, interconnection material 5 disposed between electrolyte segments and in contact with the inner electrode, and electronically conducting, porous, metal fiber current collector felts 7 disposed on top of the non-porous interconnect material and outer electrode segments, where both the non-porous interconnect material and the porous metal felts are disposed circumferentially about the cell, transversely to the axial length of the cell and the inner electrode is continuous for the entire axial length of the cell combination.

A novel concentration and viability detection method for Brettanomyces using the Cellometer image cytometry.

PubMed

Martyniak, Brian; Bolton, Jason; Kuksin, Dmitry; Shahin, Suzanne M; Chan, Leo Li-Ying

2017-01-01

Brettanomyces spp. can present unique cell morphologies comprised of excessive pseudohyphae and budding, leading to difficulties in enumerating cells. The current cell counting methods include manual counting of methylene blue-stained yeasts or measuring optical densities using a spectrophotometer. However, manual counting can be time-consuming and has high operator-dependent variations due to subjectivity. Optical density measurement can also introduce uncertainties where instead of individual cells counted, an average of a cell population is measured. In contrast, by utilizing the fluorescence capability of an image cytometer to detect acridine orange and propidium iodide viability dyes, individual cell nuclei can be counted directly in the pseudohyphae chains, which can improve the accuracy and efficiency of cell counting, as well as eliminating the subjectivity from manual counting. In this work, two experiments were performed to demonstrate the capability of Cellometer image cytometer to monitor Brettanomyces concentrations, viabilities, and budding/pseudohyphae percentages. First, a yeast propagation experiment was conducted to optimize software counting parameters for monitoring the growth of Brettanomyces clausenii, Brettanomyces bruxellensis, and Brettanomyces lambicus, which showed increasing cell concentrations, and varying pseudohyphae percentages. The pseudohyphae formed during propagation were counted either as multiple nuclei or a single multi-nuclei organism, where the results of counting the yeast as a single multi-nuclei organism were directly compared to manual counting. Second, a yeast fermentation experiment was conducted to demonstrate that the proposed image cytometric analysis method can monitor the growth pattern of B. lambicus and B. clausenii during beer fermentation. The results from both experiments displayed different growth patterns, viability, and budding/pseudohyphae percentages for each Brettanomyces species. The proposed Cellometer image cytometry method can improve efficiency and eliminate operator-dependent variations of cell counting compared with the traditional methods, which can potentially improve the quality of beverage products employing Brettanomyces yeasts.

Cloning of ES cells and mice by nuclear transfer.

PubMed

Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

2009-01-01

We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning.

Nuclear migration events throughout development

PubMed Central

Bone, Courtney R.

2016-01-01

ABSTRACT Moving the nucleus to a specific position within the cell is an important event during many cell and developmental processes. Several different molecular mechanisms exist to position nuclei in various cell types. In this Commentary, we review the recent progress made in elucidating mechanisms of nuclear migration in a variety of important developmental models. Genetic approaches to identify mutations that disrupt nuclear migration in yeast, filamentous fungi, Caenorhabditis elegans, Drosophila melanogaster and plants led to the identification of microtubule motors, as well as Sad1p, UNC-84 (SUN) domain and Klarsicht, ANC-1, Syne homology (KASH) domain proteins (LINC complex) that function to connect nuclei to the cytoskeleton. We focus on how these proteins and various mechanisms move nuclei during vertebrate development, including processes related to wound healing of fibroblasts, fertilization, developing myotubes and the developing central nervous system. We also describe how nuclear migration is involved in cells that migrate through constricted spaces. On the basis of these findings, it is becoming increasingly clear that defects in nuclear positioning are associated with human diseases, syndromes and disorders. PMID:27182060

DNA synthesis in HeLa cells and isolated nuclei after treatment with an inhibitor of spermidine synthesis, methyl glyoxal bis(guanylhydrazone).

PubMed

Krokan, H; Eriksen, A

1977-02-01

Addition of methyl glyoxal bis(guanylhydrazone) to HeLa S3 suspension cultures resulted in increased putrescine levels and decreased spermidine and spermine levels preceding a drop in incorporation of [3H]thymidine, [3H]uridine and [14C]leucine into macromolecules. When putrescine, spermidine, spermine or cadaverine was added simultaneously with methyl glyoxal bis(guanylhydrazone), the drug had no detectable effect on the synthesis of macromolecules. In nuclei isolated from cells treated with methyl glyoxal bis(guanylhydrazone) the reduction in the rate of DNA synthesis was equal to the reduction of [3H]thymidine incorporation in the corresponding whole cells. The capability of the nuclei to synthesize DNA could not be restored by adding spermidine or spermine to the system in vitro. The rate of DNA chain elongation was only reduced slightly by methyl glyoxal bis(guanylhydrazone) indicating that decreased levels of spermidine and spermine lead to a decrease in the number of replication units active in DNA synthesis within each cell.

Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

2014-09-01

We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Jian; Zheng, Wei; Wang, Zi

2014-09-08

We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

DNA (DEOXYRIBONUCLEIC ACID) SYNTHESIS FOLLOWING MICROINJECTION OF HETEROLOGOUS SPERM AND SOMATIC CELL NUCLEI INTO HAMSTER OOCYTES

EPA Science Inventory

The authors have investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in 3H-thymidine after being parthenogenetically activated by sha...

Effects of chromium picolinate on micronucleus frequency and morphology of lymphocytes in calves.

PubMed

Imamoğlu, Nalan; Uyanik, Fatma; Kocaoğlu Güçlü, Berrin; Erdem, Onur; Cem Liman, Bilal; Dönmez Altuntaş, Hamiyet

2008-11-01

We report the effects of chromium picolinate (CrPic) on micronucleus frequency, morphology of lymphocytes, and lipid peroxidation in calves. Twenty-four Holstein calves were selected for the study. They were kept in a farm and were fed a commercially available calf diet and alfalfa, ad libitum. The animals were divided into three groups of eight subjects each and were treated as follows: The first group was supplemented with a daily dose of 200 microg Cr as chromium picolinate; a second group received 400 microg Cr per day and a third group that served as control received no supplemental chromium. After 12-week supplementation, blood samples were collected to determine the micronucleus frequency, the apoptotic cell percentage, and the malondialdehyde (MDA) and blood chromium levels. In both supplemented groups, the cells had irregularly shaped and segmented nuclei. Supplementation also increased the percentage of apoptotic cells (p < 0.001) and serum MDA (p < 0.01) and slightly increased the chromium levels. The animals supplemented with 400 microg showed a significant increase of micronucleus frequency (p < 0.01). The results of this study suggest that supplementation with 200 and 400 microg chromium as chromium picolinate may lead to cytotoxicity. The higher level of supplementation may also have genotoxic effects. However, further studies investigating the mechanism of the action of CrPic are required.

A comparative study on the distribution of non-specific esterase amongst the various constituents of retinas of some vertebrates.

PubMed

Tyagi, H R; Tewari, H B

1979-01-01

A wide coverage of the retinae of a large number of animals (Calotes, Varanus, Naja, Athene, Passer, Streptopelia, Psittacula and Funambulus) from the point of view of the histoenzymological distribution of non-specific esterase amongst the various constituents reveals mostly identical patterns. They are as follows: 1. Outer segments - positive in all cases. 2. Outer plexiform layer - equipped with enzymatic activity in all the instances. 3. Inner nuclear layer - thin cytoplasmic rim of the neurons characterized by positive activity; the nuclei of the neurons are completely negative. 4. Inner plexiform layer - this layer is endowed with the enzymatic activity. 5. Ganglion cells - negative in all cases. 6. Nerve fibres of the layer of nerve fibres, situated adjasent to ganglion cells are positive in all the animals; in case of squirrel oligodandroglial cells present in the region have demonstrated activity of a high order. On of the high-lights of the present contribution is demarcation of the inner plexiform layer into three stratified zones, equipped with enzymatic activity in Calotes, Streptopelia, Naja and Funambulus. Such stratifications are not seen in Varanus, Passer and Psittacula. The significance of the various patterns and the equipment of the enzyme in various constituents at various locals have been discussed in relation to the metabolic functions, zone-wise and interzone-wise in visual processes of various animals.

