Quantification of fluorescence in situ hybridization signals by image cytometry.

In this study we aimed at the development of a cytometric system for quantification of specific DNA sequences using fluorescence in situ hybridization (ISH) and digital imaging microscopy. The cytochemical and cytometric aspects of a quantitative ISH procedure were investigated, using human peripheral blood lymphocyte interphase nuclei and probes detecting high copy number target sequences as a model system. These chromosome-specific probes were labeled with biotin, digoxigenin, or fluorescein. The instrumentation requirements are evaluated. Quantification of the fluorescence ISH signals was performed using an epi-fluorescence microscope with a multi-wavelength illuminator, equipped with a cooled charge couple device (CCD) camera. The performance of the system was evaluated using fluorescing beads and a homogeneously fluorescing specimen. Specific image analysis programs were developed for the automated segmentation and analysis of the images provided by ISH. Non-uniform background fluorescence of the nuclei introduces problems in the image analysis segmentation procedures. Different procedures were tested. Up to 95% of the hybridization signals could be correctly segmented using digital filtering techniques (min-max filter) to estimate local background intensities. The choice of the objective lens used for the collection of images was found to be extremely important. High magnification objectives with high numerical aperture, which are frequently used for visualization of fluorescence, are not optimal, since they do not have a sufficient depth of field. The system described was used for quantification of ISH signals and allowed accurate measurement of fluorescence spot intensities, as well as of fluorescence ratios obtained with double-labeled probes.     

Quantification of in situ hybridization signals in rat testes.

We performed basic research into quantifying in situ hybridization (ISH) signals in rat testis, a suitable organ for the quantification because germ cells undergo synchronized development and show stage-specific gene expression. In this model experiment, rRNA was selected as the hybridizable RNA in paraffin sections. Specimens fixed with Bouin's fixative and hybridized with digoxygenin-labeled probes could easily be analyzed quantitatively through "posterization" of the images. The amount of rRNA hybridized with the probe was greatest in early primary spermatocytes, followed by pachytene primary spermatocytes, then diplotene spermatocytes, and finally by secondary spermatocytes and spermatids. The amounts reached low levels in metaphase, anaphase, and telophase of meiotic division and early step 1 spermatids, and then slightly increased during spermiogenesis. ISH rRNA staining was a useful parameter for evaluation of the quantitative analysis of mRNA and the levels of hybridizable RNA in tissue sections.     

Quantification of methanogens by fluorescence in situ hybridization with oligonucleotide probe

To monitor anaerobic environmental engineering system, new method of quantification for methanogens was tested. It is based on the measurement of specific binding (hybridization) of 16S rRNA-targeted oligonucleotide probe Arc915, performed by fluorescence in situ hybridization (FISH) and quantified by fluorescence spectrometry. Average specific binding of Arc915 probe was 13.4±0.5 amol/cell of autofluorescent methanogens. It was 14.3, 13.3, and 12.9 amol/cell at the log phase, at stationary phase and at the period of cell lysis of batch culture, respectively. Specific binding of Arc915 probe per 1 ml of microbial sludge suspension from anaerobic digester linearly correlated with concentration of autofluorescent cells of methanogens. Coefficient of correlation was 0.95. Specific binding of oligonucleotide probe Arc915 can be used for the comparative estimation of methanogens during anaerobic digestion of organic waste. Specific binding of Arc915 probe was linear function of anaerobic sludge concentration when it was between 1.4 and 14.0 mg/ml. Accuracy of the measurements in this region was from 5 to 12%.     

