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Automated acquisition and processing of multidimensional image data in confocal in vivo microscopy.

The successful development of visualization techniques for live cell imaging leads to the development of suitable software for the acquisition and processing of multidimensional image data. This report compares several possible approaches to image acquisition and processing in confocal in vivo microscopy and suggests new alternatives to the published methods. Special attention is paid to spinning disk systems based either on a classical Nipkow disk or on the microlens principle. This study shows how to optimize image acquisition process in live cell studies using camera binning feature and how to perform object tracking using a new fast image registration method based on the graph theory.

Algorithms↗

Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells.

One of the objectives of studying endothelial cells in vitro is to evaluate neutrophil-endothelial cell interactions including potential consequences of oxidant-mediated damage to the endothelial cell. Current understanding of endothelial cell oxidative function is derived primarily from the measurement of extracellular products. We utilized 2 dyes, 2',7'-dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE), which measure hydrogen peroxide (H2O2) and superoxide anion (O2-) respectively, for their suitability to monitor oxidative mechanisms in endothelial cells and to provide a reliable measure of intracellular oxidants. Endothelial cells stained with DCFH-DA and stimulated with H2O2 exhibited an increase in the fluorescent product 2',7'-dichlorofluorescein (DCF) (measure of intracellular H2O2) which peaked at 10 min. Endothelial cells stained with HE and stimulated with H2O2 exhibited an increase in the fluorescent product ethidium bromide (EB) (measure of intracellular O2-) which lasted for approximately 60 min. Superoxide dismutase increased DCF fluorescence in endothelial cells stimulated with H2O2 by 158%. Allopurinol (xanthine oxidase inhibitor) reduced DCF and EB fluorescence by 48% and 37% respectively in endothelial cells stimulated with H2O2. Catalase completely inhibited an increase in DCF or EB fluorescence in endothelial cells stimulated with H2O2. There was a direct correlation between mean DCF and EB fluorescence intensity and the concentration of H2O2 or the number of phorbol 12-myristate 13-acetate-activated neutrophils added to endothelial cells. We conclude from these studies that DCFH-DA and HE can be used to measure intracellular H2O2 and O2- in endothelial cells and that the xanthine oxidase pathway for intracellular O2- production accounts for approximately 40% of the total intracellular O2- generated in endothelial cells after stimulation with H2O2. The combination of image cytometry and flow cytometry will be important for future evaluations of endothelial cell function.

Allopurinol↗

Ploidy differences between hormone- and chemical carcinogen-induced rat mammary neoplasms: comparison to invasive human ductal breast cancer.

To ascertain differences between solely hormone- and chemical carcinogen-induced murine mammary gland tumors (MGTs), a direct comparison of their ploidy status was assessed. Nuclear image cytometry (NIC) was used to evaluate ploidy in ductal carcinoma in situ (DCIS) and MGTs induced solely by 17beta-estradiol (E(2)) in female A-strain Copenhagen Irish hooded gene rats (ACI) and E(2) plus testosterone propionate in male Noble rats. These results were compared to ploidy data from primary MGTs induced by two synthetic carcinogens, 7,12-dimethylbenz[a]antracene and nitrosomethylurea in female Brown Lewis Norway rats and an environmental carcinogen, 6-nitrochrysene, in female Sprague-Dawley rats. Both DCIS and primary MGTs induced solely by hormones were highly aneuploid (> 84%), whereas MGTs induced by either synthetic or environmental carcinogens were primarily diploid (> 85%). Examination of 76 metaphase plates obtained from eight individual E(2)-induced ACI female rat MGTs revealed the following consistent chromosome alterations: gains in chromosomes 7, 11, 12, 13, 19, and 20 and loss of chromosome 12. On Southern blot analysis, six of nine ACI female rat primary E(2)-induced MGTs (66%) exhibited amplified copy numbers (range: 3.4-6.9 copies) of the c-myc gene. Fluorescence in situ hybridization (FISH) analysis of these MGTs revealed specific fluorescent hybridization signals for c-myc (7q33) on all three homologs of a trisomy in chromosome 7. NIC analysis of 140 successive nonfamilial sporadic invasive human ductal breast cancers (BCs) showed an aneuploid frequency of 61%, while 31 DCISs revealed a 71% aneuploid frequency. These results clearly demonstrate that the female ACI rat E(2)-induced MGTs more closely resemble invasive human DCIS and ductal BC in two pertinent aspects: they are highly aneuploid compared with chemical carcinogen-induced MGTs and exhibit a high frequency of c-myc amplification.

