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At least 343 records · Page 19Linked to original sources
PubMed1991

Three-dimensional DNA image cytometry by confocal scanning laser microscopy in thick tissue blocks.

A method for the quantification of nuclear DNA in thick tissue blocks by confocal scanning laser microscopy is presented. Tissues were stained en bloc for DNA by chromomycin A3. Three-dimensional images, 60 microns deep, were obtained by stacking up confocal fluorescent images obtained with an MRC-500 (Bio-Rad, Richmond, CA). The effects due to bleaching and attenuation by depth of fluorescence emission were corrected mathematically. The DNA contents were estimated by summing up the detected emission intensities (discretized into pixel gray levels) from each segmented nucleus. Applications to an adult rat liver and to a human in situ carcinoma of theesophagus are shown to demonstrate, respectively, the precision of the method and its potential usefulness in histopathology. Comparisons are made with DNA histograms obtained on the same materials by image cytometry on smears and by flow cytometry. Ploidy peaks obtained with the confocal method, although wider than with other methods, are well separated. Confocal image cytometry offers the invaluable advantage of preserving the tissue architecture and therefore allowing, for instance, the selection of histological regions and the evaluation of the degree of heterogeneity of a tumor.

Animals↗
PubMed1999

Ligand-receptor binding measured by laser-scanning imaging.

This report describes the integration of laser-scanning fluorometric cytometry and nonseparation ligand-binding techniques to provide new assay methods adaptable to miniaturization and high-throughput screening. Receptor-bound, cyanine dye-labeled ligands, [Cy]ligands, were discriminated from those free in solution by measuring the accumulated fluorescence associated with a receptor-containing particle. To illustrate the various binding formats accommodated by this technique, saturation- and competition-binding analyses were performed with [Cy]ligands and their cognate receptors expressed in CHO cells or as fusion proteins coated on polystyrene microspheres. We have successfully applied this technique to the analysis of G protein-coupled receptors, cytokine receptors, and SH2 domains. Multiparameter readouts from ligands labeled separately with Cy5 and Cy5.5 demonstrate the simultaneous analysis of two target receptors in a single well. In addition, laser-scanning cytometry has been used to assay enzymes such as phosphatases and in the development of single-step fluorescent immunoassays.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Fluorescent human lung macrophages analyzed by spectral confocal laser scanning microscopy and multispectral cytometry.

Numerous highly fluorescent macrophages (MPhi), designated "smoker cells," exist in the lungs of smokers and subjects who have quit smoking within 5 years. The brightly fluorescent MPhi, however, are not present in the lungs of never smokers. Some investigators have speculated that the intense fluorescence of the MPhi is due to smoke-induced changes in the autofluorescence of naturally occurring (i.e., endogenous) compounds (e.g., NADP). In contrast, other researchers have theorized that the fluorescence is due to the uptake of tobacco smoke particulates (i.e., "tar"). Studies reported herein were undertaken to test the hypothesis that the origin of the MPhi fluorescence could be profiled with the novel technologies afforded by spectral confocal laser scanning microscopy (sCLSM) and multispectral cytometry (MSC). To this end, spectral emissions were obtained by sCLSM of optical sections of live MPhi isolated from fresh surgically excised human lung tissue and in air-dried lung tissue imprints. Confirmation of spectral profiles of these single cell observations was obtained in population studies with the use of high-throughput MSC in which multispectral analyses were performed with three different lasers. Proof of concept experiments demonstrated that relatively nonfluorescent MPhi from the lungs of nonsmokers became fluorescent upon short-term ex vivo exposure to tobacco smoke tar. Summarily, the studies reported herein document that the fluorescence of human lung MPhi is due to tobacco tar.

Fluorescence↗
PubMed2006

Comparison of immunophenotyping by slide-based cytometry and by flow cytometry.

BACKGROUND: Flow cytometry (FCM) is the gold standard for immunophenotyping of peripheral blood leukocytes (PBLs). Slide-based cytometry (SBC) systems, for example the laser scanning cytometer (LSC(R), CompuCyte), can give additional information (repeated staining and scanning, morphology). In order to adequately judge the clinical usefulness of LSC for immunophenotyping it is obligatory to compare it with FCM. AIM: The aim of this study was to systematically compare immunophenotyping by both FCM and LSC methods and to test the correlation of the results. METHODS: PBLs were stained with directly labeled monoclonal antibodies with the whole blood staining method. Aliquots of the same paraformaldehyde fixed specimens were analyzed in parallel by a FACScan (BD-Biosciences) using standard protocols and by LSC with different triggers (forward scatter, CD45 FITC, or 7-AAD). For 7-AAD measurements by LSC, slides were additionally fixed with acetone before 7-AAD staining. RESULTS: Calculating the percentage distribution of PBLs obtained by LSC and by FCM showed very good correlation with regression coefficients close to 1.0 for the major populations and the lymphocyte sub-populations (neutrophils, monocytes, and lymphocytes; T-helper-, T-cytotoxic-, B-, NK-cells). The best trigger for LSC was 7-AAD. CONCLUSION: LSC can be recommended for immunophenotyping of PBLs especially in cases where only limited sample volumes are available or where additional analysis of the cells' morphology is important. The detection of rare leukocytes or weak antigens is limited; in these cases appropriate amplification steps for immunofluorescence should be engaged.