Quantification of Leuconostoc populations in mixed dairy starter cultures using fluorescence in situ hybridization

AIMS: Development of a rapid method to identify and quantify Leuconostoc populations in mesophilic starter cultures. METHODS AND RESULTS: 16S rRNA-targeted oligonucleotide probes were used in a whole cell in situ hybridization assay for the identification of the genus Leuconostoc and an undescribed Leuconostoc ribospecies. The probes were fluorescently labelled and used to quantify the Leuconostoc populations in five different mixed starter cultures. CONCLUSIONS: There was a good correlation between the results obtained using fluorescence in situ hybridization (FISH) with that of standard plate counting methods. SIGNIFICANCE AND IMPACT OF THE STUDY: To develop a FISH method capable of identifying and quantifying the Leuconostoc population in starter cultures within 1 day.     

Quantification of ratios of Bacteria and Archaea in methanogenic microbial community by fluorescence in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for Bacteria (Eub338) and Archaea (Arc915) were used for whole-cell, fluorescence in situ hybridization (FISH) to quantify the ratio of these microbial groups in an anaerobic digester. The quantity of specifically bound (hybridized) probe was measured by fluorescence spectrometry and evaluated by analysing the dissociation curve of the hybrids, by the measurement of the binding with a nonsense probe, and by the competitive inhibition of the binding of the labelled probe by the corresponding unlabelled probe. Specific binding of oligonucleotide probes with the biomass of anaerobes was 40–50% of their total binding. The ratio of Arc915 and Eub338 probes hybridized with rRNAs of the cells in anaerobic sludge was 0.50. Measurement of FISH by fluorescence spectrometry appears to be a suitable method for quantification of the microbial community of anaerobes.     

Multiple fluorescence in situ hybridization

A method for multiple fluorescence in situ hybridization is described allowing the simultaneous detection of more than three target sequences with only three fluorescent dyes (FITC, TRITC, AMCA), respectively emitting in the green, red, and blue. This procedure is based on the labeling of (DNA) probes with more than one hapten and visualisation in multiple colors. The possibility to detect multiple targets simultaneously is important for prenatal diagnosis and the detection of numerical and/or structural chromosome aberrations in tumor diagnosis. It may form the basis for an in situ hybridization based chromosome banding technique.     

Quantification of cell-specific substrate uptake by probe-defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization

A technique based on quantitative microautoradiography (QMAR) and fluorescence in situ hybridization (FISH) was developed and evaluated in order to determine the quantitative uptake of specific substrates in probe-defined filamentous bacteria directly in a complex system. The technique, QMAR-FISH, has a resolution of a single cell and is based on an improved fixation protocol and the use of an internal standard of bacteria with known specific radioactivity. The method was used to study the in situ ecophysiology of the filamentous bacteria 'Candidatus Meganema perideroedes' and Thiothrix sp. directly in an activated sludge system. The cellular uptake rate of tritium-labelled substrates revealed an average cell-specific uptake rate of 4.1 yen 10-15 mol of acetate cell-1 h-1 and 3.1 yen 10-15 mol of acetate cell-1 h-1 for the two filamentous species respectively. The two filamentous species had very similar activity in all cells along each filament. Surprisingly, the filaments within both probe-defined populations had threefold variation in activity between the different filaments, demonstrating a large variation in activity level within a single population in a complex system. The substrate affinity (Ks) for uptake of acetate of the cells within the two filamentous bacteria was determined by incubation with variable concentrations of labelled acetate. The Ks values of the 'Candidatus Meganema perideroedes' and the Thiothrix filamentous bacteria were determined to be 1.8 micro M and 2.4 micro M acetate respectively.     

Advances in fluorescence in situ hybridization

The techniques of in situ hybridization (ISH) are widely applied for analyzing the genetic make-up and RNA expression patterns of individual cells. This review focusses on a number of advances made over the last 5 years in the fluorescence ISH (FISH) field, i.e., Fiber-FISH, Multi-colour chromosome painting, Comparative Genomic Hybridization, Tyramide Signal Amplification and FISH with Polypeptide Nucleic Acid and Padlock probes.     