9,10-Dimethyl-1,2-benzanthracene↗

Intraabdominal desmoplastic small-cell tumor with divergent differentiation: clinicopathological findings and DNA ploidy.

Five cases of intraabdominal small-cell tumor with divergent differentiation are reported. All patients were of male sex. They were 10, 15, 20, 21, and 30 years of age at time of diagnosis, respectively. By light microscopy, the tumors consisted of small cells arranged in groups, nests, and clusters separated by a collagen-rich desmoplastic stroma. Immunohistochemical studies revealed the coexpression of mesenchymal, epithelial, and neural markers. Notably, all tumors coexpressed vimentin, cytokeratin, and desmin, the latter in a remarkable paranuclear dot-like fashion. In contrast to other authors, we did not find chromogranin. DNA image cytometry on four cases demonstrated two diploid and two aneuploid (hyperdiploid) cases. No correlation was found between ploidy and prognosis. One patient died from disease, another died from veno-occlusive disease after bone marrow transplantation, and the remaining patients are alive, but have progressive intraabdominal disease. Thus, our findings support the poor prognosis in this type of tumor.

Abdominal Neoplasms↗

Megakaryocyte polyploidy as a grouped geometric distribution obeying the log-normal population law.

Discrete nuclear lobe scores and flow- or image-cytometry DNA values of classically identified megakaryocytes behave as a grouped geometric distribution. This model is fully specified by a geometric mean and standard deviation (GM and GSD), the latter typically being ca 1.16 for volumes of diploid blood cell populations. Via log-normal probability paper, the 30 to 50 megakaryocytes in clinical marrow smears readily yield the ploidy model's GM and GSD which are named MPM and MPD for megakaryocyte polyploidy median and dispersion. In euthrombopoietic outbred mammals, MPMs are ca 12N ploidy units, and MPDs approximate a factor of 1.41. Both are unitless criteria. Thus, the thrombon is characterized by three populations exhibiting the high size dispersion which unmasks canonical operation of the log-normal population law: picoliter megakaryothrombocytes with their MPD ca 1.41, femtoliter thrombocytes with a volume GSD ca 1.74, and the end product of locally delivered pieces of subattoliter platelet dust with a volume GSD ca. 2.0.

Animals↗

Survival of F-reticulocytes in sickle cell disease.

Fetal hemoglobin, Hb F, is known to be an important factor for clinical course of sickle cell disease, as it suppresses polymerization of sickle hemoglobin. To investigate the effect of Hb F on the survival of sickle reticulocytes (young red cells) in circulation, Hb F levels in individual reticulocytes and mature erythrocytes were quantified via fluorescence image cytometry. We first examined unfractionated SS cells from 3 patients with different Hb F levels, and found that Hb F levels in reticulocyte populations were always lower than those in erythrocyte populations. This suggests that subsets of reticulocytes with lower Hb F levels are removed during maturation while those with higher Hb F levels tend to survive to become erythrocytes. The distribution of Hb F in reticulocytes was different among these patients and seems to strongly affect the survival of F-reticulocytes. We also analyzed density-separated fractions, and found that Hb F levels in reticulocytes found in the densest fraction were lower than those in lighter fractions. This suggests that reticulocytes with lower Hb F levels are susceptible to quick dehydration within their maturation period (1-2 days) in circulation.

Anemia, Sickle Cell↗

Estrogen increases the number of plasma cells and enhances their autoantibody production in nonautoimmune C57BL/6 mice.