Dactinomycin↗
PubMed1997

Specificity of seven monoclonal antibodies against p53 evaluated with Western blotting, immunohistochemistry, confocal laser scanning microscopy, and flow cytometry.

p53 immunostaining of histological sections shows inter- and intratumor variability in distribution and staining intensity which are usually scored semiquantitatively. In order to investigate the variation in p53 expression more accurately and its possible relation to other cellular parameters (e.g., DNA content), we have studied the possibility to measure p53 accumulation by multiparameter flow cytometry. To this end we have evaluated seven, commercially available, monoclonal antibodies (MAbs) against p53 (MAbs 1801, 240, 246, 421, 1620, Do1, and Do7) on five tumor cell lines with known p53 gene status: MCF-7 (wild-type p53 gene), COV362.cl4 and T47d (mutated p53 genes), and SAOS-2 and HL60 (no p53 mRNA). Localization of immunofluorescence was investigated with confocal laser scanning microscopy, immunofluorescence signal intensity with flow cytometry, and antibody specificity with Western blotting. Subsequently, single cell suspensions from two breast carcinomas were flow cytometrically analyzed after triple staining for p53, cytokeratin 8/18, and DNA, and compared to immunohistochemical staining. MAbs Do1 and Do7, and to a lesser extent MAb 421, accurately discriminated p53 positive from p53 negative cell lines. Even at high concentrations these MAbs yielded nuclear immunofluorescence, whereas with MAbs 1801, 240, and 246 strong cytoplasmic signals in both the p53 accumulating and p53 negative cell lines were seen. By using lower antibody concentrations the cytoplasmic immunofluorescence disappeared, but simultaneously the nuclear p53 immunostaining intensity in p53 accumulating cell lines decreased, resulting in false negative nuclei. With MAb 1620 only weak intranuclear spots were obtained in all cell lines tested. Western blotting yielded results with MAbs 1801, Do1, and Do7 in the 53 kD region of the p53 accumulating cell lines. The signal intensity obtained with MAb 1801 was much less compared to MAbs Do1 and Do7. Although all three MAbs are also described as wild-type p53 specific, only MAbs, Do1 and Do7 showed bands in the 53 kD region of cell line MCF-7. With MAb 1801 ascites and MAb 1801 supernatant an additional approximately 80 kD band was present in all cell lines tested, including SAOS-2, indicating cross reactivity of this MAb. Immunohistochemical staining of two clinical breast carcinomas confirmed the results obtained in the cell lines. Multiparameter flow cytometric analysis of these breast carcinomas with MAbs Do1 and Do7 showed intratumor heterogeneity for p53 accumulation, which was independent of DNA index heterogeneity. We conclude that MAbs Do1 and Do7 enable quantitative analysis of p53 accumulation in a multiparameter flow cytometric analysis.

Antibodies, Monoclonal↗
PubMed1995

A method to compensate for light attenuation with depth in three-dimensional DNA image cytometry using a confocal scanning laser microscope.

A method to compensate for attenuation of detected light with increased depth of the collected optical section, and its application in three-dimensional (3-D) DNA image cytometry is described. The method is based on studying the stack of 2-D histograms that can be formed from each consecutive pair of sections in a stack of optical serial sections. An attenuation factor is calculated interactively and a new compensated section series is computed. Formalin-fixed paraffin-embedded rat tissue was stained with propidium iodide. Each cell nucleus is extracted by thresholding and its total intensity is calculated. The coefficient of variation (CV) of the total intensity of all cells in each stack is computed. For comparison the CV of the same cells is computed in the uncompensated stacks. This study shows a significantly lower CV for the compensated data, thus contributing to the accuracy of DNA quantification in 3-D DNA image cytometry.

Animals↗
PubMed2006

A new approach for the analysis of testicular cells using a laser scanning cytometer.