Clinical applications of fluorescence in situ hybridization

We review here the application of fluorescence in situ hybridization with chromosome-specific probes to chromosome classification and to detection of changes in chromosome number or structure associated with genetic disease. Information is presented on probe types that are available for disease detection. We discuss the application of these probes to detection of numerical aberrations important for prenatal diagnosis and to detection and characterization of numerical and structural aberrations in metaphase spreads and in interphase nuclei to facilitate tumor diagnosis.     

Quantification of an Eikelboom type 021N bulking event with fluorescence in situ hybridization and real-time PCR

Primers targeting 16S rRNA genes were designed to detect and quantify Eikelboom type 021N organisms by real-time PCR. Eikelboom type 021N filamentous bulking was induced in a laboratory-scale sequencing batch reactor and the evolution of Eikelboom type 021N 16S rRNA and 16S rRNA genes was monitored. A significant correlation was found between the sludge volume index and the amount of these filamentous organisms present in the sludge (r ²=94.6%, n=10, P<0.01), as measured by real-time PCR. The amount of Eikelboom type 021N 16S rRNA genes increased by a factor of 21 during the experiment, while the 16S rRNA increased by a factor of 33. Moreover, Eikelboom type 021N 16S rRNA increased with increased feeding frequency. It was observed that the RNA:DNA ratio peaked before the sludge volume index increased. In parallel, a fluorescence in situ hybridization study indicated a factor of four increase in the length of Eikelboom type 021N filaments, due to a factor of two increase in both length and number of Eikelboom type 021N filaments. Further, an increase in the fraction of filaments extending outside the activated sludge flocs was observed (19–55%). Monitoring of 16S rRNA genes and 16S rRNA of Eikelboom type 021N was shown to be valuable in evaluating activated sludge settling characteristics; and measuring RNA:DNA ratios may be used as an early warning tool for sludge bulking.     

Routine fluorescence in situ hybridization in soil

The use of fluorescence in situ hybridization (FISH) to identify and enumerate soil bacteria has long been hampered by the autofluorescence of soil particles masking the bacterial signals and because the need of counting hundreds of bacteria in order to achieve statistically reliable data is time consuming. Recently, it was demonstrated that Nycodenz facilitates FISH in soil by concentrating bacteria on membrane filters and avoiding autofluorescent soil particles. We present a routine protocol for FISH in soil including the use of Nycodenz. The protocol allows fast and easy enumeration of hundreds of bacteria. We propose the use of silicon grease coated slides to treat in parallel seven samples per hybridization. Further, we developed a semi-automated approach for the enumeration of bacteria by implementing macros concatenating all steps of the image analyzes in the Image J software. Using Nycodenz, software-assisted bacterial counts statistically matched eye-counts of the same images and it was possible to count 880 DAPI stained bacteria per ten images. Fifty-five percent of these bacteria were co-labelled with the FISH probe specific for the Domain Bacteria, in accordance with recent FISH studies of bacterial populations in bulk soil. With a soil slurry protocol used for comparison, soil particles impaired automatic counts of the bacteria and FISH analysis, and only 88 DAPI stained bacteria per ten images could be counted by eye. With the Nycodenz protocol, 5 mM Na(2)EDTA used as an extractant increased the number of bacteria observed by 49%. In contrast, Tween 20 (1% or 5%) had no significant effect and increased the variability between the samples. Overall, the proposed procedure allows to process a high number of samples and to achieve a time efficient FISH characterization of soil bacterial communities.     

Telomere analysis by fluorescence in situ hybridization and flow cytometry.

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.     

Methods for topographical analysis of intra-nuclear BrdUrd-tagged fluorescence.

The observation of BrdUrd staining in the nuclei of cells from exponentially growing populations reveals different typical replicating patterns. We propose a methodological approach to order and characterize BrdUrd intranuclear distributions. First, visual ordering of the patterns is assessed using a spectral analysis coupled to a k-nearest neighbors clustering technique. Subsequently, nine topographical features are introduced to characterize the spatial distribution of BrdUrd-tagged fluorescence in the nuclei of proliferating cells. These topographical features are based on a structural approach. The localization of fluorescence spots is expressed in terms of the normalized distance from the nuclear border and its standard deviation. These topographical features are simple to calculate and easy to relate to visual experience.     

