Quantitative description of nuclear morphology in assessing resistance of sarcoma 180 to adriamycin

Resistance to adriamycin generally is explained through changes of cell/drug interactions that possibly reflect structural alterations of intracellular targets. One of the main targets of adriamycin is believed to be nuclear chromatin. In order to recognize chromatin alterations, we studied cell nuclei morphology and chromatin structure by means of digital image analysis. The studies were performed in both adriamycin-sensitive and -resistant Sarcoma 180 cell lines which were cultured under growth-stimulated and nonstimulated conditions. Using specially developed methods, we extracted parameters characterizing geometrical, optical, and structural properties of the cell nuclei from light microscopical images. The latter parameters concerned microscopical appearances of condensed chromatin and were described by features of high-optical-density regions. The results demonstrated that the quantitative criteria applied enabled the discrimination of sensitive and resistant cells. The most important parameters are the nuclear size, number, distribution, and optical density of condensed chromatin regions. In addition, the criteria permit recognition of changes related to differences in the growth conditions of the cells. The data of the image analysis suggest that adriamycin resistance in Sarcoma 180 cells is associated with characteristic patterns of cell nuclear morphology which can be described with a sufficient number of appropriate parameters. The advantages of image analysis are evident when these results are compared with the flow cytometric findings. The conclusion is that structural features of nuclear chromatin provide information essential for the assessment of drug resistance.     

Correlation between chromatin morphology as derived by digital image analysis and autoradiographic labeling pattern

In order to interpret the Feulgen-dependent chromatin morphology on a functional basis, we performed model experiments in which labeling with 14C-thymidine and 14C-uridine was used as a functional parameter. Using a relocation facility, information on either DNA or RNA, labeling intensity of a cell was added to the parameters of image analysis by measuring the same cell by scanning photometry after Feulgen staining. The Feulgen-stained nuclei were interactively sampled and automatically segmented. Most of the textural information was gained from a flat texture image obtained by subtracting the original image from a median-filtered image. In addition to the autoradiographic features, visually recognizable differences in nuclear morphology, such as the number of nucleoli and the level of condensed (inactive) and diffuse (active) regions of the chromatin, were also correlated with textural parameters. Using the supervised cluster analysis method, an attempt was made to establish a correlation between visual nuclear morphology and autoradiographic labeling intensity that improved the functional understanding of the Feulgen features. Our results further clarify the supramolecular chromatin structure and its dynamics during specific transitions in the cell cycle, namely the G0-G1, G1-S and S-G2 transitions; this information may become useful in diagnostic procedures.     

Detecting infection-related changes in peripheral blood smears with image analysis techniques

High-resolution image analysis has the potential to flag subtle changes in white blood cell morphology that may indicate the presence of certain diseases. A study was made of the feasibility of identifying patients with hematologic bacterial infections (sepsis) using measurements on Wright-Giemsa-stained peripheral blood smears. Neutrophils and lymphocytes from a group of patients with sepsis and from a control group were digitized, and parameters quantifying geometry, color, texture and shape were extracted. While color parameters differed the most between the infected and control samples, substantial differences in geometric, texture and shape parameters also were observed. Analysis of the data showed that individual neutrophils and lymphocytes from patients with sepsis were distinguishable from those of the control group with better than 84% accuracy. When average parameters were calculated from all cells of one type for each specimen, 100% accurate classification was obtained. These studies demonstrate that the image-analysis techniques used are sensitive enough to detect disease-related changes in cell morphology that are generally too subtle for reliable detection by the human eye. Future experiments will determine the specificity of this test for bacterial infections and will explore the possibility of using image analysis techniques on peripheral blood to detect and monitor a wide variety of diseases.     