The laser scanning cytometer (LSC) is a new laboratory tool that offers increased sensitivity and specificity compared to traditional technology. By combining the properties of the advantages of flow cytometry and immunohistochemistry, LSC-based analysis allows the automated evaluation of testicular cells in general and meiosis in particular. Testicular cell smears with previous staining by propidium iodide were analyzed by LSC. The results were compared with those for flow cytometry. LSC is a new, applicable methodology for analyzing spermatogenesis schedule.

Animals↗
PubMed2004

Efficacy of MitoTracker Green and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues.

BACKGROUND: Chloromethyl-X-rosamine (CMXRos) and MitoTracker Green (MTG) have proved to be useful dyes with which to measure mitochondrial function. CMXRos is a lipophilic cationic fluorescent dye that is concentrated inside mitochondria by their negative mitochondrial membrane potential (MMP). MTG fluorescence has been used as a measure of mitochondrial mass independent of MMP. The fluorescence ratio of the two dyes is a relative measure of the MMP independent of mitochondrial mass. Because MTG was recently reported to be sensitive to MMP, we have reevaluated the effects of loss of MMP on MTG and CMXRos fluorescence, using both flow cytometry and laser scanning confocal microscopy (LSCM). METHODS: Using flow cytometry, the relative fluorescence of CMXRos, R123, and MTG was determined in human lymphoblastoid cell lines (LCLs) with or without carbonyl cyanide p-trifluoromethoxylphenyl-hydrazone (FCCP), used to collapse the MMP. LSCM analysis was also used to evaluate the effect of FCCP on MTG and CMXRos fluorescence of mouse cells and viable lenses in culture. The cytotoxicity of the dyes was determined using flow analysis of endogenous NADH fluorescence. The sensitivity of MTG fluorescence to H(2)O(2) was also evaluated using flow cytometry. RESULTS: CMXRos fluorescence was dependent on MMP, whereas MTG fluorescence was not affected by MMP, using either flow or LSCM. Specific staining of mitochondria was seen with both dyes in all cell types tested, without evidence of cytotoxicity, as determined by NADH levels. H(2)O(2) damage slightly increased MTG staining of cells. CONCLUSIONS: Our results indicate that CMXRos is a nontoxic sensitive indicator of relative changes in MMP, whereas MTG is relatively insensitive to MMP and oxidative stress, using both flow and LSCM analyses, provided optimal staining conditions are used. In addition, these dyes can be useful for the study of mitochondrial morphology and function in whole tissues, using LSCM.

Aldehydes↗
PubMed2006

Proapoptotic effects of NARC 1 (= PCSK9), the gene encoding a novel serine proteinase.

BACKGROUND: NARC 1/PCSK9 encodes a novel serine proteinase known to play a role in cholesterol homeostasis. NARC 1 mRNA expression in cerebellar granule neurons (CGNs) was discovered to be induced following an apoptotic injury. Coregulation of known apoptotic mediators (caspase-3 and death receptor 6) raises the possibility that NARC 1 might be involved in the propagation of apoptotic signaling in neurons. METHODS: CGNs were transfected with EGFP-fusion constructs of wild-type and mutant NARC 1, and a laser scanning cytometry-based method of scoring cell death in transfectants was applied. Use of the poly-caspase inhibitor BAF allowed assessment of the caspase-dependence of the NARC 1 proapoptotic effect. RESULTS: Wild-type NARC 1 was found to have substantial proapoptotic effects that were only partially reversible by BAF. Mutation of the active site serine or deletion of the catalytic domain resulted in a reduced level of cell death, consistent with loss of the BAF-sensitive component of cell death. NH(2)-terminal deletion constructs of NARC 1 had effects similar to wild-type, both in the absence and presence of BAF, whereas expression of COOH-terminal deletion mutants produced a rate of cell death similar to wild-type in the absence of BAF treatment, but which lacked the capacity to be reduced by treatment with BAF. CONCLUSION: The mechanism by which NARC 1-EGFP over-expression induces cell death in cultured CGNs remains unclear. Mutation analysis established a positive correlation between the presence of the Narc 1 active site serine in the transiently expressed protein and induction of the BAF-sensitive component of the cell death phenotype. A caspase-independent component proved sufficiently complex to map discretely within the Narc 1 protein.

Amino Acid Chloromethyl Ketones↗
PubMed2005

Analysis of micronuclei in the transferrin-receptor positive reticulocytes from peripheral blood of nasopharyngeal cancer patients undergoing radiotherapy by a single-laser flow cytometer.

