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.     

Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization

Comparative genomic in situ hybridization (CGH) provides a new possibility for searching genomes for imbalanced genetic material. Labeled genomic test DNA, prepared from clinical or tumor specimens, is mixed with differently labeled control DNA prepared from cells with normal chromosome complements. The mixed probe is used for chromosomal in situ suppression (CISS) hybridization to normal metaphase spreads (CGH-metaphase spreads). Hybridized test and control DNA sequences are detected via different fluorochromes, e.g., fluorescein isothiocyanate (FITC) and tetraethylrhodamine isothiocyanate (TRITC). The ratios of FITC/TRITC fluorescence intensities for each chromosome or chromosome segment should then reflect its relative copy number in the test genome compared with the control genome, e.g., 0.5 for monosomies, 1 for disomies, 1.5 for trisomies, etc. Initially, model experiments were designed to test the accuracy of fluorescence ratio measurements on single chromosomes. DNAs from up to five human chromosome-specific plasmid libraries were labeled with biotin and digoxigenin in different hapten proportions. Probe mixtures were used for CISS hybridization to normal human metaphase spreads and detected with FITC and TRITC. An epifluorescence microscope equipped with a cooled charge coupled device (CCD) camera was used for image acquisition. Procedures for fluorescence ratio measurements were developed on the basis of commercial image analysis software. For hapten ratios 4/1, 1/1 and 1/4, fluorescence ratio values measured for individual chromosomes could be used as a single reliable parameter for chromosome identification. Our findings indicate (1) a tight correlation of fluorescence ratio values with hapten ratios, and (2) the potential of fluorescence ratio measurements for multiple color chromosome painting. Subsequently, genomic test DNAs, prepared from a patient with Down syndrome, from blood of a patient with Tcell prolymphocytic leukemia, and from cultured cells of a renal papillary carcinoma cell line, were applied in CGH experiments. As expected, significant differences in the fluorescence ratios could be measured for chromosome types present in different copy numbers in these test genomes, including a trisomy of chromosome 21, the smallest autosome of the human complement. In addition, chromosome material involved in partial gains and losses of the different tumors could be mapped to their normal chromosome counterparts in CGH-metaphase spreads. An alternative and simpler evaluation procedure based on visual inspection of CCD images of CGH-metaphase spreads also yielded consistent results from several independent observers. Pitfalls, methodological improvements, and potential applications of CGH analyses are discussed.     

Quantification of inter- and intra-nuclear variation of fluorescence in situ hybridization signals.

This study aims at the quantification of specific DNA sequences by 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. Quantification of the fluorescence ISH signals was performed using an epifluorescence microscope equipped with a multi-wavelength illuminator, and a cooled charge coupled device (CCD) camera. Specific image analysis programs were developed for the segmentation and analysis of the images provided by ISH. The fluorescence intensity distributions of the ISH spots showed large internuclear variation (CVs up to 65%) for the probes used. The variation in intensity was found to be independent of the probe, the type of labeling, and the type of immunocytochemical detection used. Variation in intensity was not caused primarily by the immunocytochemical detection method, since directly fluorescein-labeled probes showed similar internuclear variation. Furthermore, it was found that different white blood cell types, which harbor different degrees of compactness of the nuclear chromatin, showed the same variation. The intra-nuclear variation in intensity of the ISH spots on the two chromosome homologs within one nucleus was significantly smaller (approximately 20%) than the inter-nuclear variation, probably due to more constant local hybridization conditions. Due to the relatively small intranuclear variation, copy number polymorphisms of the satellite DNA sequence on chromosome 1 could readily be quantified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescein conjugates as indicators of subcellular pH. A critical evaluation

