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Expression of the cell cycle control gene, cdc25, is constitutive in the segmental founder cells but is cell-cycle-regulated in the micromeres of leech embryos.

The identifiable cells of leech embryos exhibit characteristic differences in the timing of cell division. To elucidate the mechanisms underlying these cell-specific differences in cell cycle timing, the leech cdc25 gene was isolated because Cdc25 phosphatase regulates the asynchronous cell divisions of postblastoderm Drosophila embryos. Examination of the distribution of cdc25 RNA and the zygotic expression of cdc25 in identified cells of leech embryos revealed lineage-dependent mechanisms of regulation. The early blastomeres, macromeres and teloblasts have steady levels of maternal cdc25 RNA throughout their cell cycles. The levels of cdc25 RNA remain constant throughout the cell cycles of the segmental founder cells, but the majority of these transcripts are zygotically produced. Cdc25 RNA levels fluctuate during the cell cycles of the micromeres. The levels peak during early G2, due to a burst of zygotic transcription, and then decline as the cell cycles progress. These data suggest that cells of different lineages employ different strategies of cell cycle control.

Amino Acid Sequence↗

Secondary motoneuron axons localize DM-GRASP on their fasciculated segments.

Cell surface adhesion molecules are thought to play a necessary role in axon guidance and fasciculation in the developing nervous system. We have studied a potential adhesion molecule using the zn-5 monoclonal antibody, which recognizes the surfaces of zebrafish spinal motoneurons. We show that zn-5 recognizes zebrafish DM-GRASP. DM-GRASP is a cell adhesion molecule of the immunoglobulin superfamily that mediates homophilic adhesion and neurite outgrowth in vitro. It is necessary for correct axon routing and fasciculation in the Drosophila visual system. In zebrafish, primary motoneurons pioneer the peripheral motor nerve pathways, and the axons of secondary motoneurons follow the routes established by the primary motoneuron axons. We show that, of the two classes of zebrafish spinal motoneurons, only the later growing secondary motoneurons express DM-GRASP. The secondary motoneurons restrict DM-GRASP protein to their cell bodies and fasciculated segments of their axons. Expression of DM-GRASP is transient: The protein is present during the period of axonal growth and disappears after axons have reached their muscle targets. Thus, homophilic adhesion mediated by DM-GRASP may play a role in fasciculation of secondary motoneuron axons but not in pathfinding by the pioneer axons of the primary motoneurons or in guidance of secondary motoneuron axons to their targets.

Activated-Leukocyte Cell Adhesion Molecule↗

Progesterone represses interleukin-8 and cyclo-oxygenase-2 in human lower segment fibroblast cells and amnion epithelial cells.

Labor is preceded by cervical ripening through upregulation of interleukin (IL)-1beta, IL-8, and increased prostaglandin synthesis via inducible type 2 cyclooxygenase (COX-2). Progesterone maintains myometrial quiescence during pregnancy. In this study, we examined the effects of IL-1beta and progesterone on IL-8 and prostaglandin E2 (PGE2) synthesis and IL-8 and COX-2 mRNA and promoter activity in amnion cells and lower segment fibroblast (LSF) cells. In both cell types, progesterone had no effect on basal IL-8 or PGE2 synthesis. In LSF cells, IL-1beta significantly increased IL-8 and PGE2 synthesis and COX-2 and IL-8 mRNA expression, but progesterone significantly attenuated these effects. In prelabor amnion cells, IL-1beta also increased IL-8 and PGE2 synthesis and both COX-2 and IL-8 mRNA and promoter expression; however, progesterone significantly attenuated these effects on IL-8 and PGE2 synthesis and COX-2 expression. In postlabor amnion cells, IL-1beta increased IL-8 and PGE2 synthesis and COX-2 expression, but progesterone did not attenuate the effect of IL-1beta upon IL-8 synthesis. Progesterone repression of IL-8 and COX-2 in LSF cells suggests that IL-8 and COX-2 have similar regulatory mechanisms in LSF cells and that progesterone may play a role in maintenance of cervical competence. The lack of effect of progesterone on IL-8 in postlabor cells may be the result of downregulation of the progesterone receptor during labor.