The automated micronucleus test is now accepted as a simple, objective, and accurate method for evaluating potential mutagenic effects caused by physical, chemical or biotic factors. This paper describes a single-laser flow cytometry, based on an immunomagnetic isolation technique in combination with acridine orange staining, to detect frequencies of micronucleated transferrin-receptor positive reticulocytes from human peripheral blood. Using this flow cytometric system, we detected the frequencies of micronucleated transferrin-receptor positive reticulocytes from 10 nasopharyngeal cancer patients undergoing radiotherapy and the baseline of the frequencies of micronucleated transferrin-receptor positive reticulocytes from 7 healthy donors. The results showed that the mean frequency of micronucleated transferrin-receptor positive reticulocytes from healthy donors was 0.236% and that from nasopharyngeal cancer patients before radiotherapy was 0.297%. After radiotherapy it was significantly elevated. When the cumulative dose of radiotherapy was about 20Gy, it reached a maximum of 6.905%, and then, as the cumulative dose of radiotherapy continued to increase to 30Gy, 40Gy and 50Gy, the frequency decreased to 6.258%, 5.119% and 5.007% respectively. Our results indicated that the single-laser flow cytometric system was quick, reasonable and acceptable for detecting the frequency of micronucleated transferrin-receptor positive reticulocytes from human peripheral blood.

Adult↗
PubMed2006

Automated four-color analysis of leukocytes by scanning fluorescence microscopy using quantum dots.

BACKGROUND: Scanning fluorescence microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al., Cytometry A 2004, 61:1-8). AIMS: We developed a four-color staining protocol (DNA, CD3, CD4, and CD8) for the lymphocyte phenotyping by SFM. METHODS: Organic (Alexa488, FITC, PE-Alexa610, CyChrom, APC) and inorganic (quantum dot (QD) 605 or 655) fluorochromes were used and compared in different combinations. Measurements were performed in suspension by flow cytometer (FCM) and on slide by SFM. RESULTS: Both QDs were detectable by the appropriate Axioplan-2 and FCM filters and the AxioCam BW-camera. CD4/CD8 ratios were highly correlated (P = 0.01) between the SFM and FCM. CONCLUSION: Automated SFM is an applicable tool for CD4/CD8 ratio determination in peripheral blood samples with QDs.

Benzimidazoles↗
PubMed2004

Single-particle sizing from light scattering by spectral decomposition.

A Fourier transform was applied to size an individual spherical particle from an angular light-scattering pattern. The position of the peak in the amplitude spectrum has a strong correlation with the particle size. A linear equation retrieved from regression analysis of theoretically simulated patterns provides a relation between the particle size and the location of the amplitude spectrum's peak. The equation can be successfully applied to characterize particles of size parameters that range from 8 to 180 (corresponding to particle sizes that range from 1.2 to 27.2 microm at a wavelength of 0.633 microm). The precision of particle sizing depends on the refractive index and reaches a value of 60 nm within refractive-index region from 1.35 to 1.70. We have analyzed four samples of polystyrene microspheres with mean diameters of 1.9, 2.6, 3.0, and 4.2 microm and a sample of isovolumetrically sphered erythrocytes with a scanning flow cytometer to compare the accuracy of our new method with that of others.

Algorithms↗
PubMed2006

An improved method for discrimination of cell populations in tissue sections using microscopy-based multicolor tissue cytometry.

BACKGROUND: In tissue context, researchers and pathologists lack a generally applicable standard for quantitative determination of cytological parameters. Increasing knowledge of disease-specific markers calls for an appropriate in situ tissue cytometry. METHODS: Microscopy-based multicolor tissue cytometry (MMTC) permits multicolor analysis of single cells within tissue context. RESULTS: Tissue specimens stained for CD45/CD3/CD4/CD8 were analyzed. Specificity as well as reproducibility of MMTC is demonstrated and a novel MMTC-based function to improve visual discrimination of subpopulations is introduced. CONCLUSIONS: Our data demonstrate that MMTC constitutes an important step toward automated and quantitative fluorometry of solid tissues and cell monolayers.

Algorithms↗
PubMed1991

Microscope-based multiparameter laser scanning cytometer yielding data comparable to flow cytometry data.