Fluorescein and tetramethylrhodamine conjugates to protein or dextran were used to determine subcellular pH. The pH dependence of fluorescence of fluorescein isothiocyanate (FITC) conjugates could be described by a single proton dissociation (pK'a approximately 6.8). This allowed pH to be derived accurately from spectra using the simple Henderson-Hasslebach equation. FITC and TRITC conjugates were delivered to mouse macrophage lysosomes by pinocytosis. Lysosomal pH was then determined in several different ways. First, by direct matching of the subcellular fluorescence spectrum with calibration spectra obtained in free solution. Secondly, monensin was used to equilibrate internal and extracellular pH. Subcellular pH could then be determined by the relative increase in fluorescence of the FITC conjugate without loss of probe from the lysosomes. This allowed the calibration of pH dependence with the probe in situ. Finally, macrophages were permitted to pinocytose FITC and TRITC dextran conjugates simultaneously. pH could be determined from the ratio of emissions from the two dyes within the lysosomes. Each of these different methods gave a similar value for lysosomal pH (4.8 +/- 0.1).     

Cleavage of pig embryos after labeling with fluorescent dyes

In studies on embryonic development, treated and control ova could be co-mixed before transfer to recipients if nontoxic labels for ova were available. These experiments were conducted to determine whether pig ova would continue to cleave after being stained with the fluorochromes tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC). In the first experiment, pig ova stained with TRITC and unstained control ova were transferred into opposite oviducts of recipient gilts. In the second experiment, ova stained with TRITC and ova stained with FITC were transferred into opposite oviducts of recipient gilts. Embryos were recovered 96 h after transfer (Day 6; Day 0 = onset of estrus), the presence of fluorescence was determined, and the number of nuclei per embryo was assessed. Stained ova retained sufficient fluorochrome to permit detection until the zonae pellucidae were shed. Development of embryos stained with TRITC was equal to that of unstained control embryos. However, development of embryos stained with FITC appeared slightly retarded in comparison to that of TRITC-stained embryos. These findings demonstrate the efficacy of the fluorescent staining technique for pig ova during the first six days of pregnancy.     

Preparation of fluorescent-labeled DNA and its use as a probe in molecular hybridization

A method of the fluorescent-labeled DNA preparation for visualization of the complementary nucleotide sequences has been developed. Polynucleotide probes were alkylated randomly by 4-(N-methylamino-N-2-chloroethyl)-benzylamine followed by modification with such fluorochromes as dansyl chloride or fluorescein isothiocyanate (FITC). It was found that the FITC but not dansyl-labeled polynucleotides could serve as efficient probes when about 4% of nitrogen bases were modified. The conditions minimizing the loss of the alkylated bases from DNA were determined. The procedure for hybridization with FITC-labeled DNA as a probe is described, concentration of DNA probe being about 4 ng/mm2 of the nitrocellulose filter.     

A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase-fast red reaction.

We have used naphthol-ASMX-phosphate and Fast Red TR in combination with alkaline phosphatase (APase) to produce fluorescent precipitated reaction products in a non-radioactive in situ hybridization (ISH) method. To obtain optimal and discrete localization of the strongly red fluorescent ISH signals, the enzyme precipitation procedure was optimized. The optimal reaction time and the concentrations of substrate and capture agent were determined. Furthermore, polyvinyl alcohol (PVA) was used to increase the viscosity of the reaction mixture and thus to reduce diffusion of the reaction product. Our results show that the APase-Fast Red detection method has at least the same sensitivity as currently observed in other immunofluorescent detection systems. A single copy DNA sequence of 15.8 KB could be localized with high efficiency in metaphase spreads and in interphase nuclei. Double labeling procedures, in which the FITC- and azo-dye fluorescence are combined, are also feasible. The red fluorescent ISH signals showed hardly any fading as compared with FITC fluorescence on exposure to either light from the mercury-arc lamp or laser light. Therefore, these red fluorescent signals with a virtually permanent character allow a better analysis and three-dimensional localization of such cytochemically detected genomic fractions by means of confocal scanning laser microscopy as compared with the use of FITC, TRITC, or Texas Red as label.     