Construction of repeat-free fluorescence in situ hybridization probes

FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C(0)t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes.     

Characterization of Robertsonian translocations by using fluorescence in situ hybridization.

Fluorescence in situ hybridization with five biotin-labeled probes (three alphoid probes, a probe specific for beta-satellite sequences in all acrocentric chromosomes, and an rDNA probe) was used to characterize 30 different Robertsonian translocations, including three t(13;13); one t(15;15), four t(21;21), three t(13;14), two t(13;15), two (13;21), two t(13;22), one t(14;15), eight t(14;21), two t(14;22), and two t(21;22). Of 8 de novo homologous translocations, only one t(13;13) chromosome was interpreted as dicentric, while 19 of 22 nonhomologous Robertsonian translocations were dicentric. The three monocentric nonhomologous translocations included both of the t(13;21) and one t(21;22). Two of 26 translocations studied using the beta-satellite probe showed a positive signal, while rDNA was undetectable in 10 cases studied. These results indicate that most homologous Robertsonian translocations appear monocentric, while the bulk of nonhomologous translocations show two alphoid signals. A majority of the breakpoints localized using this analysis seem to be distal to the centromere and just proximal to the beta-satellite and nuclear-organizing regions.     

An approach for quantitative assessment of fluorescence in situ hybridization (FISH) signals for applied human molecular cytogenetics.

A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies.     

Quantification of Y chromosome bearing spermatozoa of cattle using in situ hybridization.

An easy assay for quantification of Y chromosome-bearing sperm (Y-sperm) is needed, especially to monitor sperm separation techniques. In the present study a tritiated bovine male-specific DNA fragment was tested for identification of Y-sperm by in situ hybridization. A protocol for in situ hybridization to bovine sperm was developed and used to study the proportion of Y-sperm of 12 bulls. The usefulness of the method in optimization of sperm separation procedures is illustrated through analysis of fractions of sperm separated by Percoll density gradient centrifugation.     

C-banding and fluorescence in situ hybridization studies of the wheat-alien hybrid 'Agrotana'.

'Agrotana', a wheat-alien hybrid (2n = 56), is a potential source of resistance to common root rot, stem rust, wheat streak mosaic virus, and the wheat curl mite. However, the origin of 'Agrotana', reported to be durum wheat x Agropyron trichophorum (pubescent wheatgrass), is uncertain. The objective of this investigation was to determine the chromosome constitution of 'Agrotana' using C-banding and fluorescence in situ hybridization techniques. The F1 hybrid of 'Agrotana' x 'Chinese Spring' wheat showed 7 I + 21 II in 14.9% of the pollen mother cells, evidence of the presence of the A, B, and D genomes in 'Agrotana'. The hybrid had 16 heavily C-banded chromosomes, namely 4A, and 1-7B of wheat, and a translocation that probably involved wheat chromosomes 2A and 2D. In situ hybridization using biotinylated genomic DNA of Ag. trichophorum cv. Greenleaf blocked with CS DNA failed to identify the alien chromosomes in 'Agrotana', indicating that the alien chromosomes were not likely derived from pubescent wheatgrass. In situ hybridization using labelled wheat genomic DNA blocked with 'Agrotana' DNA revealed that 'Agrotana' had 40 wheat, 14 alien, and 2 (a pair) wheat-alien translocated chromosomes. There was no homology between wheat and the alien chromosomes or chromosome segments involved in the wheat-alien recombinant. Two of the seven pairs of alien chromosomes were homoeologous to each other. The ability to identify alien chromatin in wheat using labelled wheat DNA instead of labelled alien DNA will be particularly useful in chromosome engineering of wheat germplasms having alien chromatin of unknown origin.