We describe a computer-controlled 10 microns spot size laser scanning cytometer for making multiple wavelength fluorescence and scatter measurements of unconstrained cells on a surface such as a microscope slide. Designated areas of slides placed on a microscope stage are automatically scanned, and cells which generate above-threshold scatter or fluorescence values are found and individually processed to determine a list of measurement parameters. For each fluorescence or scatter measurement parameter, this list contains the integrated and peak values and bit pattern images of a scan window centered on the cell. The measurement time, the position of the cell on the slide, and two segmentation indices are also included in the list. Measurement time, cell position, and properties derived from the bit patterns are used interchangeably with integrated or peak measurement values as coordinates of multiproperty displays. Cells may be selected for counting, data display in various forms, or visual observation based on their meeting complex criteria among a chain of two property screens. Cells with selected properties may be viewed during an experiment or retrospectively. A designated specimen field may be repeatedly remeasured to perform kinetic cell studies. An argon ion and a HeNe- based laser instrument have been constructed and software has been written and evaluated with the specific goal of increasing the precision of propidium iodide-stained cellular DNA measurements. Some of the capabilities of the instrument and its current performance are described.

Algorithms↗
PubMed2004

Simultaneously multi-parameter determination of hematonosis cell apoptosis by two-photon and confocal laser scanning microscopy.

Flow cytometry (FCM), TdT-mediated dUTP Nick End Labeling (TUNEL), and DNA ladder are conventional methods to detect apoptosis of drug-treated cells. However, the assumption of cell number restricts their applications in clinics. In this paper, we report a cell-saving imaging method for quick identification of the hematonosis cell apoptosis induced by chemotherapeutic drugs. By the combination of two-photon and confocal microscopy, three main apoptosis parameters (the change of nuclear morphology, collapse of mitochondrial membrane potential, and increase of intracellular calcium) were recorded simultaneously for single As2O3-induced Molt-4 cells. The results are highly in accordance with those produced by classical flow cytometry. This work suggests that this new imaging method would be promising in the quick identification of hematonosis cell apoptosis.

Apoptosis↗
PubMed2004

Slide-based cytometry and predictive medicine: the 8th Leipziger workshop and the 1st international workshop on slide-based cytometry.

Slide-based cytometry (SBC) and related techniques offer unique tools to perform complex diagnostic procedures at very early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance, not only as a cytomics technology platform, but for patients with low blood volume such as neonates. The exact knowledge of the location of each cell on the slide allows the specimen to be restained and subsequently reanalyzed. These separate measurements can be fused to one data file (merging), increasing the information obtained per cell. Relocalization and optical evaluation of the cells, a typical feature of SBC, can be of integral importance for cytometric analysis. Predictive medicine is aimed at the detection of changes in the patient's state prior to the manifestation of deterioration or improvement. Such instances are concerned with multiorgan failure in sepsis or noninfectious posttraumatic shock in intensive care patients, or the pretherapeutic identification of high risk patients in cancer cytostatic therapy. Early anti-infectious or anti-shock therapy, as well as curative chemotherapy in combination with stem cell transplantation, may provide better survival chances for the patient as well as concomitant cost containment. Predictive medicine-guided, individualized, early reduction or cessation of therapy may lower or abrogate potential therapeutic side effects (individualized medicine). With the 8th Leipziger Workshop and the 1st International Workshop on Slide-Based Cytometry, cytomics technologies moved to more practical applications in the clinics and the clinical laboratory. This development will be continued in 2004, at the upcoming Leipziger Workshop and the International Workshop on Slide-Based Cytometry.

Cytodiagnosis↗
PubMed2002

Flow cytometry and factor analysis evaluation of confocal image sequences of morphologic and functional changes occurring at the mitochondrial level during 7-ketocholesterol-induced cell death.

OBJECTIVE: To analyze functional and morphologic alterations that occur at the mitochondrial level by flow cytometry and laser scanning confocal microscopy (CLSM) combined with factor analysis of biomedical image sequences (FAMIS). STUDY DESIGN: Under treatment of U937 cells with 7-ketocholesterol, functional alterations that occur at the mitochondrial level (especially loss of transmembrane mitochondrial potential [delta psi m]) were assessed with 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) and mitotracker red (CMXRos), whereas morphologic changes were analyzed with nonyl acridine orange (NAO). By flow cytometry, these different dyes were excited at 488 nm, whereas on CLSM, excitation of NAO and CMXRos was performed by lines of an argon laser. By CLSM, spectral sequences were performed to characterize NAO and CMXRos. FAMIS was used to transform the image sequences in factor images. RESULTS: By flow cytometry, rapid loss of delta psi m induced by 7-ketocholesterol was detected with both DiOC6(3) and CMXRos, which gave similar results. Morphologic alterations of mitochondria were revealed with NAO. The factor images obtained from confocal image sequences confirmed these results. CONCLUSION: The simultaneous use of NAO, CMXRos and FAMIS constitutes a new method to detect morphologic and functional alterations occurring at the mitochondrial level during cell death.

Cell Death↗