Regulation of plasminogen receptor expression on human monocytes and monocytoid cell lines

The capacity of human monocytoid cell lines and peripheral blood monocytes to modulate their expression of plasminogen receptors has been assessed. After PMA stimulation, THP-1 or U937 monocytoid cells were separated into adherent and nonadherent populations. Plasminogen bound to adherent cells with similar capacity and affinity as to nonstimulated cells. In contrast, the nonadherent cells bound plasminogen with 5-17-fold higher capacity (without a change in affinity). This increase was selective as urokinase bound with similar affinity and capacity to the adherent and nonadherent populations. Upregulation of plasminogen receptors on the nonadherent monocytoid cells was rapid, detectable within 30 min, and reversible, adhesion of the nonadherent cells resulted in a sixfold decrease in plasminogen binding within 90 min. The increase in plasminogen binding to the nonadherent cells was associated with a marked increase in their capacity to generate plasmin activity from cell-bound plasminogen. PMA stimulation of human peripheral blood monocytes increased their expression of plasminogen receptors by two- to fourfold. This increase was observed in both adherent and nonadherent monocytes. Freshly isolated monocytes maximally bound 5.0 x 10(5) plasminogen molecules per cell, whereas monocytes cultured for 18 h or more maximally bound 1.7 x 10(7) molecules per cell, a 30-fold difference in receptor number. These results indicate that both monocytes and monocytoid cell lines can rapidly and markedly regulate their expression of plasminogen binding sites. As enhanced plasminogen binding is correlated with an increased capacity to generate plasmin, an enzyme with broad substrate recognition, modulation of plasminogen receptors may have profound functional consequences.     

Morphometric analysis of the cell outlines of normal and polyomavirus-transformed FR 3T3 fibroblasts

A method is described for the analysis of cell shape, using an image analyzer connected to a computer to assess the cell outline. A series of parameters to assess the contribution of large cytoplasmic expansions to cell morphology and to cell spreading on a planar substratum were used to quantify the visual morphologic differences between normal (nontransformed; N.3T3) and polyomavirus-transformed (Py.3T3) Fisher rat 3T3 fibroblasts. The results show that the Py.3T3 fibroblasts are more spherical than are the N.3T3 fibroblasts and that the cytoplasmic expansions of the Py.3T3 fibroblasts are smaller than those of N.3T3, with the spreading of these two cell strains being different. These differences can be explained by the difference in cell-substratum affinity between these two cell strains.     

The use of multidimensional image-based analysis to accurately monitor cell growth in 3D bioreactor culture

The transition from traditional culture methods towards bioreactor based bioprocessing to produce cells in commercially viable quantities for cell therapy applications requires the development of robust methods to ensure the quality of the cells produced. Standard methods for measuring cell quality parameters such as viability provide only limited information making process monitoring and optimisation difficult. Here we describe a 3D image-based approach to develop cell distribution maps which can be used to simultaneously measure the number, confluency and morphology of cells attached to microcarriers in a stirred tank bioreactor. The accuracy of the cell distribution measurements is validated using in silico modelling of synthetic image datasets and is shown to have an accuracy >90%. Using the cell distribution mapping process and principal component analysis we show how cell growth can be quantitatively monitored over a 13 day bioreactor culture period and how changes to manufacture processes such as initial cell seeding density can significantly influence cell morphology and the rate at which cells are produced. Taken together, these results demonstrate how image-based analysis can be incorporated in cell quality control processes facilitating the transition towards bioreactor based manufacture for clinical grade cells.     

Quantitative image analysis of connective tissue progenitors

OBJECTIVE: To develop an image analysis system to automatically identify colony-forming units (CFUs) in in vitro cell cultures of connective tissue progenitors. This system was designed to quantitatively assess colony morphology and number of colonies in 4-cm(2) culture wells. STUDY DESIGN: Large field-of-view high-resolution fluorescence images of 4',6-diamidino-2-phenylindole (DAPI)- and alkaline phosphatase (AP)-stained bone marrow cell cultures were obtained using an epi-fluorescence microscope and automated scanning stage. Cell nuclei were identified in the DAPI-stained images after removal of fluorescent debris from the image. An Euclidean distance map (EDM) of the segmented cell nuclei was used to cluster cell nuclei into colonies. The automated system was evaluated using 40 tissue culture wells of bone marrow aspirate samples. The results of the automated analysis were compared to the manual tracings of colonies by 3 reviewers. RESULTS: The automated method agreed with all 3 reviewers on average 87.5% of the time. Additionally, reviewers identified other colonies not outlined by the reviewers on average 2.7 times more than the automated method. CONCLUSION: The automated method is a less biased method for identifying CFUs than individual reviewers, it provides more quantitative information about colony morphology than can be obtained manually and it is less time consuming.     

