A large field CCD system for quantitative imaging of microarrays.

We describe a charge-coupled device (CCD) imaging system for microarrays capable of acquiring quantitative, high dynamic range images of very large fields. Illumination is supplied by an arc lamp, and filters are used to define excitation and emission bands. The system is linear down to fluorochrome densities <<1 molecule/microm2. The ratios of the illumination intensity distributions for all excitation wavelengths have a maximum deviation approximately +/-4% over the object field, so that images can be analyzed without computational corrections for the illumination pattern unless higher accuracy is desired. Custom designed detection optics produce achromatic images of the spectral region from approximately 450 to approximately 750 nm. Acquisition of a series of images of multiple fluorochromes from multiple arrays occurs under computer control. The version of the system described in detail provides images of 20 mm square areas using a 27 mm square, 2K x 2K pixel, cooled CCD chip with a well depth of approximately 10(5) electrons, and provides ratio measurements accurate to a few percent over a dynamic range in intensity >1000. Resolution referred to the sample is 10 microm, sufficient for obtaining quantitative multicolor images from >30,000 array elements in an 18 mm x 18 mm square.

Comparative genomic hybridization.

Comparative Genomic Hybridization (CGH) is a powerful molecular cytogenetic technique that permits assessment of DNA copy number on a genome-wide scale. Of note, this methodology uses tumor DNA as a probe for fluorescence in situ hybridization (FISH) to normal metaphase chromosomes and does not require dividing cells from the tumor specimen. This unit provides protocols for CGH, for preparation of metaphase chromosomes, tumor and normal DNAs for FISH and for the microscopy and image analysis of CGH experiments.

Segmentation of confocal microscope images of cell nuclei in thick tissue sections.

Segmentation of intact cell nuclei from three-dimensional (3D) images of thick tissue sections is an important basic capability necessary for many biological research studies. However, segmentation is often difficult because of the tight clustering of nuclei in many specimen types. We present a 3D segmentation approach that combines the recognition capabilities of the human visual system with the efficiency of automatic image analysis algorithms. The approach first uses automatic algorithms to separate the 3D image into regions of fluorescence-stained nuclei and unstained background. This includes a novel step, based on the Hough transform and an automatic focusing algorithm to estimate the size of nuclei. Then, using an interactive display, each nuclear region is shown to the analyst, who classifies it as either an individual nucleus, a cluster of multiple nuclei, partial nucleus or debris. Next, automatic image analysis based on morphological reconstruction and the watershed algorithm divides clusters into smaller objects, which are reclassified by the analyst. Once no more clusters remain, the analyst indicates which partial nuclei should be joined to form complete nuclei. The approach was assessed by calculating the fraction of correctly segmented nuclei for a variety of tissue types: Caenorhabditis elegans embryos (839 correct out of a total of 848), normal human skin (343/362), benign human breast tissue (492/525), a human breast cancer cell line grown as a xenograft in mice (425/479) and invasive human breast carcinoma (260/335). Furthermore, due to the analyst's involvement in the segmentation process, it is always known which nuclei in a population are correctly segmented and which not, assuming that the analyst's visual judgement is correct.

High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays.