Light scattering microscopy measurements of single nuclei compared with GPU-accelerated FDTD simulations

NASA Astrophysics Data System (ADS)

Stark, Julian; Rothe, Thomas; Kieß, Steffen; Simon, Sven; Kienle, Alwin

2016-04-01

Single cell nuclei were investigated using two-dimensional angularly and spectrally resolved scattering microscopy. We show that even for a qualitative comparison of experimental and theoretical data, the standard Mie model of a homogeneous sphere proves to be insufficient. Hence, an accelerated finite-difference time-domain method using a graphics processor unit and domain decomposition was implemented to analyze the experimental scattering patterns. The measured cell nuclei were modeled as single spheres with randomly distributed spherical inclusions of different size and refractive index representing the nucleoli and clumps of chromatin. Taking into account the nuclear heterogeneity of a large number of inclusions yields a qualitative agreement between experimental and theoretical spectra and illustrates the impact of the nuclear micro- and nanostructure on the scattering patterns.

Light scattering microscopy measurements of single nuclei compared with GPU-accelerated FDTD simulations.

PubMed

Stark, Julian; Rothe, Thomas; Kieß, Steffen; Simon, Sven; Kienle, Alwin

2016-04-07

Single cell nuclei were investigated using two-dimensional angularly and spectrally resolved scattering microscopy. We show that even for a qualitative comparison of experimental and theoretical data, the standard Mie model of a homogeneous sphere proves to be insufficient. Hence, an accelerated finite-difference time-domain method using a graphics processor unit and domain decomposition was implemented to analyze the experimental scattering patterns. The measured cell nuclei were modeled as single spheres with randomly distributed spherical inclusions of different size and refractive index representing the nucleoli and clumps of chromatin. Taking into account the nuclear heterogeneity of a large number of inclusions yields a qualitative agreement between experimental and theoretical spectra and illustrates the impact of the nuclear micro- and nanostructure on the scattering patterns.

Platelet adhesion and human umbilical vein endothelial cell cytocompatibility of biodegradable segmented polyurethanes prepared with 4,4'-methylene bis(cyclohexyl isocyanate), poly(caprolactone) diol and butanediol or dithioerythritol as chain extenders.

PubMed

Chan-Chan, L H; Vargas-Coronado, R F; Cervantes-Uc, J M; Cauich-Rodríguez, J V; Rath, R; Phelps, E A; García, A J; San Román Del Barrio, J; Parra, J; Merhi, Y; Tabrizian, M

2013-08-01

Biodegradable segmented polyurethanes were prepared with poly(caprolactone) diol as a soft segment, 4,4'-methylene bis(cyclohexyl isocyanate) (HMDI) and either butanediol or dithioerythritol as chain extenders. Platelet adhesion was similar in all segmented polyurethanes studied and not different from Tecoflex® although an early stage of activation was observed on biodegradable segmented polyurethane prepared with dithioerythritol. Relative viability was higher than 80% on human umbilical vein endothelial cells in contact with biodegradable segmented polyurethane extracts after 1, 2 and 7 days. Furthermore, both biodegradable segmented polyurethane materials supported human umbilical vein endothelial cell adhesion, spreading, and viability similar to Tecoflex® medical-grade polyurethane. These biodegradable segmented polyurethanes represent promising materials for cardiovascular applications.

Leech segmental repeats develop normally in the absence of signals from either anterior or posterior segments

NASA Technical Reports Server (NTRS)

Seaver, E. C.; Shankland, M.

2000-01-01

We have investigated whether the development of segmental repeats is autonomous in the embryo of the leech Helobdella robusta. The segmental tissues of the germinal band arise from progeny of five stem cells called teloblasts. Asymmetric divisions of the teloblasts form chains of segment founder cells (called primary blast cells) that divide in a stereotypical manner to produce differentiated descendants. Using two distinct techniques, we have looked for potential interactions between neighboring blast cell clones along the anterior-posterior axis. In one technique, we prevented the birth of primary blast cells by injection of DNase I into the teloblast, thereby depriving the last blast cell produced before the ablation of its normal posterior neighbors. We also ablated single blast cells with a laser microbeam, which allowed us to assess potential signals acting on either more anterior or more posterior primary blast cell clones. Our results suggest that interactions along the anterior-posterior axis between neighboring primary blast cell clones are not required for development of normal segmental organization within the blast cell clone. We also examined the possibility that blast cells receive redundant signals from both anterior and posterior neighboring clones and that either is sufficient for normal development. Using double blast cell laser ablations to isolate a primary blast cell clone by removal of both its anterior and its posterior neighbor, we found that the isolated clone still develops normally. These results reveal that the fundamental segmental repeat in the leech embryo, the primary blast cell clone, can develop normally in the apparent absence of signals from adjacent repeats along the anterior-posterior axis.

Persistence, period and precision of autonomous cellular oscillators from the zebrafish segmentation clock

PubMed Central

Webb, Alexis B; Lengyel, Iván M; Jörg, David J; Valentin, Guillaume; Jülicher, Frank; Morelli, Luis G; Oates, Andrew C

2016-01-01

In vertebrate development, the sequential and rhythmic segmentation of the body axis is regulated by a “segmentation clock”. This clock is comprised of a population of coordinated oscillating cells that together produce rhythmic gene expression patterns in the embryo. Whether individual cells autonomously maintain oscillations, or whether oscillations depend on signals from neighboring cells is unknown. Using a transgenic zebrafish reporter line for the cyclic transcription factor Her1, we recorded single tailbud cells in vitro. We demonstrate that individual cells can behave as autonomous cellular oscillators. We described the observed variability in cell behavior using a theory of generic oscillators with correlated noise. Single cells have longer periods and lower precision than the tissue, highlighting the role of collective processes in the segmentation clock. Our work reveals a population of cells from the zebrafish segmentation clock that behave as self-sustained, autonomous oscillators with distinctive noisy dynamics. DOI: http://dx.doi.org/10.7554/eLife.08438.001 PMID:26880542

Dynamic chromosomal rearrangements in Hodgkin's lymphoma are due to ongoing three-dimensional nuclear remodeling and breakage-bridge-fusion cycles.

PubMed

Guffei, Amanda; Sarkar, Rahul; Klewes, Ludger; Righolt, Christiaan; Knecht, Hans; Mai, Sabine

2010-12-01

Hodgkin's lymphoma is characterized by the presence of mono-nucleated Hodgkin cells and bi- to multi-nucleated Reed-Sternberg cells. We have recently shown telomere dysfunction and aberrant synchronous/asynchronous cell divisions during the transition of Hodgkin cells to Reed-Sternberg cells.1 To determine whether overall changes in nuclear architecture affect genomic instability during the transition of Hodgkin cells to Reed-Sternberg cells, we investigated the nuclear organization of chromosomes in these cells. Three-dimensional fluorescent in situ hybridization revealed irregular nuclear positioning of individual chromosomes in Hodgkin cells and, more so, in Reed-Sternberg cells. We characterized an increasingly unequal distribution of chromosomes as mono-nucleated cells became multi-nucleated cells, some of which also contained chromosome-poor 'ghost' cell nuclei. Measurements of nuclear chromosome positions suggested chromosome overlaps in both types of cells. Spectral karyotyping then revealed both aneuploidy and complex chromosomal rearrangements: multiple breakage-bridge-fusion cycles were at the origin of the multiple rearranged chromosomes. This conclusion was challenged by super resolution three-dimensional structured illumination imaging of Hodgkin and Reed-Sternberg nuclei. Three-dimensional super resolution microscopy data documented inter-nuclear DNA bridges in multi-nucleated cells but not in mono-nucleated cells. These bridges consisted of chromatids and chromosomes shared by two Reed-Sternberg nuclei. The complexity of chromosomal rearrangements increased as Hodgkin cells developed into multi-nucleated cells, thus indicating tumor progression and evolution in Hodgkin's lymphoma, with Reed-Sternberg cells representing the highest complexity in chromosomal rearrangements in this disease. This is the first study to demonstrate nuclear remodeling and associated genomic instability leading to the generation of Reed-Sternberg cells of Hodgkin's lymphoma. We defined nuclear remodeling as a key feature of Hodgkin's lymphoma, highlighting the relevance of nuclear architecture in cancer.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon

Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin duringmore » nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.« less

Nuclear-cytoplasmic localization of acetyl coenzyme A synthetase-1 in the rat brain

PubMed Central

Ariyannur, Prasanth S.; Moffett, John R.; Madhavarao, Chikkathur N; Arun, Peethambaran; Vishnu, Nisha; Jacobowitz, David M.; Hallows, William C.; Denu, John M.; Namboodiri, Aryan M.A.