Increased light scattering resolution facilitates multidimensional flow cytometric analysis

Multidimensional flow cytometry identifies cell populations as clusters in a space created by the analysis of multiple parameters simultaneously. Optimal use of this multidimensional space requires each of the individual parameters to provide additional information for cell population discrimination as well as maximum utilization of the dynamic range available for each parameter. In this study we improve the visualization of the information present in light scattering signals from leukocytes to facilitate multidimensional flow cytometric analysis. Optimization of cell preparation techniques are essential to obtain high resolution light scattering signals that give complete separation of the granulocytes, monocytes, and granular and nongranular lymphocytes. The angle at which the forward scattered light was collected was modified to enhance the separation between leukocyte populations. Although orthogonal light scattering signals separate granular and nongranular lymphocytes, the resolution and dynamic range could not be displayed using linear or logarithmic functions. By applying a polynomial function to the orthogonal light scattering signals, all leukocyte populations could be displayed while maintaining high resolution. The combination of high resolution light scattering with a nonlinear display resulted in an equally spaced distribution of the cell populations distinguished by correlating forward and orthogonal light scattering signals. Using this approach, peripheral blood neutrophils, eosinophils, basophils, monocytes, and granular and nongranular lymphocytes were shown to occupy distinct locations in the correlation of orthogonal and forward light scattering. Surprisingly, the basophilic granulocytes were located close to granular lymphocytes and monocytes rather than near neutrophils and eosinophils.     

Regulation of human monocyte cell-surface and soluble CD23 (Fc epsilon RII) by granulocyte-macrophage colony-stimulating factor and IL-3.

We have investigated the regulation of expression of cell-surface and soluble CD23 (sCD23) by purified human peripheral blood monocytes and in cultures of human whole blood. IL-3, IL-4, and GM-CSF were found to markedly enhance the expression of CD23 on the surface of elutriated monocytes and to increase levels of sCD23 in monocyte-culture supernatants. The induction of CD23 expression by monocytes was confirmed at the mRNA level by Northern blot analysis. The ability of GM-CSF, IL-3, or IL-4 to induce cell-surface CD23 on monocytes was inhibited by specific neutralizing antibodies to the corresponding cytokine. IL-3 and GM-CSF induced maximal surface CD23 expression on monocytes by 24 to 48 h, followed by a slight decline at 72 and 96 h. In contrast, IL-4 induced a progressive increase in monocyte CD23 expression that reached a maximum at approximately 72 h. IL-4, GM-CSF, and IFN-gamma increased both surface and soluble CD23 expression by the monocytic cell line U937, whereas IL-3 had no effect. The plasma from fresh human whole blood or nonstimulated whole blood cultured for 24 to 48 h contained detectable sCD23, and addition of IL-3, IL-4, or GM-CSF to these cultures resulted in increased levels of this molecule. Two-color flow cytometry revealed that IL-3, but not GM-CSF, also enhanced CD23 expression by B cells enriched from PBMC, although the effect of IL-3 was weak in comparison with that of IL-4. These findings may have important implications for the in vivo therapeutic use of these cytokines.     

Rapid multiparameter analysis of cell stimulation in mixed lymphocyte culture reactions.

A flow-cytofluorometric method, based on the differential stability of deoxyribonucleic acid versus ribonucleic acid with the metachromatic dye, acridine orange, simultaneously measures the following parameters of stimulation in mixed lymphocyte cultures: (a) number of nonstimulated cells; (b) total number of stimulated lymphocytes; (c) number of stimulated lymphocytes in G1, S and G2 + M phases of the cell cycle; (d) number of macrophages; (e) number of dead cells. The progress of lymphocyte stimulation may also be measured by a parameter representing ribonucleic acid accumulation per cell. The method is rapid, avoids cell rinsing, fixation and centrifugation and is applicable to microcultures. Multiparameter analysis of cell stimulation which provides simultaneous measurements of lymphocyte proliferation and accumulation of ribonucleic acid per cell may prove to be a more sensitive assay of histocompatibility than tests based only on cell proliferation (tritiated thymidine incorporation).     

CD14 contributes to the adherence of human monocytes to cytokine-stimulated endothelial cells.