The immunological consequences of chronic estrogen exposure in normal individuals are not known, particularly in relation to B cells. In this study, by employing ELIspot, image cytometry, flow cytometry, cytology, and ELISA, we show that long-term exposure of normal mice to estrogen activates B cells to produce higher numbers of not only immunoglobulin-producing cells, but also autoantibody-producing cells. Estrogen promoted a decrease in B220(+) splenic lymphocytes, but resulted in a 10-fold increase in plasma cells. Further, the output of immunoglobulins including autoantibodies from individual plasma cells from estrogen-exposed mice was markedly increased, suggesting B cell hyperactivity. Importantly, our findings show that treatment of normal mice, solely with estrogen, can override B cell tolerance and promote autoreactive B cells in normal individuals.

Animals↗

Interphase AgNOR quantity and DNA content in endometrial adenocarcinoma.

The relationship between the amount of silver-stained nucleolar organizer regions (AgNORs) and the DNA content of interphase neoplastic cells in 63 cases of human endometrial adenocarcinoma was studied. Interphase AgNOR content was determined as the mean AgNOR area per nucleus by image cytometry in silver-stained samples from paraffin-embedded sections. DNA content was evaluated by cytofluorometry on single cells from the same specimen and expressed in c units as DNA ploidy. The mean AgNOR area ranged from 1.26 to 4.48 microns2 and the modal DNA ploidy from 1.90 to 4.00c. No significant associations were found between mean AgNOR area and modal DNA ploidy when analyzed either by linear regression (beta = 0.18; P = 0.15) or as dichotomized variables by chi 2 analysis (chi 2 = 0.82; P = 0.37). On a homogeneous subset of 45 cases of stage I adenocarcinomas we also evaluated the prognostic relevance of AgNOR content and DNA ploidy. In monovariate analysis both parameters were significantly related to patient survival. When the mean AgNOR area and the modal DNA ploidy were entered into regression analysis together with the depth of myometrial invasion and histological grading, they showed an independent, high prognostic value.

Adenocarcinoma↗

Intrinsic optical signaling denoting neuronal damage in response to acute excitotoxic insult by domoic acid in the hippocampal slice.

Using the seafood contaminant domoic acid (an AMPA/kainate receptor agonist), we demonstrate a distinct excitotoxic sequence of events leading to acute neuronal damage in the hippocampal slice as measured by (1) loss of the evoked CA1 field potential, (2) irreversible changes in light transmittance, (3) histopathology, and (4) lucifer yellow injection of single CA1 pyramidal neurons. Change in light transmittance (LT) through the submerged slice indirectly measures altered cell volume, both neuronal and glial. At 37 degrees C, a 1-min superfusion of 10 mu M domoate induced a prolonged reversible increase in LT, primarily in the dendritic regions of CA1 and dentate granule cells (GC), but not in the CA3 region. Spectral analysis (400-800 nm) revealed a wide-band transmittance increase, indicating cell swelling as a major source of the intrinsic signal. The evoked field potential recorded in the CA1 cell body region (PYR) was lost as LT peaked, but completely recovered upon return to the baseline LT level. Increasing domoate exposure to 10 min elicited a different and distinct LT sequence in CA1 and dentate regions. An initial LT increase in dendritic regions evolved in an irreversible decrease in LT. At the same time, LT irreversibly increased in cell body regions (CA1 PYR and GC) and the evoked field potential was irretrievably lost. Also, there was histological damage to cell body and dendritic regions of CA1 and granule cells. Injection of lucifer yellow into single CA1 neurons in slices displaying the irreversible LT sequence revealed extensive dendritic beading, whereas CA1 cells in control slices displayed a smoothly contoured arbor. Consistent with acute neuronal damage, the optical changes generated by domoate did not require extracellular Ca2+, and lowering the temperature protected the slice from irreversible damage to CA1 and GC regions. Although glial changes may also occur, we conclude that imaging light transmittance reveals dynamic and compartmentalized excitotoxic changes in neuronal volume. Beading of the dendritic arbor increases light scatter, thereby decreasing LT and highlighting damaged dendritic regions.

