RESUMO
The article reviews the existing methods of multicolor FISH on nuclear targets, first of all, interphase chromosomes. FISH proper and image acquisition are considered as two related components of a single process. We discuss (1) M-FISH (combinatorial labeling + deconvolution + wide-field microscopy); (2) multicolor labeling + SIM (structured illumination microscopy); (3) the standard approach to multicolor FISH + CLSM (confocal laser scanning microscopy; one fluorochrome - one color channel); (4) combinatorial labeling + CLSM; (5) non-combinatorial labeling + CLSM + linear unmixing. Two related issues, deconvolution of images acquired with CLSM and correction of data for chromatic Z-shift, are also discussed. All methods are illustrated with practical examples. Finally, several rules of thumb helping to choose an optimal labeling + microscopy combination for the planned experiment are suggested.
Assuntos
Núcleo Celular/ultraestrutura , Hibridização in Situ Fluorescente/métodos , Interfase/genética , Núcleo Celular/genética , Coloração Cromossômica , Cor , Humanos , Microscopia de FluorescênciaRESUMO
The rapid spread of the use of new 24-color karyotyping techniques has preceded their standardization. This is best documented by the fact that the exact resolution limits have not yet been defined. Indeed, it is shown here that a substantial proportion of interchromosomal aberrations will be missed by all multicolor karyotyping systems currently in use. We demonstrate that both the sensitivity and the specificity of 24-color karyotyping critically depend on the fluorochrome composition of chromosomes involved in an interchromosomal rearrangement. As a solution, we introduce a conceptual change in probe labeling. Seven-fluorochrome sets that overcome many of the current limitations are described, and examples of their applications are shown. The criteria presented here for an optimized probe-set design and for the estimation of resolution limits should have important consequences for pre- and postnatal diagnostics and for research applications.
Assuntos
Aberrações Cromossômicas/genética , Cor , Sondas de DNA/metabolismo , Corantes Fluorescentes/metabolismo , Hibridização in Situ Fluorescente/métodos , Cariotipagem/métodos , Bandeamento Cromossômico/métodos , Sondas de DNA/genética , Feminino , Humanos , Indóis/metabolismo , Masculino , Sensibilidade e Especificidade , Translocação Genética/genéticaRESUMO
We describe the generation of a complete set of human chromosome-specific painting probes depleted in repetitive sequences. These probes yield highly specific signals when hybridized without the addition of a blocking agent, such as Cot-1 DNA, and without probe preannealing prior to hybridization. Fluorescent intensities and signal-to-background ratios for these probes are comparable to those of untreated probes hybridized with Cot-1 DNA. We demonstrate the suitability of these probes for applications with very complex probe sets, such as multiplex-FISH.
Assuntos
Coloração Cromossômica/métodos , Cromossomos Humanos/genética , Sondas de DNA/genética , Sequências Repetitivas de Ácido Nucleico , Ligação Competitiva , DNA/genética , Genoma Humano , Humanos , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Multiplex-FISH (M-FISH) is a recently developed technique by which each of the two dozen human chromosomes-the 22 autosomes and the X and Y sex chromosomes-can be stained or "painted" with uniquely distinctive colors. Using a combinatorial labeling technique and a specially designed filter set, each DNA probe can be identified by its unique spectral signature. Here we present several significant optimizations of the M-FISH technology. First, a new strategy for labeling the probes is described which allows for easy and fast production of the complex M-FISH probe mix. Second, a newly developed, completely motorized microscope equipped with an eight-position filter wheel and a new generation of filter sets is presented that allows fully automatic imaging of a complete metaphase spread within seconds. Third, to determine the characteristic spectral signatures for all different combinations of fluorochromes, we developed a novel multichannel image analysis method. The spectral analysis is solely guided by the image information itself and does not require any user interaction. A complete analysis of a metaphase spread can be accomplished in less than 3 min. Sophisticated built-in quality controls were developed, and the value of visual inspection of M-FISH images as a simple means of controlling the computer-generated chromosome classification are illustrated. In addition, we discuss advantages of adding new fluorochromes to the traditionally used five fluorochromes.
Assuntos
Aberrações Cromossômicas , DNA de Neoplasias/análise , Rearranjo Gênico , Hibridização in Situ Fluorescente/métodos , Bandeamento Cromossômico/métodos , Corantes Fluorescentes , Humanos , Processamento de Imagem Assistida por Computador/métodos , Indóis , Microscopia de Fluorescência/métodosRESUMO
Scanning electron microscopy revealed that exposure of hydra polyps to DMSO at concentrations used for permeabilizing tissue results in striking changes in epithelial cell morphology. Epithelial cells from treated polyps rounded up in shape and formed numerous large blebs at the cell surface. Along the borders of epithelial cells numerous small projections became detectable. The DMSO-induced changes at the cell surface corresponded to drastic changes in the intracellular organization. No evidence could be found for DMSO induced opening of cell junctions and/or opening of the interstitial space. The results demonstrate that DMSO affects the morphology and intracellular organization of hydra epithelial cells. Thus, caution is necessary in interpreting cell behavior in DMSO treated tissue.