Monocyte adherence to endothelial cells (EC) is selectively increased during inflammation. The mechanisms underlying monocyte-EC interaction indicated the involvement of surface-adhesion molecules on monocytes and EC. In earlier studies we noticed that the monocyte-specific mAb, designated mAb 63D3, in contrast to mAb against the beta 2-integrin molecules, inhibited the monocyte binding to monolayers of rIL-1 alpha-stimulated venous EC. The aim of the present study was to further characterize the Ag recognized by mAb 63D3 and to investigate the specific contribution of this Ag to the adherence of monocytes to cultured human macrovascular venous or arterial EC. Flow cytometric analysis demonstrated that the 63D3 Ag is expressed exclusively on the surface of peripheral blood monocytes. SDS-PAGE analysis of mAb 63D3 immunoprecipitates of 125I-labeled human monocyte surface proteins revealed that the target Ag for mAb 63D3 is a 52- to 55-kDa molecule identical to the myeloid differentiation protein CD14. Stimulation of EC with rIL-1 alpha or rTNF-alpha for 4 or 24 h or rIFN-gamma for 24 h increased (p less than 0.005) the number of monocytes bound to both types of EC. This cytokine-induced increase in monocyte adherence was significantly (p less than 0.0005) inhibited when the monocytes were coated with various mAb against CD14. The binding of monocytes to nonstimulated venous or arterial EC was not inhibited by anti-CD14 mAb. Our results lead to the conclusion that CD14 molecules, which on basis of their structure and m.w. are not related to the beta 2-integrin family of heterodimeric leukocyte adhesion molecules, participate in the binding of monocytes to cytokine-stimulated EC.     

显微摄像条件对细胞形态测试结果的影响

目的:研究显微摄像条件对细胞形态测试结果的影响情况。方法:以苏木精-伊红(HE)染色的胃壁细胞为研究对象,分别在不同光亮度、对比度、饱和度和锐度的成像条件下进行显微摄像,采用图像分析软件(Image-Pro Plus 6.0)测试胃壁细胞的色度学和几何形态学参数,并对测试结果进行分析比较。结果:不同光亮度、对比度、饱和度组的胃壁细胞红、绿、蓝三基色差异均有统计学意义(P<0.05),其中红、绿、蓝基色值在高光亮度和高对比度组最高,同时红基色值在高饱和组最高,而绿、蓝基色值在高饱和组最低(P<0.05);不同锐度的胃壁细胞红、绿、蓝三基色差异没有统计学意义(P>0.05)。不同光亮度、对比度、饱和度和锐度的胃壁细胞的面积、周长、平均直径差异均没有统计学意义(P>0.05)。结论:光亮度、对比度和饱和度对细胞色度学参数影响明显,而对几何形态学参数无明显影响。     

Determination of yeast viability during a stress-model alcoholic fermentation using reagent-free microscopy image analysis

A dedicated microscopy imaging system including automated positioning, focusing, image acquisition, and image analysis was developed to characterize a yeast population with regard to cell morphology. This method was used to monitor a stress-model alcoholic fermentation with Saccharomyces cerevisiae. Combination of dark field and epifluorescence microscopy after propidium iodide staining for membrane integrity showed that cell death went along with important changes in cell morphology, with a cell shrinking, the onset of inhomogeneities in the cytoplasm, and a detachment of the plasma membrane from the cell wall. These modifications were significant enough to enable a trained human operator to make the difference between dead and viable cells. Accordingly, a multivariate data analysis using an artificial neural network was achieved to build a predictive model to infer viability at single-cell level automatically from microscopy images without any staining. Applying this method to in situ microscope images could help to detect abnormal situations during a fermentation course and to prevent cell death by applying adapted corrective actions.     

A quantitative analysis of the human bone marrow granulocytic cell lineage using the SAMBA 200 cell image processor. I. The normal maturation sequence

A quantitative image analysis of the human normal bone marrow granulocytic line was performed using the SAMBA 200 image analyzer. The steps of image acquisition, preprocessing, segmentation and parametrization are described. Forty-one parameters were computed on 941 cell images belonging to the various maturation stages. The automated classification of these cells based upon a stepwise linear discriminant analysis resulted in 77% correctly classified cells; the five most discriminating parameters were the nuclear area, the nuclear convexity degree, the average cytoplasmic hue, the regularity of the nuclear boundary and the average cytoplasmic luminance. The evolution of the parameters correlates well with the cytologic evolution and the biochemical and functional events during the maturation process. It can be inferred from our results that the maturation sequence can be subdivided into two phases according to the evolution of the cell profiles. The first phase, from myeloblast to myelocyte, is discontinuous and appears as the critical point with regard to the expression of genes. The second phase, from myelocyte to polymorphonuclear cell, is a continuous sequence of transformations leading to the functional granulocyte.     