Animals↗

Cell segmentation, tracking, and mitosis detection using temporal context.

The Large Scale Digital Cell Analysis System (LSDCAS) developed at the University of Iowa provides capabilities for extended-time live cell image acquisition. This paper presents a new approach to quantitative analysis of live cell image data. By using time as an extra dimension, level set methods are employed to determine cell trajectories from 2D + time data sets. When identifying the cell trajectories, cell cluster separation and mitotic cell detection steps are performed. Each of the trajectories corresponds to the motion pattern of an individual cell in the data set. At each time frame, number of cells, cell locations, cell borders, cell areas, and cell states are determined and recorded. The proposed method can help solving cell analysis problems of general importance including cell pedigree analysis and cell tracking. The developed method was tested on cancer cell image sequences and its performance compared with manually-defined ground truth. The similarity Kappa Index is 0.84 for segmentation area and the signed border positioning segmentation error is 1.6 +/- 2.1 microm.

Algorithms↗

Topological proteomics, toponomics, MELK-technology.

MELK is an ultrasensitive topological proteomics technology analysing proteins on the single cell level (Multi-Epitope-Ligand-'Kartographie'). It can trace out large scale protein patterns with subcellular resolution, mapping the topological position of many proteins simultaneously in a cell. Thereby, it addresses higher level order in a proteome, referred to as the toponome, coding cell functions by topologically and timely determined webs of interacting proteins. The resulting cellular protein maps provide new structures in the proteome: single combinatorial protein patterns (s-CPP), and combinatorial protein pattern motifs (CPP-motifs), bound to superior units. They are images of functional protein networks, which are specific signatures of tissues, cell types, cell states and diseases. The technology unravels hierarchies of proteins related to particular cell functions or dysfunctions, thus identifying and prioritising key proteins within cell and tissue protein networks. Interlocking MELK with the drug screening machinery provides new clues related to the selection of target proteins, and functionally relevant hits and drug leads. The present chapter summarizes the steps that have contributed to the establishment of the technology.

Humans↗

Vinblastine and 5-fluorouracil sensitivity of xenografts of four pancreatic ductal adenocarcinomas: is there a correlation with histological and cytological tumour differentiation?

In a search for nuclear parameters which may predict chemosensitivity of ductal adenocarcinoma of the pancreas, the growth of four xenografted pancreatic carcinomas in response to chemotherapeutic agents was correlated with histological and cytological features of tumour differentiation. Histologically, the tumours were classified according to their ability to form glands into poorly (PaTu-2, PaTu-3), moderately (Panc-1) and well differentiated (PaTu-39) ductal adenocarcinomas. Cytologically, similar segregation of tumours was possible using the 'nuclear form factor', which was one of four nuclear parameters analysed by image cytometry on Feulgen stained tumour imprints. Histological and cytological differentiation correlated closely with tumour growth. One week after a single intraperitoneal injection of either vinblastine or 5-fluorouracil, both drugs inhibited the growth of PaTu-2 and PaTu-3 significantly. The growth of Panc-1 was only affected by vinblastine, while neither drug had an effect on PaTu-39. The results suggest that the response of pancreatic ductal adenocarcinoma to chemotherapeutic drugs may be, to some extent, predicted by histological and cytological differentiation features. However, within these lines, each tumour may show a specific response pattern.

Animals↗

Determination of DNA ploidy and ABH antigen reactivity in both frozen and formalin-fixed bladder tumor tissue.

In a prospective series of patients with transitional-cell bladder carcinomas, DNA ploidy and ABH antigen reactivity were determined in both frozen tissue and formalin-fixed paraffin blocks. ABH antigen measurement in frozen tissue was hampered by methodological problems, especially insufficient morphology, which made quantification difficult. In only 38/55 (69%) cases was the same ABH reactivity found. The knowledge of secretor status was not helpful in the interpretation of these results. In 53/59 (90%) cases the same DNA ploidy was achieved, resulting in good correlation between flow cytometric DNA measurements in formalin-fixed and frozen tissue. The deviations occurred mainly in cases exhibiting a tetraploid DNA profile. DNA assessments using image cytometry on imprints of fresh tissue was a rapid and reliable method in our hands, with identical results being obtained in 32/36 (89%) cases.