2011-01-01

Acetyl coenzyme A synthetase 1 (AceCS1) catalyzes the synthesis of acetyl coenzyme A from acetate and coenzyme A, and is thought to play diverse roles ranging from fatty acid synthesis to gene regulation. Using an affinity purified antibody generated against an 18-mer peptide sequence of AceCS1, and a polyclonal antibody directed against recombinant AceCS1 protein, we examined the expression of AceCS1 in the rat brain. AceCS1 immunoreactivity in the adult rat brain was present predominantly in cell nuclei, with only light to moderate cytoplasmic staining in some neurons, axons and oligodendrocytes. Some non-neuronal cell nuclei were very strongly immunoreactive, including those of some oligodendrocytes, whereas neuronal nuclei ranged from unstained to moderately stained. Both antibodies stained some neuronal cell bodies and axons, especially in the hindbrain. AceCS1 immunoreactivity was stronger and more widespread in the brains of 18 day old rats than in adults, with increased expression in oligodendrocytes and neurons, including cortical pyramidal cells. Expression of AceCS1 was substantially upregulated in neurons throughout the brain after controlled cortical impact injury. The strong AceCS1 expression observed in the nuclei of CNS cells during brain development and after injury is consistent with a role in nuclear histone acetylation and therefore the regulation of chromatin structure and gene expression. The cytoplasmic staining observed in some oligodendrocytes, especially during postnatal brain development, suggests an additional role in CNS lipid synthesis and myelination. Neuronal and axonal localization implicates AceCS1 in cytoplasmic acetylation reactions in some neurons. PMID:20533355

Effects of arbuscular mycorrhizal colonization and phosphorus application on nuclear ploidy in Allium porrum plants.

PubMed

Fusconi, Anna; Lingua, Guido; Trotta, Antonio; Berta, Graziella

2005-07-01

Arbuscular mycorrhizal (AM) colonization can strongly affect the plant cell nucleus, causing displacement from the periphery to the center of the cell, hypertrophy and polyploidization. The hypertrophy response has been shown in a variety of AM plants whilst polyploidization has been reported only in Lycopersicon esculentum, a multiploid species with a small genome. In order to determine whether polyploidization is a general plant response to AM colonization, analyses were performed on Allium porrum, a plant with a large genome, which is much less subject to polyploidization than L. esculentum. The ploidy status of leaves, complete root systems and four zones of the adventitious roots was investigated in relation to phosphorus content, AM colonization and root differentiation in A. porrum plants grown under two different regimes of phosphate nutrition in order to distinguish direct effects of the fungus from those of improved nutrition. Results showed the presence of two nuclear populations (2C and 4C) in all treatments and samples. Linear regression analyses suggested a general negative correlation between phosphorus content and the proportion of 2C nuclei. The percentage of 2C nuclei (and consequently that of 4C nuclei), was also influenced by AM colonization, differentiation and ageing of the root cells, which resulted in earlier occurrence, in time and space, of polyploid nuclei.

Distribution of Vesicular Glutamate Transporter 2 and Ionotropic Glutamate Receptors in the Auditory Ganglion and Cochlear Nuclei of Pigeons (Columba livia).

PubMed

Karim, M R; Atoji, Y

2016-02-01

Glutamate is a principal excitatory neurotransmitter in the auditory system. Our previous studies revealed localization of glutamate receptor mRNAs in the pigeon cochlear nuclei, suggesting the existence of glutamatergic input from the auditory nerve to the brainstem. This study demonstrated localization of mRNAs for vesicular glutamate transporter 2 (vGluT2) and ionotropic glutamate receptors (AMPA, kainate and NMDA) in the auditory ganglion (AG) and cochlear nuclei (magnocellular, angular and laminar nuclei). VGluT2 mRNA was intensely expressed in AG and intensely or moderately in the cochlear nuclei. The AG and cochlear nuclei showed intense-to-moderate mRNA signals for GluA2, GluA3, GluA4, GluK4 and GluN1. These results suggest that the pigeon AG neurons receives glutamatergic input from hair cells and in turn projects to the magnocellular and angular nuclei. Glutamate may play a pivotal role in the excitatory synapse transmission in the peripheral auditory pathway of birds. © 2015 Blackwell Verlag GmbH.

Extracting Fluorescent Reporter Time Courses of Cell Lineages from High-Throughput Microscopy at Low Temporal Resolution

PubMed Central

Downey, Mike J.; Jeziorska, Danuta M.; Ott, Sascha; Tamai, T. Katherine; Koentges, Georgy; Vance, Keith W.; Bretschneider, Till

2011-01-01

The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted to different cell types and fluorescent markers. PMID:22194797

An investigation of the viscoelastic properties and the actin cytoskeletal structure of triple negative breast cancer cells.

PubMed

Hu, Jingjie; Zhou, Yuxiao; Obayemi, John D; Du, Jing; Soboyejo, Winston O

2018-05-30

An improved understanding of the evolution of cell structure and viscoelasticity with cancer malignancy could enable the development of a new generation of biomarkers and methods for cancer diagnosis. Hence, in this study, we present the viscoelastic properties (moduli and viscosities) and the actin cytoskeletal structures of triple negative breast cancer (TNBC) cells with different metastatic potential. These include: MCF-10A normal breast cells (studied as a control); MDA-MB-468 cells (less metastatic TNBC cells), and MDA-MB-231 cells (highly metastatic TNBC cells). A combination of shear assay and digital imaging correlation (DIC) techniques is used to measure the local viscoelastic properties of live breast cells subjected to constant shear stress. The local moduli and viscosities of the nuclei and cytoplasm are characterized using a generalized Maxwell model, which is used to determine the time-dependent creep responses of cells. The nuclei are shown to be stiffer and more viscous than the cytoplasms of the normal breast cells and TNBC cells. The MCF-10A normal breast cells are found to be twice as stiff as the less metastatic MDA-MB-468 breast cancer cells and over ten times stiffer than the highly metastatic MDA-MB-231 breast cancer cells. Similar trends are also observed in the viscosities of the nuclei and the cytoplasms. The measured differences in cell viscoelastic properties are also associated with significant changes in the cell cytoskeletal structure, which is studied using confocal fluorescence microscopy. This reveals significant differences in the levels of actin expression and organization in TNBC cells as they become highly metastatic. Our results suggest that the shear assay measurements of cell viscoelastic properties may be used as effective biomarkers for TNBC diagnosis and screening. Copyright © 2018 Elsevier Ltd. All rights reserved.

AutoCellSeg: robust automatic colony forming unit (CFU)/cell analysis using adaptive image segmentation and easy-to-use post-editing techniques.

PubMed

Khan, Arif Ul Maula; Torelli, Angelo; Wolf, Ivo; Gretz, Norbert

2018-05-08

In biological assays, automated cell/colony segmentation and counting is imperative owing to huge image sets. Problems occurring due to drifting image acquisition conditions, background noise and high variation in colony features in experiments demand a user-friendly, adaptive and robust image processing/analysis method. We present AutoCellSeg (based on MATLAB) that implements a supervised automatic and robust image segmentation method. AutoCellSeg utilizes multi-thresholding aided by a feedback-based watershed algorithm taking segmentation plausibility criteria into account. It is usable in different operation modes and intuitively enables the user to select object features interactively for supervised image segmentation method. It allows the user to correct results with a graphical interface. This publicly available tool outperforms tools like OpenCFU and CellProfiler in terms of accuracy and provides many additional useful features for end-users.

Robust Nucleus/Cell Detection and Segmentation in Digital Pathology and Microscopy Images: A Comprehensive Review.

PubMed

Xing, Fuyong; Yang, Lin

2016-01-01

Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to interobserver variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literature. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast, fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation.

Expression of HIWI in human esophageal squamous cell carcinoma is significantly associated with poorer prognosis

PubMed Central

2009-01-01

Background HIWI, the human homologue of Piwi family, is present in CD34+ hematopoietic stem cells and germ cells, but not in well-differentiated cell populations, indicating that HIWI may play an impotent role in determining or maintaining stemness of these cells. That HIWI expression has been detected in several type tumours may suggest its association with clinical outcome in cancer patients. Methods With the methods of real-time PCR, western blot, immunocytochemistry and immunohistochemistry, the expression of HIWI in three esophageal squamous cancer cell lines KYSE70, KYSE140 and KYSE450 has been characterized. Then, we investigated HIWI expression in a series of 153 esophageal squamous cell carcinomas using immunohistochemistry and explored its association with clinicopathological features. Results The expression of HIWI was observed in tumour cell nuclei or/and cytoplasm in 137 (89.5%) cases, 16 (10.5%) cases were negative in both nuclei and cytoplasm. 86 (56.2%) were strongly positive in cytoplasm, while 49 (32.0%) were strongly positive in nuclei. The expression level of HIWI in cytoplasm of esophageal cancer cells was significantly associated with histological grade (P = 0.011), T stage (P = 0.035), and clinic outcome (P < 0.001), while there was no correlation between the nuclear HIWI expression and clinicopathological features. Conclusion The expression of HIWI in the cytoplasm of esophageal cancer cells is significantly associated with higher histological grade, clinical stage and poorer clinical outcome, indicating its possible involvement in cancer development. PMID:19995427

Localization of DNA methyltransferase-1 during oocyte differentiation, in vitro maturation and early embryonic development in cow

PubMed Central

Lodde, V.; Modina, S.C.; Franciosi, F.; Zuccari, E.; Tessaro, I.; Luciano, A.M.