Gene dosage variations occur in many diseases. In cancer, deletions and copy number increases contribute to alterations in the expression of tumour-suppressor genes and oncogenes, respectively. Developmental abnormalities, such as Down, Prader Willi, Angelman and Cri du Chat syndromes, result from gain or loss of one copy of a chromosome or chromosomal region. Thus, detection and mapping of copy number abnormalities provide an approach for associating aberrations with disease phenotype and for localizing critical genes. Comparative genomic hybridization (CGH) was developed for genome-wide analysis of DNA sequence copy number in a single experiment. In CGH, differentially labelled total genomic DNA from a 'test' and a 'reference' cell population are cohybridized to normal metaphase chromosomes, using blocking DNA to suppress signals from repetitive sequences. The resulting ratio of the fluorescence intensities at a location on the 'cytogenetic map', provided by the chromosomes, is approximately proportional to the ratio of the copy numbers of the corresponding DNA sequences in the test and reference genomes. CGH has been broadly applied to human and mouse malignancies. The use of metaphase chromosomes, however, limits detection of events involving small regions (of less than 20 Mb) of the genome, resolution of closely spaced aberrations and linking ratio changes to genomic/genetic markers. Therefore, more laborious locus-by-locus techniques have been required for higher resolution studies. Hybridization to an array of mapped sequences instead of metaphase chromosomes could overcome the limitations of conventional CGH (ref. 6) if adequate performance could be achieved. Copy number would be related to the test/reference fluorescence ratio on the array targets, and genomic resolution could be determined by the map distance between the targets, or by the length of the cloned DNA segments. We describe here our implementation of array CGH. We demonstrate its ability to measure copy number with high precision in the human genome, and to analyse clinical specimens by obtaining new information on chromosome 20 aberrations in breast cancer.

Efficient, interactive, and three-dimensional segmentation of cell nuclei in thick tissue sections.

Segmentation of intact cell nuclei in three-dimensional (3D) images of thick tissue sections is an important basic capability necessary for many biological research studies. Because automatic algorithms do not correctly segment all nuclei in tissue sections, interactive algorithms may be preferable for some applications. Existing interactive segmentation algorithms require the analyst to draw a border around the nucleus under consideration in all successive two-dimensional (2D) planes of the 3D image. The present paper describes an algorithm with two main advantages over the existing method. First, the analyst draws borders only in 2D planes that cut approximately through the center of the nucleus under consideration so that the nuclear borders generally are most distinct. Second, the analyst draws only five borders around each nucleus, and then the algorithm interpolates the entire surface. The algorithm results in segmented objects that correspond to individual, visually identifiable nuclei. The segmented surfaces, however, may not exactly represent the true nuclear surface. An optional, automatic surface optimization algorithm can be applied to reduce this error.

Molecular cytometry of cancer.

The application of molecular probes to diagnosis and prognosis of malignancies has redefined our perceptions of disease, allowing diagnosis by genotypic rather than phenotypic criteria. DNA analysis is especially useful when applied to pathological material in situ, because this allows the pathologist to combine information from both morphological and molecular observations. DNA in situ hybridization is a useful approach for the molecular pathologist, especially when combined with cytometric analysis. Potential clinical applications for in situ hybridization and the recently described technique of comparative genomic hybridization in tumor diagnosis and prognosis are described.

Computer image analysis of comparative genomic hybridization.

We describe and evaluate the image-processing and analysis techniques we have developed for the quantitative analysis of comparative genomic hybridization (CGH; Science 258:818, 1992). In a typical CGH application, two genomic DNA samples are simultaneously hybridized to metaphase chromosomes and detected with different fluorochromes. The primary data in CGH are contained in the intensity ratios of the fluorochromes as a function of position on the chromosomes, which reflect variation in DNA copy number ratio between the two DNA samples. Analysis involves chromosome segmentation, intensity normalization, background corrections, and calculation of the fluorescence intensity profiles and the ratio profile along the chromosome's length. Profiles from several copies of the same chromosome in different metaphases are averaged to reduce the noise. Confidence intervals are calculated and displayed for the mean profiles. The techniques were evaluated by examining the variability found in comparisons of two normal genomic DNAs, where the ratio was expected to be constant, and by measuring the ratios obtained for cell lines with cytogenetically documented copy number changes involving several chromosomal segments. The limits of sensitivity of CGH analysis were investigated by simulation. Guidelines for the interpretation of CGH data and indications of areas for future development of the analytical techniques are also presented.

Semiautomated DNA probe mapping using digital imaging microscopy: I. System development.

