Assuntos
Cromossomos Humanos/ultraestrutura , Cromossomos/ultraestrutura , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente/métodos , Telômero/ultraestrutura , Algoritmos , Southern Blotting , Sondas de DNA , Humanos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodosRESUMO
The DNA of human chromosomes terminates in several kilobases of telomere repeats that are gradually lost with; age and with replication in vitro. Defective telomere maintenance has been shown to be causally linked to cell cycle exit and apoptosis. In order to overcome the limitations imposed by Southern blotting, we have established a quantitative fluorescence in situ hybridization (Q-FISH) technique. This technique allows estimation of telomere length in specific chromosome arms from metaphase cell preparations. Furthermore, we have extended quantitative in situ hybridization to flow cytometry (flow FISH) in order to obtain information on the mean telomere repeat content in suspended cells. Telomere length in granulocytes, monocytes, CD8 and CD4 T lymphocytes and natural killer cells was found to differ slightly in the peripheral blood of adults. However, strikingly longer telomeres were observed in B lymphocytes (approximately 1.3 kb longer), suggesting a functional role for telomere maintenance in this cell subset. In summary, Q-FISH and flow FISH represent new methods for measuring telomere length in single cells and allow studies of telomere dynamics in haematopoietic subpopulations at various stages of normal and abnormal antigen responses.
Assuntos
Células-Tronco Hematopoéticas/ultraestrutura , Hibridização in Situ Fluorescente/métodos , Telômero/ultraestrutura , Adulto , Animais , Linfócitos B/fisiologia , Feminino , Citometria de Fluxo , Humanos , Camundongos , Linfócitos T/fisiologia , Telomerase/metabolismoRESUMO
The loss of telomere repeats has been causally linked to in vitro replicative senescence of human diploid fibroblasts (HDFs). In order to study the mechanism(s) by which telomere shortening signals cell senescence, we analyzed the telomere length at specific chromosome ends at cumulative population doublings in polyclonal and clonal HDFs by quantitative fluorescence in situ hybridization. The rate of telomere shortening at individual telomeres varied between 50 and 150 bp per population doubling and short telomeres with an estimated 1-2 kb of telomere repeats accumulated prior to senescence. The average telomere length in specific chromosome ends was remarkably similar between clones. However, some exceptions with individual telomeres measuring 0.5-1 kb were observed. In the fibroblast clones, the onset of replicative senescence was significantly correlated with the mean telomere fluorescence but, strikingly, not with chromosomes with the shortest telomere length. The accumulation of short telomeres in late passages of cultured HDFs is compatible with selection of cells on the basis of telomere length and limited recombination between telomeres prior to senescence.
Assuntos
Senescência Celular/fisiologia , Mapeamento Cromossômico , Fibroblastos/citologia , Pele/citologia , Telômero/ultraestrutura , Divisão Celular , Cromossomos Humanos Par 17 , Células Clonais , Feto , Fibroblastos/ultraestrutura , Humanos , Hibridização in Situ Fluorescente , Masculino , Metáfase , Telômero/fisiologiaRESUMO
BACKGROUND: The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>10(5)) and does not provide information on the telomere length of individual chromosomes. Here, we describe a digital image microscopy system for measurements of the fluorescence intensity derived from telomere repeat sequences in metaphase cells following quantitative fluorescence in situ hybridization (Q-FISH). METHODS: Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T(2)AG(3))(n) sequences and the DNA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, the integrated fluorescence intensity value for each telomere, which is proportional to the number of hybridized probes, is calculated and presented to the user. RESULTS: Indirect tests of our method were performed using simulated as well as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was found that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10-15%) can be obtained. CONCLUSIONS: We demonstrate that accurate and repeatable fluorescence intensity measurements can be made from Q-FISH images that provide information on the length of telomere repeats at individual chromosomes from limited number of cells.