Amnion↗

Variability in the terminations of GABAergic chandelier cell axons on initial segments of pyramidal cell axons in the monkey sensory-motor cortex.

Chandelier cell axons were studied in the sensory-motor cortex of adult monkeys. The axonal fields of Golgi-impregnated chandelier cells in layer II in motor cortex are flattened sagittally. The vertical terminal portions of the axons varied both in length and in the numbers converging to form terminations of greater or lesser complexity. Golgi-impregnated plexuses were embedded in plastic and resectioned serially at 2.5-3.0 micrograms. A single axonal field could have as many as 400 terminal rows. All lie 3-13 micrograms beneath pyramidal cell somata. These terminations are not randomly distributed but instead, form clusters. Further resectioning the plastic sections for electron microscopy revealed that all the terminations are on the initial axon segments of pyramidal cells and all form symmetric synaptic contacts. In immunocytochemical material stained for glutamic acid decarboxylase (GAD), the enzyme involved in the synthesis of GABA, GAD-positive boutons were found to form symmetric synaptic contacts with a variety of postsynaptic elements including the axon hillocks and axon initial segments of pyramidal cells. Serial reconstructions from electron micrographs revealed GAD-positive terminals synapsing with the axon initial segment of pyramidal cells joined by cytoplasmic bridges and forming vertically oriented rows identical to those of chandelier cell terminals identified positively in the resectioned Golgi material. The GAD-positive terminals forming initial segment synapses were never continuous with GAD-positive terminals forming axo hillock synapses. The latter probably arise from basket cell axons. Initial segments of pyramidal cell axons in layers II and III were contacted by more GAD-positive terminals than the initial segments of pyramidal cell axons in layer V. The largest pyramidal cells in layer III received the most synapses. Many larger pyramidal cells, identified as callosally projecting cells by the retrograde transport of horseradish peroxidase (HRP), were shown in serial electron micrographs to possess large numbers of initial segment synapses, comparable to those seen in the immunocytochemical material. Serial reconstructions of pyramidal cell axons from axon hillock to the first myelin internode in resectioned Golgi, immunocytochemical and HRP material showed that the number of synapses varied from 2 to 52 for layers II and III and from 2 to 26 for layer V. The number of synapses on the axon hillocks varied from zero to 12. The variability in these terminations may be an important factor in the shaping of the functional properties of the pyramidal cells.

Animals↗

Cell image segmentation with kernel-based dynamic clustering and an ellipsoidal cell shape model.

In this paper, we propose a novel approach to cell image segmentation under severe noise conditions by combining kernel-based dynamic clustering and a genetic algorithm. Our method incorporates a priori knowledge about cell shape. That is, an elliptical cell contour model is introduced to describe the boundary of the cell. Our method consists of the following components: (1) obtain the gradient image; (2) use the gradient image to obtain points which possibly belong to cell boundaries; (3) adjust the parameters of the elliptical cell boundary model to match the cell contour using a genetic algorithm. The method is tested on images of noisy human thyroid and small intestine cells.

Algorithms↗

Segmentation of cell nuclei in tissue section analysis.

Image segmentation is a critical step in digital picture analysis, especially for that of tissue sections. As the morphology of the cell nuclei provides important biological information, their segmentation is of particular interest. The known segmentation methods are not adequate for segmenting cell nuclei of tissue sections; the reason for this lies in the optical properties of their images. We have developed new gradient methods of segmentation of previously presegmented images by taking these properties into account and by using the approximately circular shape of the cell nuclei as a priori information. In our first technique, the segment method, the images of the nuclei are divided into eight segments, special gradient filters being defined for each segment. This has enabled us to improve the gradient image. After searching for local maxima, the contours of nuclei can be found. In the second method, the method of transformation into the polar coordinate system (PCS), the a priori information serves to define a circular direction field for gradient computation and contour finding. In contrast with the first method, which offers a rapid, general idea about the nuclear shape, the PCS method permits precise segmentation and morphological analysis of the cell nuclei.