2009-01-01

DNA methyltransferase-1 (Dnmt1) is involved in the maintenance of DNA methylation patterns and is crucial for normal mammalian development. The aim of the present study was to assess the localization of Dnmt1 in cow, during the latest phases of oocyte differentiation and during the early stages of segmentation. Dnmt1 expression and localization were assessed in oocytes according to the chromatin configuration, which in turn provides an important epigenetic mechanism for the control of global gene expression and represents a morphological marker of oocyte differentiation. We found that the initial chromatin condensation was accompanied by a slight increase in the level of global DNA methylation, as assessed by 5-methyl-cytosine immunostaining followed by laser scanning confocal microscopy analysis (LSCM). RT-PCR confirmed the presence of Dnmt1 transcripts throughout this phase of oocyte differentiation. Analogously, Dnmt1 immunodetection and LSCM indicated that the protein was always present and localized in the cytoplasm, regardless the chromatin configuration and the level of global DNA methylation. Moreover, our data indicate that while Dnmt1 is retained in the cytoplasm in metaphase II stage oocytes and zygotes, it enters the nuclei of 8–16 cell stage embryos. As suggested in mouse, the functional meaning of the presence of Dnmt1 in the bovine embryo nuclei could be the maintainement of the methylation pattern of imprinted genes. In conclusion, the present work provides useful elements for the study of Dnmt1 function during the late stage of oocyte differentiation, maturation and early embryonic development in mammals. PMID:22073356

Development of representative magnetic resonance imaging-based atlases of the canine brain and evaluation of three methods for atlas-based segmentation.

PubMed

Milne, Marjorie E; Steward, Christopher; Firestone, Simon M; Long, Sam N; O'Brien, Terrence J; Moffat, Bradford A

2016-04-01

To develop representative MRI atlases of the canine brain and to evaluate 3 methods of atlas-based segmentation (ABS). 62 dogs without clinical signs of epilepsy and without MRI evidence of structural brain disease. The MRI scans from 44 dogs were used to develop 4 templates on the basis of brain shape (brachycephalic, mesaticephalic, dolichocephalic, and combined mesaticephalic and dolichocephalic). Atlas labels were generated by segmenting the brain, ventricular system, hippocampal formation, and caudate nuclei. The MRI scans from the remaining 18 dogs were used to evaluate 3 methods of ABS (manual brain extraction and application of a brain shape-specific template [A], automatic brain extraction and application of a brain shape-specific template [B], and manual brain extraction and application of a combined template [C]). The performance of each ABS method was compared by calculation of the Dice and Jaccard coefficients, with manual segmentation used as the gold standard. Method A had the highest mean Jaccard coefficient and was the most accurate ABS method assessed. Measures of overlap for ABS methods that used manual brain extraction (A and C) ranged from 0.75 to 0.95 and compared favorably with repeated measures of overlap for manual extraction, which ranged from 0.88 to 0.97. Atlas-based segmentation was an accurate and repeatable method for segmentation of canine brain structures. It could be performed more rapidly than manual segmentation, which should allow the application of computer-assisted volumetry to large data sets and clinical cases and facilitate neuroimaging research and disease diagnosis.

Direct visualization of anatomic subfields within the superior aspect of the human lateral thalamus by MRI at 7T.

PubMed

Kanowski, M; Voges, J; Buentjen, L; Stadler, J; Heinze, H-J; Tempelmann, C

2014-09-01

The morphology of the human thalamus shows high interindividual variability. Therefore, direct visualization of landmarks within the thalamus is essential for an improved definition of electrode positions for deep brain stimulation. The aim of this study was to provide anatomic detail in the thalamus by using inversion recovery TSE imaging at 7T. The MR imaging protocol was optimized on 1 healthy subject to segment thalamic nuclei from one another. Final images, acquired with 0.5(2)-mm2 in-plane resolution and 3-mm section thickness, were compared with stereotactic brain atlases to assign visualized details to known anatomy. The robustness of the visualization of thalamic nuclei was assessed with 4 healthy subjects at lower image resolution. Thalamic subfields were successfully delineated in the dorsal aspect of the lateral thalamus. T1-weighting was essential. MR images had an appearance very similar to that of myelin-stained sections seen in brain atlases. Visualized intrathalamic structures were, among others, the lamella medialis, the external medullary lamina, the reticulatum thalami, the nucleus centre médian, the boundary between the nuclei dorso-oralis internus and externus, and the boundary between the nuclei dorso-oralis internus and zentrolateralis intermedius internus. Inversion recovery-prepared TSE imaging at 7T has a high potential to reveal fine anatomic detail in the thalamus, which may be helpful in enhancing the planning of stereotactic neurosurgery in the future. © 2014 by American Journal of Neuroradiology.

The luteinizing hormone-releasing hormone pathways in rhesus (Macaca mulatta) and pigtailed (Macaca nemestrina) monkeys: new observations on thick, unembedded sections.

PubMed

Silverman, A J; Antunes, J L; Abrams, G M; Nilaver, G; Thau, R; Robinson, J A; Ferin, M; Krey, L C

1982-11-01

Immunocytochemical procedures on thick, unembedded tissue sections were used to study the localization of LHRH neurons and fibers in the diencephalon and mesencephalon of rhesus and pigtailed macaques. Cell bodies were visualized in large numbers. Much of their dendritic arborization was also filled with reaction product. Cell bodies were present in the preoptic area, the periventricular hypothalamic zone from the level of the anterior hypothalamus to the premammillary nuclei, the infundibular nucleus, supraoptic nucleus, several septal nuclei, the nervus terminalis, and the amygdala. The localization of LHRH cells in several of these areas represents new observations. LHRH axons were observed to innervate the portal vessels in the median eminence, the organum vasculosum of the lamina terminalis, the median eminence, the organum vasculosum of the lamina terminalis, the medial mammillary nuclei, the epithalamus, and the amygdala. These observations are discussed in relationship to the regulation of gonadotropin secretion in the primate.

Crystalline inclusions in the cytoplasm and nuclei of cells of acute myeloid leukaemia.

PubMed

Pearson, E C

1989-01-01

In a survey by electron microscopy of peripheral blood and/or bone marrow from 230 adult patients with acute myeloid leukaemia, five were observed to contain crystalline inclusions in the cytoplasm of the leukaemic cells and a sixth contained crystals in the nuclei. In four cases, two of FAB type M2 and two of M4, the cytoplasmic crystals were hexagonal in section and 1-2 micron long. Two examples showed internal periodicities in the range 3.3-4.0 nm when the electronmicrographs were analysed by optical diffractometry. A single case of M1 contained smaller trapezoidal crystals with a 4.9nm periodicity. The sixth patient, with unusual cytological abnormalities and a rare t(3; 6) chromosomal translocation, contained six-sided crystals in the nuclei of some relatively undifferentiated cells. To the best of our knowledge such intranuclear crystals have not previously been reported in leukaemia. The relevance of the crystals to the leukaemic process is discussed.

Anatomy of the human hypothalamus (chiasmatic and tuberal region).

PubMed

Braak, H; Braak, E

1992-01-01

The hypothalamus sensu stricto consists of the chiasmatic, the tuberal and the mamillary region. The present study is confined to the poorly myelinated chiasmatic and tuberal region. Both regions harbor many nuclear grays with relatively clear-cut boundaries embedded in an ill-defined nerve cell assembly referred to as the hypothalamic gray. Prominent components of the chiasmatic region are the magnocellular neurosecretory complex (supraoptic nucleus, paraventricular nucleus, accessory neurosecretory nucleus), the sexually dimorphic intermediate nucleus, the suprachiasmatic and retrochiasmatic nuclei. The dominating structure of the tuberal region is the complex of the ventromedial, posteromedial and dorsomedial nuclei supplemented by the periventricular and infundibular nuclei. Lateral portions of the tuber cinereum harbor the lateral tuberal nucleus and the tuberomamillary nucleus. The lateral tuberal nucleus exhibits pronounced cell loss in Huntington's chorea and is also severely involved in cases of dementia with argyrophilic grains. The large nerve cells of the tuberomamillary nucleus show particularly severe affection in both Alzheimer's (intraneuronal neurofibrillary changes) and Parkinson's disease (Lewy bodies).

Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

PubMed Central

Winnacker, E L

1975-01-01

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA. PMID:1117487

Cytological evidence for chromosome elimination in wheat x Imperata cylindrica hybrids.

PubMed

Komeda, Norio; Chaudhary, Harinder K; Suzuki, Go; Mukai, Yasuhiko

2007-06-01

Haploid induction of wheat by crossing with Imperata cylindrica pollen is an efficient method for doubled haploid breeding. We investigated the process of wheat haploid formation after crossing with I. cylindrica. Our cytological observations of zygotes showed the successful fertilization of parental gametes. Wheat haploids were formed by complete elimination of I. cylindrica chromosomes. Missegregation of I. cylindrica chromosomes was observed in the first cell division of zygote. At metaphase I. cylindrica chromosomes did not congress onto the equatorial plate. The sister chromosomes did not move toward the poles during anaphase, though their cohesion was released normally. I. cylindrica chromosomes were still in the cytoplasm at telophase and eliminated from daughter nuclei. After two-celled stage, we could find no I. cylindrica chromosome in the nuclei but micronuclei containing I. cylindrica chromatin in the cytoplasm. These observations indicate that I. cylindrica chromosomes are completely eliminated from nuclei in the first cell division probably due to lack of functional kinetochores.

CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation

PubMed Central

2013-01-01

The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening. PMID:23938087

CellSegm - a MATLAB toolbox for high-throughput 3D cell segmentation.

PubMed

Hodneland, Erlend; Kögel, Tanja; Frei, Dominik Michael; Gerdes, Hans-Hermann; Lundervold, Arvid

2013-08-09

: The application of fluorescence microscopy in cell biology often generates a huge amount of imaging data. Automated whole cell segmentation of such data enables the detection and analysis of individual cells, where a manual delineation is often time consuming, or practically not feasible. Furthermore, compared to manual analysis, automation normally has a higher degree of reproducibility. CellSegm, the software presented in this work, is a Matlab based command line software toolbox providing an automated whole cell segmentation of images showing surface stained cells, acquired by fluorescence microscopy. It has options for both fully automated and semi-automated cell segmentation. Major algorithmic steps are: (i) smoothing, (ii) Hessian-based ridge enhancement, (iii) marker-controlled watershed segmentation, and (iv) feature-based classfication of cell candidates. Using a wide selection of image recordings and code snippets, we demonstrate that CellSegm has the ability to detect various types of surface stained cells in 3D. After detection and outlining of individual cells, the cell candidates can be subject to software based analysis, specified and programmed by the end-user, or they can be analyzed by other software tools. A segmentation of tissue samples with appropriate characteristics is also shown to be resolvable in CellSegm. The command-line interface of CellSegm facilitates scripting of the separate tools, all implemented in Matlab, offering a high degree of flexibility and tailored workflows for the end-user. The modularity and scripting capabilities of CellSegm enable automated workflows and quantitative analysis of microscopic data, suited for high-throughput image based screening.

GABA Immunoreactivity in Auditory and Song Control Brain Areas of Zebra Finches

PubMed Central

Pinaud, Raphael; Mello, Claudio V.

2009-01-01

Inhibitory transmission is critical to sensory and motor processing and is believed to play a role in experience-dependent plasticity. The main inhibitory neurotransmitter in vertebrates, GABA, has been implicated in both sensory and motor aspects of vocalization in songbirds. To understand the role of GABAergic mechanisms in vocal communication, GABAergic elements must be characterized fully. Hence, we investigated GABA immunohistochemistry in the zebra finch brain, emphasizing auditory areas and song control nuclei. Several nuclei of the ascending auditory pathway showed a moderate to high density of GABAergic neurons including the cochlear nuclei, nucleus laminaris, superior olivary nucleus, mesencephalic nucleus lateralis pars dorsalis, and nucleus ovoidalis. Telencephalic auditory areas, including field L subfields L1, L2a and L3, as well as the caudomedial nidopallium (NCM) and mesopallium (CMM), contained GABAergic cells at particularly high densities. Considerable GABA labeling was also seen in the shelf area of caudodorsal nidopallium, and the cup area in the arcopallium, as well as in area X, the lateral magnocellular nucleus of the anterior nidopallium, the robust nucleus of the arcopallium and nidopallial nucleus HVC. GABAergic cells were typically small, most likely local inhibitory interneurons, although large GABA-positive cells that were sparsely distributed were also identified. GABA-positive neurites and puncta were identified in most nuclei of the ascending auditory pathway and in song control nuclei. Our data are in accordance with a prominent role of GABAergic mechanisms in regulating the neural circuits involved in song perceptual processing, motor production, and vocal learning in songbirds. PMID:17466487

Automatic cell detection and segmentation from H and E stained pathology slides using colorspace decorrelation stretching

NASA Astrophysics Data System (ADS)

Peikari, Mohammad; Martel, Anne L.

2016-03-01

Purpose: Automatic cell segmentation plays an important role in reliable diagnosis and prognosis of patients. Most of the state-of-the-art cell detection and segmentation techniques focus on complicated methods to subtract foreground cells from the background. In this study, we introduce a preprocessing method which leads to a better detection and segmentation results compared to a well-known state-of-the-art work. Method: We transform the original red-green-blue (RGB) space into a new space defined by the top eigenvectors of the RGB space. Stretching is done by manipulating the contrast of each pixel value to equalize the color variances. New pixel values are then inverse transformed to the original RGB space. This altered RGB image is then used to segment cells. Result: The validation of our method with a well-known state-of-the-art technique revealed a statistically significant improvement on an identical validation set. We achieved a mean F1-score of 0.901. Conclusion: Preprocessing steps to decorrelate colorspaces may improve cell segmentation performances.

3D Imaging of Retinal Pigment Epithelial Cells in the Living Human Retina

PubMed Central

Liu, Zhuolin; Kocaoglu, Omer P.; Miller, Donald T.

2016-01-01

Purpose Dysfunction of the retinal pigment epithelium (RPE) underlies numerous retinal pathologies, but biomarkers sensitive to RPE change at the cellular level are limited. In this study, we used adaptive optics optical coherence tomography (AO-OCT) in conjunction with organelle motility as a novel contrast mechanism to visualize RPE cells and characterize their 3-dimensional (3D) reflectance profile. Methods Using the Indiana AO-OCT imaging system (λc = 790 nm), volumes were acquired in the macula of six normal subjects (25–61 years). Volumes were registered in 3D with subcellular accuracy, layers segmented, and RPE and photoreceptor en face images extracted and averaged. Voronoi and two-dimensional (2D) power spectra analyses were applied to the images to quantify RPE and cone packing and cone-to-RPE ratio. Results Adaptive optics OCT revealed two distinct reflectance patterns at the depth of the RPE. One is characterized by the RPE interface with rod photoreceptor tips, the second by the RPE cell nuclei and surrounding organelles, likely melanin. Increasing cell contrast by averaging proved critical for observing the RPE cell mosaic, successful in all subjects and retinal eccentricities imaged. Retinal pigment epithelium mosaic packing and cell thickness generally agreed with that of histology and in vivo studies using other imaging modalities. Conclusions We have presented, to our knowledge, the first detailed characterization of the 3D reflectance profile of individual RPE cells and their relation to cones and rods in the living human retina. Success in younger and older eyes establishes a path for testing aging effects in larger populations. Because the technology is based on OCT, our measurements will aid in interpreting clinical OCT images. PMID:27472277

In vitro response of retinal pigment epithelial cells exposed to chitosan materials prepared with different cross-linkers.

PubMed

Lai, Jui-Yang; Li, Ya-Ting; Wang, Tsu-Pin

2010-01-01

The interaction between cells and biopolymers is the evaluation indicator of the biocompatibility of materials. The purpose of this work was to examine the responses of retinal pigment epithelial (RPE) cells to genipin (GP) or glutaraldehyde (GTA) cross-linked chitosan by means of cell viability assays, cytokine expression analyses, and apoptosis assays. Evaluations of non-cross-linked chitosan were conducted simultaneously for comparison. Both GP and GTA treated samples with the same extent of cross-linking (around 80%) were prepared by varying cross-linking time. Our results showed that GP cross-linking was carried out by either radical polymerization of the monomers or S(N)2 nucleophilic substitution reaction involving the replacement of the ester group on the monomer with a secondary amide linkage. On the other hand, GTA could react with free amino groups of chitosan, leading to the formation of either the Schiff bases or the Michael-type adducts with terminal aldehydes. The biocompatibility of non-cross-linked chitosan membranes was demonstrated by the absence of any signs of toxicity or inflammation reaction. The present study showed that the ARPE-19 cells exposed to GTA cross-linked chitosan membranes had significantly higher cytotoxicity, interleukin-6 levels, and number of TUNEL-positive nuclei than did those exposed to GP treated samples. In addition, the materials modified with GTA trigger apoptosis at an early stage and may induce toxicity in the RPE cells later. The findings suggest that while the chitosan molecules bridged by GP are satisfactorily cytocompatible, the counterparts treated by GTA do not seem to be tolerated. In terms of material safety, the GP cross-linked chitosan may be compatible with human RPE cells and may have a potential application as delivery carriers in the treatment of posterior segment diseases.