Assuntos
DNA/análise , Microscopia de Fluorescência/métodos , Telômero/química , Algoritmos , Calibragem , Carbocianinas , Humanos , Hibridização in Situ Fluorescente , Indóis , Cariotipagem , Linfócitos/ultraestrutura , Masculino , Metáfase , Tamanho da Partícula , Sequências Repetitivas de Ácido Nucleico , Processamento de Sinais Assistido por Computador , Telômero/ultraestruturaRESUMO
Human ovarian surface epithelial (HOSE) cells transfected with the E6 and E7 oncogenes of the human papilloma virus (PV) do not express measurable telomerase activity. Relative to untransfected control cells, HOSE-PV cells have an extended in vitro lifespan characterized by a very high frequency of telomeric associations (TAs) of chromosomes. In order to study the role of telomere shortening in the formation of TAs, we studied the telomere length in 120 dicentric chromosomes in HOSE-PV cells by using quantitative fluorescence in situ hybridization. Forty percent of the dicentric chromosomes had no fluorescence signal at the junction site, and in the remainder the fluorescence at the junction was less than at corresponding unjoined ends. These observations support a critical role of telomere shortening in the development of TAs and the subsequent genetic instability observed in a majority of tumor cells.
Assuntos
Sequências Repetitivas de Ácido Nucleico/genética , Proteínas Repressoras , Telômero/genética , Células Cultivadas , Feminino , Humanos , Hibridização in Situ Fluorescente/métodos , Proteínas Oncogênicas Virais/genética , Ovário , Papillomaviridae/genética , Proteínas E7 de Papillomavirus , Telômero/químicaRESUMO
Human chromosomes terminate in a series of T2AG3 repeats, which, together with associated proteins, are essential for chromosome stability. In somatic cells, these sequences are known to be gradually lost through successive cells divisions; however, information about changes on specific chromosomes is not available. Individual telomeres could mediate important biological effects as was shown in yeast, in which loss of a single telomere results in cell-cycle arrest and chromosome loss. We now demonstrate by quantitative fluorescence in situ hybridization (Q-FISH; ref. 7) that the number of T2AG3 repeats on specific chromosome arms is very similar in different tissues from the same donor and varies only to some extent between donors. In all sixteen individuals studied, telomeres on chromosome 17p were shorter than the median telomere length--a finding confirmed by analysis of terminal restriction fragments from sorted chromosomes. These observations provide evidence of chromosome-specific factors regulating the number of T2AG3 repeats in individual telomeres and raise the possibility that the relatively short telomeres on chromosome 17p contribute to the frequent loss of 17p alleles in human cancers.
Assuntos
Cromossomos Humanos Par 17 , Telômero , Adulto , Células da Medula Óssea , Carbocianinas , Células Cultivadas , Fibroblastos/citologia , Corantes Fluorescentes , Humanos , Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Indóis , Metáfase , Sequências Repetitivas de Ácido NucleicoRESUMO
The ultra-long telomeres that have been observed in mice are not in accordance with the concept that critical telomere shortening is related to aging and immortalization. Here, we have used quantitative fluorescence in situ hybridization to estimate (T2AG3)n lengths of individual telomeres in various mouse strains. Telomere lengths were very heterogeneous, but specific chromosomes of bone marrow cells and skin fibroblasts from individual mice had similar telomere lengths. We estimate that the shortest telomeres are around 10 kb in length, indicating that each mouse cell has a few telomeres with (T2AG3)n lengths within the range of human telomeres. These short telomeres may be critical in limiting the replicative potential of murine cells.