Animals↗

Structural characteristics of developing Nitella internodal cell walls.

The Nilella intermodal cell is formed by a division of the segment cell, the latter being a direct derivative of the shoot apical cell. The internodal cell is remarkable in that it elongates from an initial length of about 20 microns to a mature length of about 60 millimeters. The structures of the apical and segment cells, and the internodal cells in all stages of development were examined with the techniques of interference, polarization, and electron microscopy. The apical and segment cells were found to be isotropic. The upper part of the segment cell, destined to form a node, shows a curious pitted structure that was characteristic of certain node structures. The lower part of the segment cell, destined to become an internodal cell, shows a vague transverse arrangement of fibrils at the inner wall surface. The internodal cells, from the time they are first formed, show negative birefringence and a transverse arrangement of microfibrils at the inner wall surface. The elongation of the internodal cell is characterized by a rise, dip, and rise in both the optical thickness and retardation of the cell wall. The dip in both these variables coincides with the attainment of the maximum relative elongation rate. After the cessation of elongation, wall deposition continues, but the fibrils at .the inner surface of the wall are now seen to occur in fields of nearly parallel microfibrils. These fields, with varying fibrillar directions, may partly overlap each other or may merge with one another. Unlike the growing wall, this wall which is deposited after the end of elongation is isotropic.

Cell Differentiation↗

Bone marrow cell scene segmentation by computer-aided color cytophotometry.

Computer scene segmentation of touching cell images in bone marrow, on the basis of color information, is achieved using digitized scans at three different wavelengths of light. With trivariate histograms and Euler's coordinate transformation, it is possible cytophotometrically to isolate, on the basis of chromatic differences, individual heterogeneous cells located in cell groups. The ability of the described computer methods to isolate correctly the touching cell images is determined by visual comparison of the cells as seen in the microscope and the computer-generated displays of the scanned and segmented scenes.

Bone Marrow Cells↗

Renewal of fatty acids in the membranes of visual cell outer segments.

The renewal of fatty acids in the visual cells and pigment epithelium of the frog retina was studied by autoradiographic analysis of animals injected with tritiated palmitic, stearic, or arachidonic acids. Most of the radioactive material could be extracted from the retina with chloroform-methanol, indicating that the fatty acids had been esterified in lipids. Analysis of the extracts, after injection of [(3)H]palmitic acid, revealed that the radioactivity was predominantly in phospholipid. Palmitic acid was initially concentrated in the pigment epithelium, particularly in oil droplets which are storage sites for vitamin A esterified with fatty acid. The cytoplasm, but not the nucleus of these cells, was also heavily labeled. Radioactive fatty acid was bound immediately to the visual cell outer segment membranes, including detached rod membranes which had been phagocytized by the pigment epithelium. This is believed to be due to fatty acid exchange in phospholipid molecules already situated in the membranes. Gradually, the concentration of radioactive material in the visual cell outer segment membranes increased, apparently as a result of the addition of new phospholipid molecules, possibly augmented by the transfer from the pigment epithelium of esterified vitamin A. Injected fatty acid became particularly concentrated in new membranes which are continually assembled at the base of rod outer segments. This localized concentration was short-lived, apparently due to the rapid renewal of fatty acid. The results support the conclusion that rods renew the lipids of their outer segments by membrane replacement, whereas both rods and cones renew the membrane lipids by molecular replacement, including fatty acid exchange and replacement of phospholipid molecules in existing membranes.

Animals↗

The effect of ulinastatin, a human protease inhibitor, on the transfusion-induced increase of plasma polymorphonuclear granulocyte elastase.