Evaluation of image analysis and laser granulometry for microbial cell sizing

A direct cell size measurement technique and an image analysis based sizing method were developed. The former consisted of a manual size measurement of the two-dimensional cell images on a video screen, with automatic data recording. This method was chosen as the reference. The latter, a semiautomatic method took advantage of a commercial computer program designed for image processing and particle morphology analysis. It gave average and median size values which were compatible with the manual method. However, the performance of these time consuming methods is limited. Hence, the laser granulometry technique, intrinsically far more powerful while capable of analysing millions of sample objects in a short time delay, was applied. The comparison revealed that this method gives too low size values, particularly in disagreement with the known dimensions of the bacterial (Zymomonas mobilis) cells. A size correction method was developed to realign the granulometry results ofZ. mobilis cell samples with those of the direct manual measurement method.     

Morphological characterization and viability assessment of Trichoderma reesei by image analysis

The production of cellulase from the filamentous fungus Trichoderma reesei is a critical step in the industrial process leading to cellulose ethanol. As a result of the lack of quantitative analysis tools, the intimate relationship that exists between the morphological and physiological states of the microorganism, the shear field in the bioreactor, and the process performance is not yet fully understood. A semiautomatic image analysis protocol was developed to characterize the mycelium morphology and to estimate its percentage viability during the fermentation process based on four morphological types (unbranched, branched, entangled, and clumped microorganisms). Pictures taken under bright field microscopy combined with images of fluorescein diacetate stained fungi were used to assess the morphological parameters and the percentage viability of microorganisms simultaneously. The method was tested during the course of fed-batch fermentation in a reciprocating plate bioreactor. The use of the image analysis protocol was found to be successful in quantifying the variations in the morphology and the viability of T. reesei throughout the fermentation.     

Comparison of morphological characteristics of Streptomyces natalensis by image analysis and focused beam reflectance measurement

A morphological interpretation is presented for data collected during growth of a filamentous organism, using a focused beam reflectance measurement (FBRM) system. The morphology of the organism was also obtained using conventional semiautomatic image analysis to support the interpretation of the FBRM data. The model organism employed is the filamentous soil-borne actinomycete Streptomyces natalensis, which produces the antifungal agent pimaricin. The organism was cultivated both in shake flasks and in a bench-scale stirred tank bioreactor. It was found that FBRM could be used to track changes in the morphology of the organism throughout the course of its growth on both scales. These changes were highlighted using both the median chord length and length-weighted mean chord length obtained from the chord length distribution measured with the FBRM probe. The ability of the FBRM probe to respond to changes in both the size and morphology of mycelial aggregates was supported by standard image analysis parameters, including equivalent diameter, convex area, and compactness.     

Effect of hyperbaric stress on yeast morphology: study by automated image analysis

The effects of hyperbaric stress on the morphology of Saccharomyces cerevisiae were studied in batch cultures under pressures between 0.1 MPa and 0.6 MPa and different gas compositions (air, oxygen, nitrogen or carbon dioxide), covering aerobic and anaerobic conditions. A method using automatic image analysis for classification of S. cerevisiae cells based on their morphology was developed and applied to experimental data. Information on cell size distribution and bud formation throughout the cell cycle is reported. The results show that the effect of pressure on cell activity strongly depends on the nature of the gas used for pressurization. While nitrogen and air to a maximum of 0.6 MPa of pressure were innocuous to yeast, oxygen and carbon dioxide pressure caused cell inactivation, which was confirmed by the reduction of bud cells with time. Moreover, a decrease in the average cell size was found for cells exposed for 7.5 h to 0.6 MPa CO₂.     

Morphological analysis of<Emphasis Type="Italic"> Yarrowia lipolytica</Emphasis> under stress conditions through image processing

Stress conditions (thermal and oxidative) were applied to Yarrowia lipolytica culture. A rearrangement in cell metabolism as well as dimorphism was observed under these conditions. An image analysis procedure was employed for morphology characterization, and a net increase of around 25% on hyphae formation was detected. A significant increment in total hyphal length was detected, compared with the control system. The results obtained lead to the consideration of a possible relationship between dimorphism and a cell response mechanism to stress conditions.