FISH and chips: Automation of fluorescent dot counting in interphase cell nuclei

Netten, Hans; Young, Ian T.; Vliet, Lucas J. van; Tanke, Hans J.; Vroljik, Hans; Sloos, Willem C. R.

doi:10.1002/(sici)1097-0320(19970501)28:1<1::aid-cyto1>3.0.co;2-k

Cited by 111 publications

(84 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Then a 3D Top Hat filter is applied to enhance the spots against the background and decrease the level of background noise. This filter is defined by: Top Hat(Im) 5 Im 2 Max7(Min7(Im)), as previously described (25). The second step is the segmentation of the spots.…”

Section: Image Processingmentioning

confidence: 99%

Smart 3D‐fish: Automation of distance analysis in nuclei of interphase cells by image processing

et al. 2005

View full text Add to dashboard Cite

Background: Detection of fluorescent probes by fluorescence in situ hybridization in cells with preserved threedimensional nuclear structures (3D-FISH) is useful for studying the organization of chromatin and localization of genes in interphase nuclei. Fast and reliable measurements of the relative positioning of fluorescent spots specific to subchromosomal regions and genes would improve understanding of cell structure and function. Methods: 3D-FISH protocol, confocal microscopy, and digital image analysis were used. Results: New software (Smart 3D-FISH) has been developed to automate the process of spot segmentation and distance measurements in images from 3D-FISH experiments. It can handle any number of fluorescent spots and incorporate images of 4 0 ,6-diamino-2-phenylindole counterstained nuclei to measure the relative positioning of

show abstract

Section: Image Processingmentioning

confidence: 99%

Smart 3D‐fish: Automation of distance analysis in nuclei of interphase cells by image processing

et al. 2005

View full text Add to dashboard Cite

show abstract

“…Also notable are systems for detecting dots visible by fluorescence in situ hybridization (14) and comet tails indicating DNA damage (15). Software development for more sophisticated applications, such as the automated assessment of subcellular localization, has been much more challenging (11,16,17).…”

mentioning

confidence: 99%

Automated microscopy identifies estrogen receptor subdomains with large‐scale chromatin structure unfolding activity

2004

View full text Add to dashboard Cite

Background:Recently, several transcription factors were found to possess large-scale chromatin unfolding activity; these include the VP16 acidic activation domain, BRCA1, E2F1, p53, and the glucocorticoid and estrogen steroid receptors. In these studies, proteins were fluorescently labeled and targeted to a multimerized array of DNA sequences in mammalian cultured cells, and changes in the appearance and/or size of the array were observed. This type of experiment is impeded by the low throughput of traditional microscopy. Methods: We report the application of automated microscopy to provide unattended, rapid, quantitative measurements of fluorescently labeled chromosome regions. Results: The automated image collection routine produced results comparable to results previously obtained by manual methods and was significantly faster. Using this

show abstract

“…In typical studies, a background pixel consists of 5000 photons, a DAPI-stained nucleus pixel is an additional 3000 photons, and a labeled chromosome pixel can be an additional 10000 photons. These numbers are at least a factor of 100 smaller than the values that are obtained for images acquired through a conventional CCD camera at room-level illumination [2]. Several investigators have evaluated the hybridization efficiencies of interphase FISH using probes specific for chromosomes 13, 18, 21, X and Y.…”

mentioning

confidence: 96%

Fluorescence Dot Counting Efficiency in Radiance Mapped Images

Shah

Merchant

2006

Microsc Microanal

View full text Add to dashboard Cite

Chromosomal aberrations are a variation from the normal, either in structure or number of chromosomes, which result from an exchange of genetic material between two or more chromosomes or from a rearrangement of genetic sequences contained in a single chromosome. The analysis of such aberrations can be useful both in a clinical and in a toxicological context. A particularly useful cytogenetic technique for the analysis of aberrations is Fluorescence in situ Hybridization (FISH) [1]. FISH relies on the use of fluorescent dyes that are attached to the DNA probes and when excited by one wavelength of light, emit light at a second, longer, wavelength. FISH signals in interphase cells become visible as colored dots. In general, the emission spectra of most fluorophores are broad and partially overlapping. In typical FISH study, three fluorescent dyes are used, one to stain the cell nuclei, and two to detect DNA sequences. Although FISH is widely used for genetic analyses, its reliability and accuracy depends on the types of probes, cells and their fixation. The major disadvantage associated with the use of fluorescent dyes is the weak signal emanating from a cell or its labeled constituents. In typical studies, a background pixel consists of 5000 photons, a DAPI-stained nucleus pixel is an additional 3000 photons, and a labeled chromosome pixel can be an additional 10000 photons. These numbers are at least a factor of 100 smaller than the values that are obtained for images acquired through a conventional CCD camera at room-level illumination [2]. Several investigators have evaluated the hybridization efficiencies of interphase FISH using probes specific for chromosomes 13, 18, 21, X and Y. The results reported vary from 60% to 98% for XY detection and 42% to 88% for autosomal aneuploidy detection depending on the type of cells and the choice of probes [3]. These results can be acceptable for rapid detection of chromosome aneuploidies, however, when the cells sought represent a very low proportion of the total cell population, a very high efficiency is desired while maximizing specificity and sensitivity.In this paper, we present an approach to improve the efficiency of detecting fluorescent signals in FISH images by recovering the radiance map of the CCD camera. In general, there exists a nonlinear relationship between the true radiance of the fluorescent probe and the detected pixel value. We use a simple technique to recover the response curve by using a set of images taken with varying but known exposure durations [4]. This allows us to generate an image with increased dynamic range that covers the full extent of the radiance captured by all the images. We further analyze the distribution of each of the fluorescent signals and remap the image values to generate a high contrast image with compressed dynamic range. This leads to images where saturated values are attenuated while the weak signals are amplified. We used 20 interphase FISH slides labeled with DAPI, FITC, and Texas Red to validate the efficiency of our...

show abstract

FISH and chips: Automation of fluorescent dot counting in interphase cell nuclei

Cited by 111 publications

References 18 publications

Smart 3D‐fish: Automation of distance analysis in nuclei of interphase cells by image processing

Smart 3D‐fish: Automation of distance analysis in nuclei of interphase cells by image processing

Automated microscopy identifies estrogen receptor subdomains with large‐scale chromatin structure unfolding activity

Fluorescence Dot Counting Efficiency in Radiance Mapped Images

Contact Info

Product

Resources

About