Ulinastatin (UTI), a human protease inhibitor, inhibits polymorphonuclear granulocyte elastase (PMNE) release from granulocytes stimulated by several pathologic inflammatory processes. Blood transfusions increase plasma PMNE concentrations. We evaluated the effects of UTI on the transfusion-induced increase in PMNE. Thirty-nine patients undergoing gastrectomy were divided into four groups: Group A (n = 8), blood transfusion (-), UTI (-); Group B (n = 9), blood transfusion (+), UTI(-); Group C (n = 12), blood transfusion (-), UTI(+); and Group D (n = 10), blood transfusion (+), UTI(+). UTI of 300,000 U was infused in Groups C and D. White blood cell count (WBC), segmented cell count, plasma PMNE concentration, and PMNE release (PMNE concentration/segmented cell) were measured before, at the end of, and at 1 and 3 days after the operation. Although no significant differences were seen in WBC and segmented cell count among the groups, plasma PMNE concentration and PMNE release in Group B (transfusion without UTI) were significantly more than in Group A (no treatment) and Group D (transfusion with UTI) at the end of the operation, but the concentrations of the variables were not significantly different between Groups A and C (only UTI treatment). These results indicate that a single injection of 300,000 U of UTI inhibited transfusion-induced PMNE increase at the end of the operation. Thus, the present results may facilitate future studies testing the effects of UTI given at larger doses, or by a continuous administration, on the prevention of organ derangement after blood transfusion.

Adult↗

Efficient, interactive, and three-dimensional segmentation of cell nuclei in thick tissue sections.

Segmentation of intact cell nuclei in three-dimensional (3D) images of thick tissue sections is an important basic capability necessary for many biological research studies. Because automatic algorithms do not correctly segment all nuclei in tissue sections, interactive algorithms may be preferable for some applications. Existing interactive segmentation algorithms require the analyst to draw a border around the nucleus under consideration in all successive two-dimensional (2D) planes of the 3D image. The present paper describes an algorithm with two main advantages over the existing method. First, the analyst draws borders only in 2D planes that cut approximately through the center of the nucleus under consideration so that the nuclear borders generally are most distinct. Second, the analyst draws only five borders around each nucleus, and then the algorithm interpolates the entire surface. The algorithm results in segmented objects that correspond to individual, visually identifiable nuclei. The segmented surfaces, however, may not exactly represent the true nuclear surface. An optional, automatic surface optimization algorithm can be applied to reduce this error.

Algorithms↗

Supervised learning-based cell image segmentation for p53 immunohistochemistry.

In this paper, we present two new algorithms for cell image segmentation. First, we demonstrate that pixel classification-based color image segmentation in color space is equivalent to performing segmentation on grayscale image through thresholding. Based on this result, we develop a supervised learning-based two-step procedure for color cell image segmentation, where color image is first mapped to grayscale via a transform learned through supervised learning, thresholding is then performed on the grayscale image to segment objects out of background. Experimental results show that the supervised learning-based two-step procedure achieved a boundary disagreement (mean absolute distance) of 0.85 while the disagreement produced by the pixel classification-based color image segmentation method is 3.59. Second, we develop a new marker detection algorithm for watershed-based separation of overlapping or touching cells. The merit of the new algorithm is that it employs both photometric and shape information and combines the two naturally in the framework of pattern classification to provide more reliable markers. Extensive experiments show that the new marker detection algorithm achieved 0.4% and 0.2% over-segmentation and under-segmentation, respectively, while reconstruction-based method produced 4.4% and 1.1% over-segmentation and under-segmentation, respectively.

Algorithms↗

Acetazolamide inhibition of basolateral Cl-/HCO3- exchange in rabbit renal proximal tubule S3 segment.