An RNA Recognition Motif-Containing Protein Functions in Meiotic Silencing by Unpaired DNA

PubMed Central

Samarajeewa, Dilini A.; Manitchotpisit, Pennapa; Henderson, Miranda; Xiao, Hua; Rehard, David G.; Edwards, Kevin A.; Shiu, Patrick K. T.; Hammond, Thomas M.

2017-01-01

Meiotic silencing by unpaired DNA (MSUD) is a biological process that searches pairs of homologous chromosomes (homologs) for segments of DNA that are unpaired. Genes found within unpaired segments are silenced for the duration of meiosis. In this report, we describe the identification and characterization of Neurospora crassa sad-7, a gene that encodes a protein with an RNA recognition motif (RRM). Orthologs of sad-7 are found in a wide range of ascomycete fungi. In N. crassa, sad-7 is required for a fully efficient MSUD response to unpaired genes. Additionally, at least one parent must have a functional sad-7 allele for a cross to produce ascospores. Although sad-7-null crosses are barren, sad-7Δ strains grow at a wild-type (wt) rate and appear normal under vegetative growth conditions. With respect to expression, sad-7 is transcribed at baseline levels in early vegetative cultures, at slightly higher levels in mating-competent cultures, and is at its highest level during mating. These findings suggest that SAD-7 is specific to mating-competent and sexual cultures. Although the role of SAD-7 in MSUD remains elusive, green fluorescent protein (GFP)-based tagging studies place SAD-7 within nuclei, perinuclear regions, and cytoplasmic foci of meiotic cells. This localization pattern is unique among known MSUD proteins and raises the possibility that SAD-7 coordinates nuclear, perinuclear, and cytoplasmic aspects of MSUD. PMID:28667016

A Fast, Automatic Segmentation Algorithm for Locating and Delineating Touching Cell Boundaries in Imaged Histopathology

PubMed Central

Qi, Xin; Xing, Fuyong; Foran, David J.; Yang, Lin

2013-01-01

Summary Background Automated analysis of imaged histopathology specimens could potentially provide support for improved reliability in detection and classification in a range of investigative and clinical cancer applications. Automated segmentation of cells in the digitized tissue microarray (TMA) is often the prerequisite for quantitative analysis. However overlapping cells usually bring significant challenges for traditional segmentation algorithms. Objectives In this paper, we propose a novel, automatic algorithm to separate overlapping cells in stained histology specimens acquired using bright-field RGB imaging. Methods It starts by systematically identifying salient regions of interest throughout the image based upon their underlying visual content. The segmentation algorithm subsequently performs a quick, voting based seed detection. Finally, the contour of each cell is obtained using a repulsive level set deformable model using the seeds generated in the previous step. We compared the experimental results with the most current literature, and the pixel wise accuracy between human experts' annotation and those generated using the automatic segmentation algorithm. Results The method is tested with 100 image patches which contain more than 1000 overlapping cells. The overall precision and recall of the developed algorithm is 90% and 78%, respectively. We also implement the algorithm on GPU. The parallel implementation is 22 times faster than its C/C++ sequential implementation. Conclusion The proposed overlapping cell segmentation algorithm can accurately detect the center of each overlapping cell and effectively separate each of the overlapping cells. GPU is proven to be an efficient parallel platform for overlapping cell segmentation. PMID:22526139

Dynamics of the slowing segmentation clock reveal alternating two-segment periodicity

PubMed Central

Shih, Nathan P.; François, Paul; Delaune, Emilie A.; Amacher, Sharon L.

2015-01-01

The formation of reiterated somites along the vertebrate body axis is controlled by the segmentation clock, a molecular oscillator expressed within presomitic mesoderm (PSM) cells. Although PSM cells oscillate autonomously, they coordinate with neighboring cells to generate a sweeping wave of cyclic gene expression through the PSM that has a periodicity equal to that of somite formation. The velocity of each wave slows as it moves anteriorly through the PSM, although the dynamics of clock slowing have not been well characterized. Here, we investigate segmentation clock dynamics in the anterior PSM in developing zebrafish embryos using an in vivo clock reporter, her1:her1-venus. The her1:her1-venus reporter has single-cell resolution, allowing us to follow segmentation clock oscillations in individual cells in real-time. By retrospectively tracking oscillations of future somite boundary cells, we find that clock reporter signal increases in anterior PSM cells and that the periodicity of reporter oscillations slows to about ∼1.5 times the periodicity in posterior PSM cells. This gradual slowing of the clock in the anterior PSM creates peaks of clock expression that are separated at a two-segment periodicity both spatially and temporally, a phenomenon we observe in single cells and in tissue-wide analyses. These results differ from previous predictions that clock oscillations stop or are stabilized in the anterior PSM. Instead, PSM cells oscillate until they incorporate into somites. Our findings suggest that the segmentation clock may signal somite formation using a phase gradient with a two-somite periodicity. PMID:25968314

Syncytia in plants: cell fusion in endosperm-placental syncytium formation in Utricularia (Lentibulariaceae).

PubMed

Płachno, Bartosz J; Swiątek, Piotr

2011-04-01

The syncytium formed by Utricularia is extremely unusual and perhaps unique among angiosperm syncytia. All typical plant syncytia (articulated laticifers, amoeboid tapetum, the nucellar plasmodium of river weeds) are formed only by fusion of sporophytic cells which possess the same genetic material, unlike Utricularia in which the syncytium possesses nuclei from two different sources: cells of maternal sporophytic nutritive tissue and endosperm haustorium (both maternal and paternal genetic material). How is this kind of syncytium formed and organized and is it similar to other plant syncytial structures? We used light and electron microscopy to reconstruct the step-by-step development of the Utricularia syncytia. The syncytia of Utricularia developed through heterotypic cell fusion involving the digestion of the cell wall, and finally, heterokaryotic multinucleate structures were formed, which possessed different-sized nuclei that were not regularly arranged in the cytoplasm. We showed that these syncytia were characterized by hypertrophy of nuclei, abundant endoplasmic reticulum and organelles, and the occurrence of wall ingrowths. All these characters testify to high activity and may confirm the nutritive and transport functions of the syncytium for the developing embryo. In Utricularia, the formation of the syncytium provides an economical way to redistribute cell components and release nutrients from the digested cell walls, which can now be used for the embryo, and finally to create a large surface for the exchange of nutrients between the placenta and endosperm.

Single-nucleus RNA-seq of differentiating human myoblasts reveals the extent of fate heterogeneity

PubMed Central

Zeng, Weihua; Jiang, Shan; Kong, Xiangduo; El-Ali, Nicole; Ball, Alexander R.; Ma, Christopher I-Hsing; Hashimoto, Naohiro; Yokomori, Kyoko; Mortazavi, Ali

2016-01-01

Myoblasts are precursor skeletal muscle cells that differentiate into fused, multinucleated myotubes. Current single-cell microfluidic methods are not optimized for capturing very large, multinucleated cells such as myotubes. To circumvent the problem, we performed single-nucleus transcriptome analysis. Using immortalized human myoblasts, we performed RNA-seq analysis of single cells (scRNA-seq) and single nuclei (snRNA-seq) and found them comparable, with a distinct enrichment for long non-coding RNAs (lncRNAs) in snRNA-seq. We then compared snRNA-seq of myoblasts before and after differentiation. We observed the presence of mononucleated cells (MNCs) that remained unfused and analyzed separately from multi-nucleated myotubes. We found that while the transcriptome profiles of myoblast and myotube nuclei are relatively homogeneous, MNC nuclei exhibited significant heterogeneity, with the majority of them adopting a distinct mesenchymal state. Primary transcripts for microRNAs (miRNAs) that participate in skeletal muscle differentiation were among the most differentially expressed lncRNAs, which we validated using NanoString. Our study demonstrates that snRNA-seq provides reliable transcriptome quantification for cells that are otherwise not amenable to current single-cell platforms. Our results further indicate that snRNA-seq has unique advantage in capturing nucleus-enriched lncRNAs and miRNA precursors that are useful in mapping and monitoring differential miRNA expression during cellular differentiation. PMID:27566152

Unsupervised segmentation of H and E breast images

NASA Astrophysics Data System (ADS)

Hope, Tyna A.; Yaffe, Martin J.

