Bioimaging最新文献

Using examples of hybridization both to RNA and to DNA sequences we demonstrate that whole-mount Drosophila embryos are excellent objects with which to study questions about nuclear structure and function by combining FISH and confocal laser scanning microscopy. A fluorescently labelled 29-base oligonucleotide was used to probe transcription at a locus known to be strongly induced upon heat shock. The transcript from this locus apparently serves to stabilize and protect nuclear proteins which may be needed for nuclear processes after heat or chemical stress. We found that less than 5% of the protein is colocalized with the transcript under normal growth conditions, but more than 50% is sequestered by the transcript during heat shock. DNA probes to genes in the bithorax complex were used to examine the relationship between homologous pairing and gene expression in late stage gastrulating embryos. Analysis of the disposition of probes cloned in P1 vectors in embryos from mid-blastoderm throughout gastrulation allowed us to conclude that polarized nuclear organization breaks down after the blastoderm stage. Homologous pairing of the bithorax complex genes proceeds during gastrulation so that at the time of germ band retraction the two alleles are always in close proximity independent of expression of the gene or the region along the anterior–posterior axis of the body. Finally, we demonstrate that smaller DNA targets can be visualized in whole mount embryos by enhancement of the FISH signal by tyramide-fluorophore deposition.

Fluorescence in situ hybridization allows the enumeration of chromosomal abnormalities in interphase cell nuclei. This process is called dot counting. To estimate the distribution of chromosomes per cell, a large number of cells have to be analysed, particularly when the frequency of aberrant cells is low. Automation of dot counting is desirable because manual counting is tedious, fatiguing, and time consuming. We have developed a completely automated fluorescence microscope system that counts fluorescent hybridization dots for one probe in interphase cell nuclei. This system works with two fluorescent dyes—one for the DNA hybridization dots and one for the cell nucleus. A fully automated scanning procedure has been used for the image acquisition. After an image is acquired it has to be analysed in order to find the nuclei and to detect the dots. This article focuses upon the dot detection procedure. Three different algorithms are presented. The problems of ‘overlapping’ dots and split dots are discussed. The automated dot counter has been tested on a number of normal specimens where DAPI was used for the nucleus counter stain and a centromeric probe was used to mark the chromosome 12. The slides contained lymphocytes from cultured blood. The performance of the different algorithms has been evaluated and compared with manually obtained results. The automated counting results approximate the results of manual counting.

Fluorescence in situ hybridization (FISH) has become a widely used technique for the cytogenetic analysis of cells and genomes. Today, FISH techniques allow DNA and RNA probes to be localized in tissue or cellular preparations with speed, specificity, simplicity and safety. Improvements in probe labelling and detection, fluorescence microscopy and the introduction of digital imaging have all combined to enable FISH to be applied not only to answer questions about genome organization, structure and function but also to play an important diagnostic role in the clinical laboratory.

Karyotype analysis by means of chromosome banding techniques is the pillar of cytogenetic diagnostics, both in the clinical and in the cancer cytogenetic laboratory. Using chromosome banding alone, however, a disturbingly high number of chromosomal aberrations cannot be characterized comprehensively. We have therefore developed a novel karyotyping approach, termed spectral karyotyping, that is based on the simultaneous hybridization of 24 combinatorially labelled human chromosome painting probes. The visualization of all human chromosomes in different colours is achieved by spectral imaging. Spectral imaging combines fluorescence microscopy, CCD-imaging and Fourier spectroscopy to visualize, simultaneously, the entire spectrum at all image points. Here we describe the principle of spectral imaging, define software and hardware requirements, and present relevant applications of the technique to cytogenetics and cytology.

High resolution physical mapping of clonal DNA fragments with kilobase (kb) resolution can now be performed rapidly by fluorescence in situ hybridization (FISH) onto individual DNA molecules (DNA fibers). We developed a sensitive procedure termed ‘quantitative DNA fiber mapping’ which consists of three steps: preparation of DNA fibers, hybridization of non-isotopically labeled probes and determination of the relative mapping position by fluorescence image analysis. The DNA fibers are produced by binding linearized DNA molecules with one or both ends to a solid substrate followed by homogeneous stretching of the molecule by the action of a receding meniscus during drying (‘molecular combing’). In a slight variation of this protocol, we deposit circular DNA molecules. Substrates for DNA immobilization are glass slides, coverslips or thin sheets of mica derivatized with amino-silane. Probes are prepared to counterstain the DNA fibers, from the clones to be mapped and for specific landmarks along linear or circular DNA molecules such as cloning vector sequences. Following hybridization and immunocytochemical detection of bound probes, images are analysed and relative distances are recorded for map assembly. Here, we describe our experience with substrate preparation, molecular combing and mapping of cloned or enzymatically synthesized probes ranging in size from 1.2 kb to 100 kb along DNA molecules that are between 17 kb and 1200 kb in size.

Epifluorescence filter sets and computer software for the detection and discrimination of up to 27 different DNA probes using multiplex-fluorescence in situ hybridization (M-FISH) have been developed recently. This paper focuses on a more detailed description of the specific software developed for the analysis of M-FISH experiments. Crucial steps in the evaluation process such as standardization of image acquisition, chromosome segmentation, fluorescence background estimation, accurate measurement of fluorescence intensities and definition of thresholds will be described. The application of M-FISH and comparative genomic hybridization (CGH) for the detection of chromosomal abnormalities in squamous cell cancer of the head and neck is also reported. Finally, we try to define an acceptable ‘standard’ for a fully automated multicolor imaging system.

In this paper the aspects of image acquisition, processing and analysis for DNA-fibre mapping are described. As the nature and the quality of the fibre-FISH signals (given its resolution range of 1–500 kb) may vary to a great extent, an interactive approach was chosen for the selection and analysis of the fibres. The accuracy of this fibre-FISH mapping approach was compared with restriction mapping on the basis of a map of seven cosmid contigs from the thyroglobulin gene, which spans about 300 kb. The results were in full agreement with restriction mapping. Standard errors for sizes of the cosmids, gaps, and overlaps were obtained between 2.0 and 6.2 kb. By alternately labelling the clones of the DNA map a colour barcode can be composed which eases the identification of gene rearrangements, as is illustrated on two patients with a deletion in the Duchenne muscular dystrophy (DMD) gene. The time needed for straightening a fibre and defining the distances between the different cosmids is dominated by the amount of human interaction and typically takes 1–2 min. From this study it is clear that fibre-FISH analysis is well suited for mapping cosmid contigs and defining breakpoints in patient material with the same or better accuracy as restriction mapping and PCR analysis.