ABO Blood-Group System↗

Automated image analysis DNA cytometry in testicular cancer.

The value of automated DNA cytometry for differentiation of testis cancer was evaluated in 54 seminomas, 13 HCG-positive seminomas, and 48 embryonal carcinomas. Slices of paraffin embedded tissue were enzymatically digested and stained with Feulgen SITS after fixation on glass slides. Automated DNA cytometry was performed with a Modular Image Analysis Computer (MIAC). DNA histogram phenotpye and computed DNA indices were correlated with the different tumor types. The ratio of hypertriploid to hypotriploid increased from HCG-positive seminoma over embryonal carcinoma to seminoma. The following mathematical DNA indices were found to correlate with tumor type: mean ploidy, 2c deviation index, 5c exceeding rate, variation coefficient of the GO/1 fraction and DNA nucleus diameter correlation.

Carcinoma, Embryonal↗

[DNA content of the tumor cell. A new prognostic parameter in hepatocellular carcinoma?].

Hepatocellular carcinoma is a heterogeneous disease with considerable differences in malignant behaviour. Some relevant factors for prognosis are known. In this study we analysed DNA ploidy as a potential prognostic parameter. With DNA image cytometry we were able to differentiate between diploid, hypotriploid, triploid, hypertriploid, tetraploid and aneuploid tumours. The best prognosis was for patients with diploid, hypotriploid and tetraploid tumours with a median survival time of 41 months in contrast to 3 months for patients with triploid, hypertriploid or aneuploid tumours. There was a strong correlation between histomorphological parameters and the DNA content. The DNA content of tumour cells may be considerable clinical relevance in hepatocellular carcinoma regarding the decision as to whether or not to perform a resection. In patients with prognostically unfavorable parameters adjuvant oncological therapy may improve the prognosis.

Adult↗

Nuclear morphometry and DNA densitometry of human gliomas by image analysis.

In 48 patients with gliomas in whom complete clinical follow-up was obtained, DNA ploidy was evaluated by using formalin-fixed paraffin-embedded tissues and by means of image analysis. The mean DNA indices, determined by averaging DNA indices of all tumor cells in a tumor, were mainly affected by mean DNA indices of the nuclei of SG2M phase tumor cell (including S phase and G2M phase cells) (SG2M DNA indices) and that mean DNA indices correlated with the SG2M phase fraction. The SG2M DNA indices and the percentage of tumor cells with S phase and G2M phase were higher in high grade gliomas including anaplastic glioma and glioblastoma multiforme than in low grade gliomas. Patients with G2M-hypertetraploid tumors demonstrated a shorter time to tumor progression than those with G2M-tetraploid in high grade glioma. Morphometrically, the nuclei of SG2M phase glioma cells were larger and more deformity than those of G0G1 phase (including G0 phase and G1 phase cells) cells. The G2M-hypertetraploid tumors were highly malignant and demonstrated large nuclei, greater nuclear deformity, and a higher proliferative potential. The G2M-tetraploid gliomas demonstrated a shorter time to tumor progression in cases whose the SG2M fraction was large. In contrast, G2M-hypotetraploid gliomas revealed an insignificant trend towards a longer time to tumor progression than those associated with tetraploid and hypertetraploid gliomas. We emphasize herein the prognostic importance of the SG2M phase cell, as well as other proliferation indices.

Adolescent↗

Methods in quantitative image analysis.