2017-03-01

Heterogeneity of ductal carcinoma in situ (DCIS) continues to be an important topic. Combining biomarker and hematoxylin and eosin (HE) morphology information may provide more insights than either alone. We are working towards a computer-based identification and description system for DCIS. As part of the system we are developing a region of interest finder for further processing, such as identifying DCIS and other HE based measures. The segmentation algorithm is designed to be tolerant of variability in staining and require no user interaction. To achieve stain variation tolerance we use unsupervised learning and iteratively interrogate the image for information. Using simple rules (e.g., "hematoxylin stains nuclei") and iteratively assessing the resultant objects (small hematoxylin stained objects are lymphocytes), the system builds up a knowledge base so that it is not dependent upon manual annotations. The system starts with image resolution-based assumptions but these are replaced by knowledge gained. The algorithm pipeline is designed to find the simplest items first (segment stains), then interesting subclasses and objects (stroma, lymphocytes), and builds information until it is possible to segment blobs that are normal, DCIS, and the range of benign glands. Once the blobs are found, features can be obtained and DCIS detected. In this work we present the early segmentation results with stains where hematoxylin ranges from blue dominant to red dominant in RGB space.

Using wavelet denoising and mathematical morphology in the segmentation technique applied to blood cells images.

PubMed

Boix, Macarena; Cantó, Begoña

2013-04-01

Accurate image segmentation is used in medical diagnosis since this technique is a noninvasive pre-processing step for biomedical treatment. In this work we present an efficient segmentation method for medical image analysis. In particular, with this method blood cells can be segmented. For that, we combine the wavelet transform with morphological operations. Moreover, the wavelet thresholding technique is used to eliminate the noise and prepare the image for suitable segmentation. In wavelet denoising we determine the best wavelet that shows a segmentation with the largest area in the cell. We study different wavelet families and we conclude that the wavelet db1 is the best and it can serve for posterior works on blood pathologies. The proposed method generates goods results when it is applied on several images. Finally, the proposed algorithm made in MatLab environment is verified for a selected blood cells.

Fast and robust segmentation of white blood cell images by self-supervised learning.

PubMed

Zheng, Xin; Wang, Yong; Wang, Guoyou; Liu, Jianguo

2018-04-01

A fast and accurate white blood cell (WBC) segmentation remains a challenging task, as different WBCs vary significantly in color and shape due to cell type differences, staining technique variations and the adhesion between the WBC and red blood cells. In this paper, a self-supervised learning approach, consisting of unsupervised initial segmentation and supervised segmentation refinement, is presented. The first module extracts the overall foreground region from the cell image by K-means clustering, and then generates a coarse WBC region by touching-cell splitting based on concavity analysis. The second module further uses the coarse segmentation result of the first module as automatic labels to actively train a support vector machine (SVM) classifier. Then, the trained SVM classifier is further used to classify each pixel of the image and achieve a more accurate segmentation result. To improve its segmentation accuracy, median color features representing the topological structure and a new weak edge enhancement operator (WEEO) handling fuzzy boundary are introduced. To further reduce its time cost, an efficient cluster sampling strategy is also proposed. We tested the proposed approach with two blood cell image datasets obtained under various imaging and staining conditions. The experiment results show that our approach has a superior performance of accuracy and time cost on both datasets. Copyright © 2018 Elsevier Ltd. All rights reserved.

The DNA of ciliated protozoa.

PubMed Central

Prescott, D M

1994-01-01

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons. Images PMID:8078435

(abstract) Effects of Radiation and Oxidative Stress on Development and Morphology of Intestinal Cells

NASA Technical Reports Server (NTRS)

Honda, Shuji; Nelson, Gregory; Schubert, Wayne

1993-01-01

Intestinal cells when subjected to oxidative stress or radiation exhibit abnormal nuclear divisions observed as: 1) supernumerary cell divisions in anterior intestinal cells or 2) incomplete nuclear division and the persistence of anaphase bridges between daughter nuclei. Two oxygen sensitive mutants, mev-1 and rad-8 were observed to exhibit spontaneous supernumerary nuclear divisions at low frequency. N2 can be induced to undergo these divisions by treatment with the superoxide dismutase (SOD) inhibitor diethyl dithicarbamate or with the free radical generator methyl viologen. By contrast, the free radical generator bleomycin produces anaphase bridges in N2 intestinal nuclei at high frequency. Intestinal anaphase bridges can be induced by ionizing radiation and their formation is dependent on dose and radiation type.

Dynamics of growth zone patterning in the milkweed bug Oncopeltus fasciatus

PubMed Central

Weiss, Aryeh; Williams, Terri A.; Nagy, Lisa M.

2017-01-01

We describe the dynamic process of abdominal segment generation in the milkweed bug Oncopeltus fasciatus. We present detailed morphological measurements of the growing germband throughout segmentation. Our data are complemented by cell division profiles and expression patterns of key genes, including invected and even-skipped as markers for different stages of segment formation. We describe morphological and mechanistic changes in the growth zone and in nascent segments during the generation of individual segments and throughout segmentation, and examine the relative contribution of newly formed versus existing tissue to segment formation. Although abdominal segment addition is primarily generated through the rearrangement of a pool of undifferentiated cells, there is nonetheless proliferation in the posterior. By correlating proliferation with gene expression in the growth zone, we propose a model for growth zone dynamics during segmentation in which the growth zone is functionally subdivided into two distinct regions: a posterior region devoted to a slow rate of growth among undifferentiated cells, and an anterior region in which segmental differentiation is initiated and proliferation inhibited. PMID:28432218

Classification of Normal and Apoptotic Cells from Fluorescence Microscopy Images Using Generalized Polynomial Chaos and Level Set Function.

PubMed

Du, Yuncheng; Budman, Hector M; Duever, Thomas A

2016-06-01

Accurate automated quantitative analysis of living cells based on fluorescence microscopy images can be very useful for fast evaluation of experimental outcomes and cell culture protocols. In this work, an algorithm is developed for fast differentiation of normal and apoptotic viable Chinese hamster ovary (CHO) cells. For effective segmentation of cell images, a stochastic segmentation algorithm is developed by combining a generalized polynomial chaos expansion with a level set function-based segmentation algorithm. This approach provides a probabilistic description of the segmented cellular regions along the boundary, from which it is possible to calculate morphological changes related to apoptosis, i.e., the curvature and length of a cell's boundary. These features are then used as inputs to a support vector machine (SVM) classifier that is trained to distinguish between normal and apoptotic viable states of CHO cell images. The use of morphological features obtained from the stochastic level set segmentation of cell images in combination with the trained SVM classifier is more efficient in terms of differentiation accuracy as compared with the original deterministic level set method.

In situ optical microscopy of the martensitic phase transformation of lithium

NASA Astrophysics Data System (ADS)

Krystian, M.; Pichl, W.

2000-12-01

The phase transformation of lithium was investigated by in situ optical microscopy in a helium cryostat. The martensite microstructure is composed of irregular segments which grow in rapid bursts from many nuclei to a final size of 10 to 20 μm and then are immobilized. A major part of the segments is arranged in groups of parallel lamellas. A theoretical consideration of lattice compatibility predicts the existence of an almost perfectly coherent habit-plane interface between bcc and 9R in lithium. Therefore, the irregular microstructure is interpreted by the presence of the disordered polytype phase. Comparison with an earlier investigation in comparably impure lithium indicates a strong influence of impurities on the transformation mechanism. The connections between the low-temperature phase diagram, the geometrical compatibility condition, and the martensite microstructure are discussed.

Nuclei of plants as a sink for flavanols.

PubMed

Feucht, W; Polster, J

2001-01-01

Onion cepa (L.) and Tsuga canadensis (L.) Carr. were investigated histochemically on the association of flavanols to nuclei. The young roots of Onion cepa are totally devoid of flavanol structures. Therefore, the excised roots tips were directly incubated into different solutions of flavanols. After 3 h of incubation a flavanol binding on the nuclei was recognizable, as seen by a yellowish-brown tanning reaction. Still to ensure the presence of flavanols on the nuclei, subsequent staining with the p-dimethylaminocinnamaldehyde reagent (DMACA) resulted in an intense blue colouration. Tsuga canadensis has significant amounts of vacuolar flavanol deposits in all parts of the tree as indicated by the DMACA reagent. It is obvious that also the nuclei were associated strongly with flavanols which can be demonstrated particularly elegant in the cells of the seed wings by histochemical methods. However, the mode of flavanol release from the original deposits is not yet clear.