Algorithms have been developed to help automate the mapping of DNA sequences along metaphase chromosomes using fluorescence in situ hybridization (FISH). Custom algorithms computationally define chromosome boundaries and compute chromosomal medial axes. A dynamic regional thresholding (DRT) algorithm is described that allows reliable detection of hybridization domains, even when they differ substantially in size and intensity. Chromosomal locations are calculated by determining the fractional location of each hybridization probe along the medial axis of a metaphase chromosome relative to the short arm (FLpter). These algorithms were tested on simulated data and by analysis of the location of probes that had been previously mapped by other techniques. These algorithms allow probes to be mapped rapidly along human chromosomes with a precision of 2-3 Mb.

Semiautomated DNA probe mapping using digital imaging microscopy: II. System performance.

This paper describes an evaluation of a semiautomated, multicolor image-analysis system to map cloned probes along metaphase chromosomes. Mapping with this system consists of fluorescence in situ hybridization (FISH) for probe localization, automatic acquisition of multicolor images showing total chromosomal DNA and probe location(s), and automatic determination of the fractional locations of the probes along the chromosomes relative to the short arm telomere (FLpter). The system was evaluated by mapping ten phage and ten cosmid probes previously mapped to chromosome 3 with other procedures. The standard deviations of FLpter measurements averaged 3.4 Mb and 2.6 Mb for phage and cosmid probes, respectively. With this variation, the order of two probes mapped in separate hybridizations could be determined with 95% confidence when their separation was greater than 2.5 Mb. In all cases, the probe locations and order were consistent with previous mapping data. FLpter values were converted to band locations using measurements of the band locations made using digital imaging microscopy. This proved superior to conversions made using ISCN ideograms.

Automatic fluorescence metaphase finder speeds translocation scoring in FISH painted chromosomes.

A fluorescence metaphase finder was constructed with commercially available hardware and a standard Unix workstation. Its accuracy was measured in terms of the number of false positive and false negative detected metaphases on a variety of different slide preparations. The metaphase finder was used in a translocation scoring experiment in which metaphase preparations of human peripheral blood lymphocytes were hybridized with whole chromosome probes to chromosomes #1, #2, and #4. The automatic finder presented metaphases to the cytogeneticist, centered in the eyepieces at x63. The cytogeneticist's scores of analyzable metaphases and of painted chromosomes involved in rearrangements were recorded. The time for the analysis was recorded and compared to the time to analyze a similar number of cells in a purely visual experiment in which the cytogeneticist scanned for cells and analyzed them, both at x63. The results showed that, neglecting the machine time spent scanning unattended, the amount of time required for the analysis was reduced by a factor of three. Furthermore, in this experiment the metaphase finder found more scorable metaphases than the cytogeneticist found by visual scanning. Machine-assisted scoring had additional, less quantifiable, benefits; notably that digital images of metaphases sometimes assisted the analysis of chromosome rearrangements, that cells could be revisited easily, and that the analysis was much less fatiguing.

Physical mapping of chromosome 17 cosmids by fluorescence in situ hybridization and digital image analysis.

We used fluorescence in situ hybridization and digital image analysis to localize cosmids along human chromosome 17. Seventy-one cosmids were selected at random from a chromosome 17 library constructed from a partial Sau3AI digest of flow-sorted chromosomes from a mouse-human hybrid cell line. Sixty-three of these (89%) gave a signal only on chromosome 17. The 40 cosmids producing the most distinct hybridization signals in metaphase and interphase cells were precisely mapped using digital image analysis. An additional 20 cosmids, previously mapped by linkage analysis, were also mapped. The order of these probes determined by metaphase mapping was consistent with the order determined by linkage analysis.

Comparative genomic hybridization: a rapid new method for detecting and mapping DNA amplification in tumors.

Recent evidence indicates that many more genes than the currently known oncogenes may undergo amplification in tumors. We have developed a new technique, Comparative Genomic Hybridization (CGH), which allows rapid detection of DNA amplification anywhere in the tumor genome and maps the amplified sequences on normal chromosomes. CGH is based on a competitive in situ hybridization of differentially labeled tumor DNA and normal DNA to a normal human metaphase spread. Regions of gain of DNA sequences are seen as an increased color ratio of two fluorochromes used to detect the labeled DNAs. Over 20 different regions of amplification have been identified using CGH.

Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors.

Comparative genomic hybridization produces a map of DNA sequence copy number as a function of chromosomal location throughout the entire genome. Differentially labeled test DNA and normal reference DNA are hybridized simultaneously to normal chromosome spreads. The hybridization is detected with two different fluorochromes. Regions of gain or loss of DNA sequences, such as deletions, duplications, or amplifications, are seen as changes in the ratio of the intensities of the two fluorochromes along the target chromosomes. Analysis of tumor cell lines and primary bladder tumors identified 16 different regions of amplification, many in loci not previously known to be amplified.

Comparison of strategies to detect and quantitate uniquely marked cells in intra- and inter-species hemopoietic chimeras.

Evaluation of the outcome of successful bone marrow transplantation and indepth studies of transplantation biology rely increasingly upon detection and enumeration of donor hemopoietic cells in the transplanted recipients. The ability to detect and enumerate low levels of donor engraftment in interphase cell subpopulations in hemopoietic chimeras is particularly important for studies of mixed lineage chimerism, early relapse manifestations, and engraftment of subpopulations present at low frequency. We describe and compare the sensitivity and specificity of DNA-based detection strategies (fluorescence in situ hybridization, in vitro DNA amplification using the polymerase chain reaction) and flow cytometric analysis of cell surface markers to detect cells carrying marker DNA or proteins in syngeneic (mouse-to-mouse) and xenogeneic (mouse-to-human, monkey, sheep) backgrounds. DNA-based detection strategies offer advantages of rapid analysis and enumeration of target cell frequencies with detection sensitivities approximating 10(-4). The sensitivity of immunofluorescence-linked flow cytometric-based detection of nucleated leukocytes approached 10(-3), whereas flow cytometric-based detection of fixed human erythrocytes was feasible at cell frequencies of 10(-5). Data described in this manuscript should facilitate selection of appropriate methodologies for assessment of hemopoietic chimerism following transplantation.

Proposed standard for image cytometry data files.

A number of different types of computers running a variety of operating systems are presently used for the collection and analysis of image cytometry data. In order to facilitate the development of sharable data analysis programs, to allow for the transport of image cytometry data from one installation to another, and to provide a uniform and controlled means for including textual information in data files, this document describes a data storage format that is proposed as a standard for use in image cytometry. In this standard, data from an image measurement are stored in a minimum of two files. One file is written in ASCII to include information about the way the image data are written and optionally, information about the sample, experiment, equipment, etc. The image data are written separately into a binary file. This standard is proposed with the intention that it will be used internationally for the storage and handling of biomedical image cytometry data. The method of data storage described in this paper is similar to those methods published in American Association of Physicists in Medicine (AAPM) Report Number 10 and in ACR-NEMA Standards Publication Number 300-1985.

Pyridine nucleotide analog interference with metabolic processes in mitogen-stimulated human T lymphocytes.

The differential metabolic effects of three nicotinamide analogs, 6-aminonicotinamide, 3-aminobenzamide, and 5-methylnicotinamide, were analyzed in mitogen-stimulated preparations of human T lymphocytes. Mitogen stimulation with the phorbol ester TPA and a monoclonal antibody to the T3 cell surface antigen caused an increase in cellular NAD and ATP levels and a marked increase in glucose metabolism as demonstrated by an increase in cellular levels of glucose 6-phosphate and a sevenfold increase in radioactive CO2 formation from [l-14C]glucose. 6-Aminonicotinamide had drastic inhibitory effects on the mitogen-stimulated increases in NAD and ATP levels as well as on the metabolism of glucose. Treatment of the mitogen-stimulated cells with 6-aminonicotinamide also caused a marked increase in cellular levels of 6-phosphogluconate, suggesting inhibition of the hexose monophosphate shunt at 6-phosphogluconate dehydrogenase. Radioactive CO2 formation from [6-14C]glucose showed that metabolism through the tricarboxylic acid cycle was not used to compensate for the inhibition of the hexose monophosphate shunt pathway. Treatment of cells with 3-aminobenzamide had the opposite effect of 6-aminonicotinamide in that cellular NAD levels increased, presumable due to inhibition of poly(ADP-ribose) polymerase. 3-Aminobenzamide did not interfere with ATP or glucose 6-phosphate levels and did not cause significant elevations of 6-phosphogluconate. Thus, 6-aminonicotinamide appears to have direct inhibitory effects on the synthesis of both pyridine nucleotides and poly(ADP-ribose), whereas 3-aminobenzamide has its major inhibitory effect on poly(ADP-ribose) synthesis. 5-Methylnicotinamide also interferes with the mitogen-stimulated increase in NAD levels but not as effectively as 6-aminonicotinamide. The alterations in pyridine nucleotide metabolism resulting from treatment with these nicotinamide analogs can produce drastic and diverse alterations in pathways of glucose utilization and energy generation.