The main steps of image analysis are image capturing, image storage (compression), correcting imaging defects (e.g. non-uniform illumination, electronic-noise, glare effect), image enhancement, segmentation of objects in the image and image measurements. Digitisation is made by a camera. The most modern types include a frame-grabber, converting the analog-to-digital signal into digital (numerical) information. The numerical information consists of the grey values describing the brightness of every point within the image, named a pixel. The information is stored in bits. Eight bits are summarised in one byte. Therefore, grey values can have a value between 0 and 256 (2(8)). The human eye seems to be quite content with a display of 5-bit images (corresponding to 64 different grey values). In a digitised image, the pixel grey values can vary within regions that are uniform in the original scene: the image is noisy. The noise is mainly manifested in the background of the image. For an optimal discrimination between different objects or features in an image, uniformity of illumination in the whole image is required. These defects can be minimised by shading correction [subtraction of a background (white) image from the original image, pixel per pixel, or division of the original image by the background image]. The brightness of an image represented by its grey values can be analysed for every single pixel or for a group of pixels. The most frequently used pixel-based image descriptors are optical density, integrated optical density, the histogram of the grey values, mean grey value and entropy. The distribution of the grey values existing within an image is one of the most important characteristics of the image. However, the histogram gives no information about the texture of the image. The simplest way to improve the contrast of an image is to expand the brightness scale by spreading the histogram out to the full available range. Rules for transforming the grey value histogram of an existing image (input image) into a new grey value histogram (output image) are most quickly handled by a look-up table (LUT). The histogram of an image can be influenced by gain, offset and gamma of the camera. Gain defines the voltage range, offset defines the reference voltage and gamma the slope of the regression line between the light intensity and the voltage of the camera. A very important descriptor of neighbourhood relations in an image is the co-occurrence matrix. The distance between the pixels (original pixel and its neighbouring pixel) can influence the various parameters calculated from the co-occurrence matrix. The main goals of image enhancement are elimination of surface roughness in an image (smoothing), correction of defects (e.g. noise), extraction of edges, identification of points, strengthening texture elements and improving contrast. In enhancement, two types of operations can be distinguished: pixel-based (point operations) and neighbourhood-based (matrix operations). The most important pixel-based operations are linear stretching of grey values, application of pre-stored LUTs and histogram equalisation. The neighbourhood-based operations work with so-called filters. These are organising elements with an original or initial point in their centre. Filters can be used to accentuate or to suppress specific structures within the image. Filters can work either in the spatial or in the frequency domain. The method used for analysing alterations of grey value intensities in the frequency domain is the Hartley transform. Filter operations in the spatial domain can be based on averaging or ranking the grey values occurring in the organising element. The most important filters, which are usually applied, are the Gaussian filter and the Laplace filter (both averaging filters), and the median filter, the top hat filter and the range operator (all ranking filters). Segmentation of objects is traditionally based on threshold grey values. (AB

Algorithms↗

Flow and image cytometric study of pancreatic neuroendocrine tumours: frequent DNA aneuploidy and an association with the clinical outcome.

Eighteen pancreatic neuroendocrine (NE) tumours were analysed for nuclear DNA content by image cytometry (ICM) and flow cytometry (FCM). The DNA indices (DIs) obtained by ICM were somewhat higher than those obtained by FCM, but a major disagreement was present only in 1 case. Thirteen patients had been followed up at least for 6 years after the diagnosis or until death. At 6 years of follow-up all 4 patients with a tumour with a DI greater than or equal to 1.8 by ICM had died from their NE tumour or had metastatic disease, whereas all 9 patients with a smaller DI had no evidence of the disease (P = 0.001). The DIs calculated from the FCM data also correlated well with the final outcome (P = 0.01). A high incidence of DNA aneuploidy was found by both methods in histologically and clinically benign NE tumours; 12 (67%) were DNA aneuploid by FCM and 16 (89%) by ICM. It is concluded that pancreatic NE tumours are frequently DNA aneuploid, and both cytometric DNA methods give prognostic information in these tumours. The presence of DNA aneuploidy should not be considered as a sign of malignant behaviour in pancreatic NE tumours, whereas a large DI is associated with poor prognosis.

Adult↗