The major nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common epitopes also present in nuclear lamins

NASA Technical Reports Server (NTRS)

Tong, C. G.; Dauwalder, M.; Clawson, G. A.; Hatem, C. L.; Roux, S. J.

1993-01-01

The major nucleoside triphosphatase (NTPase) activities in mammalian and pea (Pisum sativum L.) nuclei are associated with enzymes that are very similar both biochemically and immunochemically. The major NTPase from rat liver nuclei appears to be a 46-kD enzyme that represents the N-terminal portion of lamins A and C, two lamina proteins that apparently arise from the same gene by alternate splicing. Monoclonal antibody (MAb) G2, raised to human lamin C, both immunoprecipitates the major (47 kD) NTPase in pea nuclei and recognizes it in western blot analyses. A polyclonal antibody preparation raised to the 47-kD pea NTPase (pc480) reacts with the same lamin bands that are recognized by MAb G2 in mammalian nuclei. The pc480 antibodies also bind to the same lamin-like bands in pea nuclear envelope-matrix preparations that are recognized by G2 and three other MAbs known to bind to mammalian lamins. In immunofluorescence assays, pc480 and anti-lamin antibodies stain both cytoplasmic and nuclear antigens in plant cells, with slightly enhanced staining along the periphery of the nuclei. These results indicate that the pea and rat liver NTPases are structurally similar and that, in pea nuclei as in rat liver nuclei, the major NTPase is probably derived from a lamin precursor by proteolysis.

A new level set model for cell image segmentation

NASA Astrophysics Data System (ADS)

Ma, Jing-Feng; Hou, Kai; Bao, Shang-Lian; Chen, Chun

2011-02-01

In this paper we first determine three phases of cell images: background, cytoplasm and nucleolus according to the general physical characteristics of cell images, and then develop a variational model, based on these characteristics, to segment nucleolus and cytoplasm from their relatively complicated backgrounds. In the meantime, the preprocessing obtained information of cell images using the OTSU algorithm is used to initialize the level set function in the model, which can speed up the segmentation and present satisfactory results in cell image processing.

Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina

PubMed Central

Lahne, Manuela; Li, Jingling; Marton, Rebecca M.

2015-01-01

Loss of retinal neurons in adult zebrafish (Danio rerio) induces a robust regenerative response mediated by the reentry of the resident Müller glia into the cell cycle. Upon initiating Müller glia proliferation, their nuclei migrate along the apicobasal axis of the retina in phase with the cell cycle in a process termed interkinetic nuclear migration (INM). We examined the mechanisms governing this cellular process and explored its function in regenerating the adult zebrafish retina. Live-cell imaging revealed that the majority of Müller glia nuclei migrated to the outer nuclear layer (ONL) to divide. These Müller glia formed prominent actin filaments at the rear of nuclei that had migrated to the ONL. Inhibiting actin filament formation or Rho-associated coiled-coil kinase (Rock) activity, which is necessary for phosphorylation of myosin light chain and actin myosin-mediated contraction, disrupted INM with increased numbers of mitotic nuclei remaining in the basal inner nuclear layer, the region where Müller glia typically reside. Double knockdown of Rho-associated coiled-coil kinase 2a (Rock2a) and Rho-associated coiled-coil kinase 2b (Rock2b) similarly disrupted INM and reduced Müller glial cell cycle reentry. In contrast, Rock inhibition immediately before the onset of INM did not affect Müller glia proliferation, but subsequently reduced neuronal progenitor cell proliferation due to early cell cycle exit. Long-term, Rock inhibition increased the generation of mislocalized ganglion/amacrine cells at the expense of rod and cone photoreceptors. In summary, INM is driven by an actin-myosin-mediated process controlled by Rock2a and Rock2b activity, which is required for sufficient proliferation and regeneration of photoreceptors after light damage. SIGNIFICANCE STATEMENT The human retina does not replace lost or damaged neurons, ultimately causing vision impairment. In contrast, zebrafish are capable of regenerating lost neurons. Understanding the mechanisms that regulate retinal regeneration in these organisms will help to elucidate approaches to stimulate a similar response in humans. In the damaged zebrafish retina, Müller glia dedifferentiate and proliferate to generate neuronal progenitor cells (NPCs) that differentiate into the lost neurons. We show that the nuclei of Müller glia and NPCs migrate apically and basally in phase with the cell cycle. This migration is facilitated by the actin cytoskeleton and Rho-associated coiled-coil kinases (Rocks). We demonstrate that Rock function is required for sufficient proliferation and the regeneration of photoreceptors, likely via regulating nuclear migration. PMID:26609156

Actin-Cytoskeleton- and Rock-Mediated INM Are Required for Photoreceptor Regeneration in the Adult Zebrafish Retina.

PubMed

Lahne, Manuela; Li, Jingling; Marton, Rebecca M; Hyde, David R

2015-11-25

Loss of retinal neurons in adult zebrafish (Danio rerio) induces a robust regenerative response mediated by the reentry of the resident Müller glia into the cell cycle. Upon initiating Müller glia proliferation, their nuclei migrate along the apicobasal axis of the retina in phase with the cell cycle in a process termed interkinetic nuclear migration (INM). We examined the mechanisms governing this cellular process and explored its function in regenerating the adult zebrafish retina. Live-cell imaging revealed that the majority of Müller glia nuclei migrated to the outer nuclear layer (ONL) to divide. These Müller glia formed prominent actin filaments at the rear of nuclei that had migrated to the ONL. Inhibiting actin filament formation or Rho-associated coiled-coil kinase (Rock) activity, which is necessary for phosphorylation of myosin light chain and actin myosin-mediated contraction, disrupted INM with increased numbers of mitotic nuclei remaining in the basal inner nuclear layer, the region where Müller glia typically reside. Double knockdown of Rho-associated coiled-coil kinase 2a (Rock2a) and Rho-associated coiled-coil kinase 2b (Rock2b) similarly disrupted INM and reduced Müller glial cell cycle reentry. In contrast, Rock inhibition immediately before the onset of INM did not affect Müller glia proliferation, but subsequently reduced neuronal progenitor cell proliferation due to early cell cycle exit. Long-term, Rock inhibition increased the generation of mislocalized ganglion/amacrine cells at the expense of rod and cone photoreceptors. In summary, INM is driven by an actin-myosin-mediated process controlled by Rock2a and Rock2b activity, which is required for sufficient proliferation and regeneration of photoreceptors after light damage. The human retina does not replace lost or damaged neurons, ultimately causing vision impairment. In contrast, zebrafish are capable of regenerating lost neurons. Understanding the mechanisms that regulate retinal regeneration in these organisms will help to elucidate approaches to stimulate a similar response in humans. In the damaged zebrafish retina, Müller glia dedifferentiate and proliferate to generate neuronal progenitor cells (NPCs) that differentiate into the lost neurons. We show that the nuclei of Müller glia and NPCs migrate apically and basally in phase with the cell cycle. This migration is facilitated by the actin cytoskeleton and Rho-associated coiled-coil kinases (Rocks). We demonstrate that Rock function is required for sufficient proliferation and the regeneration of photoreceptors, likely via regulating nuclear migration. Copyright © 2015 the authors 0270-6474/15/3515612-23$15.00/0.

Investigating the spectral characteristics of backscattering from heterogeneous spheroidal nuclei using broadband finite-difference time-domain simulations

NASA Astrophysics Data System (ADS)

Chao, Guo-Shan; Sung, Kung-Bin

2010-02-01

Backscattered light spectra have been used to extract size distribution of cell nuclei in epithelial tissues for noninvasive detection of precancerous lesions. In existing experimental studies, size estimation is achieved by assuming nuclei as homogeneous spheres or spheroids and fitting the measured data with models based on Mie theory. However, the validity of simplifying nuclei as homogeneous spheres has not been thoroughly examined. In this study, we investigate the spectral characteristics of backscattering from models of spheroidal nuclei under plane wave illumination using three-dimensional finite-difference time-domain (FDTD) simulation. A modulated Gaussian pulse is used to obtain wavelength dependent scattering intensity with a single FDTD run. The simulated model of nuclei consists of a nucleolus and randomly distributed chromatin condensation in homogeneous cytoplasm and nucleoplasm. The results show that backscattering spectra from spheroidal nuclei have similar oscillating patterns to those from homogeneous spheres with the diameter equal to the projective length of the spheroidal nucleus along the propagation direction. The strength of backscattering is enhanced in heterogeneous spheroids as compared to homogeneous spheroids. The degree of which backscattering spectra of heterogeneous nuclei deviate from Mie theory is highly dependent on the distribution of chromatin/nucleolus but not sensitive to nucleolar size, refractive index fluctuation or chromatin density.