Role of nicotinamide adenine dinucleotide and adenosine triphosphate in glucocorticoid-induced cytotoxicity in susceptible lymphoid cells.

The possibility that corticosteroid cytotoxicity could be mediated by activation of poly(ADP-ribose) polymerase and consequent depletion of NAD and ATP was evaluated in steroid-sensitive S49.1 and steroid-resistant S49.143R mouse lymphoma cells and in lymphocytes from a patient with chronic lymphocytic leukemia. All cell types were shown to have the enzyme poly(ADP-ribose) polymerase and to increase activity in response to DNA strand breaks. Incubation of susceptible cells with 1 microM dexamethasone resulted in DNA strand breaks. Susceptible cells also showed a dose-dependent decrease in NAD and ATP that preceded loss of cell viability. These studies suggest that steroid-induced cytotoxicity in susceptible lymphocytes is due to the presence of DNA strand breaks that activate poly(ADP-ribose) polymerase to a sufficient degree to consume cellular pools of NAD with a consequent depletion of ATP and loss of cell viability.

Induction of the pyridine nucleotide synthesis pathway in mitogen-stimulated human T-lymphocytes.

Mitogen stimulation of purified human T-lymphocytes with the phorbol ester 12-O-tetradecanoyl, phorbol-13-acetate (TPA) and a monoclonal antibody to the T3 cell surface antigen caused a 6-11-fold increase in cellular levels of poly(ADP-ribose) polymerase, a 6-20-fold amplification of cellular NAD+ levels and a 3-21-fold increase in NADP+ levels. Treatment of the cells with a combination of the two mitogenic signals also caused a 5-20-fold increase in NMN pyrophosphorylase activity, a 3-14-fold increase in ATP-NMN adenylyl transferase activity, and a 5-13-fold increase in NAD kinase activity. This is the first report showing induction of these three enzymes as part of the mitogenic response in purified human T-lymphocytes. Maximum increases in activity of each of these three enzymes required the combined presence of TPA and monoclonal antibody to human T-cell T3 antigen anti-T3. Analysis of the relative enzyme levels indicates that NMN pyrophosphorylase is the rate-limiting enzyme for NAD synthesis and NAD kinase is the rate-limiting enzyme for NADP synthesis.

Metabolic consequences of DNA damage: DNA damage induces alterations in glucose metabolism by activation of poly (ADP-ribose) polymerase.

In this communication we show that activation of poly(ADP-ribose) polymerase by DNA damage can produce drastic alterations in carbohydrate metabolism. We examined alterations in NAD+, NADP+, ATP and glucose-6-phosphate in L1210 murine leukemia cells, following exposure to different concentrations of N-methyl-N'-nitro-N-nitrosoguanidine. Treatment of cells with 20 micrograms/ml MNNG produced rapid depletion of NAD+ and ATP. The G-6-P pool showed a biphasic change: first the pool size decreased, then increased to a level greater than that present in control cells. Nicotinamide treatment prevented the total depletion of NAD+ and this in turn helped preserve the ATP pools and prevented the biphasic alteration in G-6-P pool sizes.