Assuntos
Sequências Repetitivas de Ácido Nucleico , Telômero/genética , Animais , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos , Especificidade da EspécieRESUMO
A simple technique which automatically detects and then segments nucleated cells in Wright's giemsa-stained blood smears is presented. Our method differs from others in 1) the simplicity of our algorithms; 2) inclusion of touching (as well as nontouching) cells; and 3) use of these algorithms to segment as well as to detect nucleated cells employing conventionally prepared smears. Our method involves: 1) acquisition of spectral images; 2) preprocessing the acquired images; 3) detection of single and touching cells in the scene; 4) segmentation of the cells into nuclear and cytoplasmic regions; and 5) postprocessing of the segmented regions. The first two steps of this algorithm are employed to obtain high-quality images, to remove random noise, and to correct aberration and shading effects. Spectral information of the image is used in step 3 to segment the nucleated cells from the rest of the scene. Using the initial cell masks, nucleated cells which are just touching are detected and separated. Simple features are then extracted and conditions applied such that single nucleated cells are finally selected. In step 4, the intensity variations of the cells are then used to segment the nucleus from the cytoplasm. The success rate in segmenting the nucleated cells is between 81 and 93%. The major errors in segmentation of the nucleus and the cytoplasm in the recognized nucleated cells are 3.5% and 2.2%, respectively.
Assuntos
Algoritmos , Células Sanguíneas/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Leucemia/sangue , Automação , Corantes Azur , Núcleo Celular/ultraestrutura , Criança , Citoplasma/ultraestrutura , Coleta de Dados , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Leucemia/classificação , Leucemia/diagnósticoRESUMO
The effect of defocusing on the quality of signals from live cells detected by an automated image cytometry device, the Cell Analyzer, was examined. The influence of these effects on the ability of this device to automatically locate cells plated into a tissue culture flask was then determined by measuring the performance of cell detection and recognition procedures as a function of focus setting. Acceptable limits for deviation from the optimal focus setting (as determined by microscope objective position) were found to be similar for both these procedures, ranging from 40 microns below to 25 microns above the optimal focus position. These limits were asymmetrical about ideal focus due to a pronounced asymmetry in the effects of positive and negative defocusing on the cell signal.
Assuntos
Técnicas Citológicas/instrumentação , Microscopia/instrumentação , Animais , Linhagem Celular , Processamento de Imagem Assistida por ComputadorRESUMO
An automated image cytometry device, the Cell Analyzer, was used to locate live V79 cells plated at low densities in a tissue culture flask. Cells and other objects were detected by moving the flask in steps across a linear solid-state image sensor. The step size was selected to be small enough to allow detection of all the cells in the area being scanned but sufficiently large so that most cells would be detected on only one image line. To distinguish cells from other detected objects, a recognition algorithm utilizing 18 characteristic cell signal features was developed. The algorithm first tests whether a set of feature values falls within specified upper and lower bounds, and then applies a linear discriminant function to the remaining data to further discriminate cells from debris. False-positive errors of 5% or less were achieved with this method, whereas 15-35% of cells were misclassified as debris.
Assuntos
Algoritmos , Contagem de Células/métodos , Técnicas de Cultura/métodos , Processamento de Imagem Assistida por Computador/métodos , Animais , Linhagem Celular , CricetinaeRESUMO
An image acquisition and processing system has been developed for quantitative microscopy of absorption or fluorescence in stained cells. Three different light transducers are used in the system to exploit the best characteristics of these sensors for different biological measurements. A digital scanner, in the form of a linear array charge-coupled device (CCD), acquires data with high spatial and photometric resolution. A color (RGB) camera is employed when spectral information is required for the segmentation of cellular subcomponents. An image-intensified charged-injection device (CID) camera provides for very low light intensity measurements, primarily for fluorescence-labeled cells. Properties of these transducers, such as contrast transfer function, linearity, and photo-response nonuniformity, have been measured. Two dedicated image processing units were incorporated into the system. The front-end processor, based on a digital signal processor, provides functions such as object detection, raw image calibration, compression, artifact removal, and filtering. The second image processor is associated with the frame memory and includes a histogram processor, a dedicated arithmetic logic unit for image processing functions, and a graphics module for one-bit overlay functions. An interactive program was developed to acquire cell images and to experiment with a range of segmentation algorithms, feature extractions, and other image processing functions. The results of any image operation are displayed on the video monitor. Once a desired processing sequence is determined, the sequence may be stored to become part of a command library and can be executed thereafter as a single instruction.