Cell pH (pH(i)) and cell membrane potential (Vb) were measured in isolated S3 segments of rabbit renal proximal tubule with double-barrelled microelectrodes to search for a possible effect of the carbonic anhydrase inhibitor, acetazolamide (ACZ), on Cl-/HCO3- exchange in the basolateral cell membrane. ACZ was found to retard and reduce the pH(i) response to bath Cl- removal reversibly with half-maximal inhibition at 0.42 mmol/l and a rather flat concentration dependence (Hill coefficient approximately 0.36). To determine whether the retardation resulted from inhibition of cytoplasmic carbonic anhydrase, which might have delayed the attainment of HCO3-/CO2 equilibrium, we have measured the response of pH(i) to step changes in PCO2 in the presence and absence of ACZ. ACZ greatly retarded the pH(i) response to CO2 steps; however, the concentration dependence differed (half-maximal inhibition at 18 mumol/l) and even at maximal ACZ concentrations the response to CO2 steps was more than twice as fast as the response to Cl- replacement. Since, in addition, the ACZ inhibition of Cl-/HCO3- exchange could not be overcome by increasing PCO2 we conclude that the ACZ effect on Cl-/HCO3- exchange in rabbit proximal tubule S3 segments does not result from inhibition of cytosolic or membrane-bound carbonic anhydrase, but from a direct interaction with the exchanger molecule.

Acetazolamide↗

The renewal of photoreceptor cell outer segments.

The utilization of methionine-(3)H by retinal photoreceptor cells has been studied by radioautographic technique in the rat, mouse, and frog. In all three species, the labeled amino acid is concentrated initially in the inner segment of the cell. Within 24 hr, the radioactive material is displaced to the base of the outer segment, where it accumulates as a distinct reaction band. The reaction band then gradually moves along the outer segment and ultimately disappears at the apex of the cell, which is in contact with the retinal pigment epithelium. These findings are interpreted to indicate that the photoreceptor cell outer segment is continually renewed, by the repeated lamellar apposition of material (membranous discs) at the base of the outer segment, in conjunction with a balanced removal of material at its apex. The outer segment renewal rate is accelerated in frogs when ambient temperature is raised, and is elevated in both frogs and rats when the intensity of retinal illumination is increased.

Animals↗

Iterative thresholding for segmentation of cells from noisy images.

We introduce an iterative thresholding algorithm for the segmentation of cells from noisy cell images. The thresholding image, which is initially a constant, changes iteratively with both the previous segmentation and image local activity. Experimental results for both synthesized and real cell images are provided to demonstrate the performance of the algorithm.

Algorithms↗

Granule cell raphes in the developing mouse cerebellum.

The cerebellar cortex of many vertebrates shows a striking parasagittal compartmentation that is thought to play a role in the establishment and maintenance of functional cerebellar connectivity. Here, we demonstrate the existence of multiple parasagittal raphes of cells in the molecular layer of the developing cerebellar cortex of postnatal mouse. The histological appearance and immunostaining profile of the raphe cells suggest that they are migrating granule cells. We therefore conclude that the granule cell raphes previously described in birds also exist in a mammalian species. The raphes in mouse are visible on nuclear stains from around birth to postnatal day 6 and are frequently found at the boundaries of Purkinje cell segments that differentially express cadherins ("early-onset" parasagittal banding pattern). A similar relation between the raphe pattern and various markers for the early-onset banding pattern has been found in the chicken cerebellum. One of the cadherins mapped in the present study (OL-protocadherin) continues to be expressed in specific Purkinje cell segments until at least postnatal day 14. At this stage of development, the borders of the OL-protocadherin-positive Purkinje cell segments coincide with the borders of Purkinje cell segments that express zebrin II, a marker for the "late-onset" parasagittal banding pattern which persists in the adult cerebellum. These findings demonstrate that the early-onset banding pattern, as reflected in the complementary arrangement of raphes/Purkinje cell segments, and the late-onset pattern of zebrin II expression share at least some positional cues during development.

Animals↗

A parametric fitting algorithm for segmentation of cell images.

This paper presents a parametric fitting algorithm for segmentation of cervical and breast cell images from cytology smears. A parametric elliptical model for cells is introduced and the parameters adjusted to fit the cell shapes while minimizing a cost function. Segmentation results of noisy human cervical cell and textured breast cell images demonstrate that the proposed parametric fitting algorithm is very successful in segmentation of images of both nonoverlapped and overlapped elliptically shaped cells.

Algorithms↗