Development of multi-color FISH method for analysis of seven Bifidobacterium species in human feces

We have developed a multi-color fluorescence in situ hybridization (FISH) method which detects, by a single reaction, all seven species of Bifidobacterium (B. adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. dentium, and B. longum), the dominant bacteria in human feces. First, eight new types of oligonucleotide probe were designed, complementary with the 16S rRNA sequence specific to genus Bifidobacterium and each bifidobacterial species described above. Using whole cell hybridization, the fluorescent intensity was measured against the bacterial species targeted by each probe, to show that each probe is specific to the targeted bacteria and that the relative fluorescent intensity (RFI) as an indicator of probe accessibility is high at 61-117%. Then, bacterial species-specific probes were labeled with fluorochromes (FITC, TAMRA and Cy5) in seven different ways, singly or in combination. Using these probes, seven species of Bifidobacterium were differentially stained in mixed samples of cultured bacteria and feces from adult volunteers, proving the efficacy of this technique.     

Stabilization of fluorochromes after immunofluorescent staining of tissue and embedding in Epon

Tissue blocks or sections immunofluorescent stained before embedding, i.g., liver and kidney, can be stored for more than 3 years without demonstrable fluorescence decay. The processing steps, including poststaining dehydration by alcohols and embedding in expoxy resins, seem to stabilize the fluorochromes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) so that they fade less during illumination. This is an advantage of the pre-embedding, immunofluorescent staining technique which is combined with a lack of damage to the antigens by the plastic embedding medium.     

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.     

Stabilization of Fluorochromes After Immunofluorescent Staining of Tissue and Embedding in Epon

Tissue blocks or sections immunofluo-nt stnined before embedding, ig., liver and kidney, can be stored for more than 3 years without demonstrable fluorescence decay. The processing steps, including poststaining dehydration by alcohols and embedding in expoxy resins, seem to stabilize the fluorochromes fluo-in isotbiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) so that they fade I- during illumination. This is an advantage of the preembedding, immunofluo-nt staining technique which is combined with a lack of damage to the antigens by the plastic embedding medium.     

Producing single-stranded DNA probes with the Taq DNA polymerase: a high yield protocol

We report an efficient procedure to synthesize either single- or double-stranded probes labeled with biotin-11-dUTP, biotin-21-dUTP or digoxigenin-11-dUTP. To produce the single-stranded probe, only a single primer is utilized in a Taq polymerase amplification of 55 cycles. A cytomegalovirus probe is presented. This procedure allows easy production of nonradioactively labeled pure single-stranded probes of any desired length and specificity.     

Evaluation of five green fluorescence-emitting streptavidin-conjugated fluorochromes for use in immunofluorescence microscopy

 Fluorescein isothiocyanate (FITC) is largely used in immunofluorescence methods. We propose to analyse the quality of some recent fluorochromes using image analysis. Fluorochromes tested include FITC and dichlorotriazinylaminofluorescein (DTAF), dipyrrometheneboron difluoride (BODIPY), Rhodol Green and cyanine 2. RAMOS cells were immunolabelled against the proliferating cell nuclear antigen (PCNA) revealed by the biotin-streptavidin technique. Slides were mounted in anhydrous glycerol or in buffered glycerol (pH 7.0 or pH 8.5). No antifading medium was added. Cell fluorescence emission intensity and bleaching characteristics were measured. Rhodol Green exhibited the highest fluorescence intensity and the best photobleaching resistance. Although BODIPY also resisted well during the photobleaching assay, its fluorescence intensity was weak. FITC, DTAF and cyanine 2 showed intermediate fluorescence intensity and a fast decay of fluorescence. Among the green emitting fluorochromes tested, Rhodol Green appeared to be the best. Accepted: 1 April 1996     

Evaluation of fluorochromes for flow cytometric detection of Cryptosporidium parvum oocysts labelled by fluorescent in situ hybridization

Oligonucleotide probes specific to Cryptosporidium parvum (CRY1) were conjugated with a range of fluorochromes. The fluorescence after in situ hybridization (FISH) labelling of oocysts and controls was assessed. The objective was to determine the most suitable conjugate for FISH labelling, followed by analysis with a 488 nm laser flow cytometer. The most promising candidate was fluorescein isothiocyanate but only when linked to the CRY1 probe via an 18-carbon spacer arm consisting of six ethylene glycol moieties. The use of the spacer increased fluorescent signals fivefold compared with an equivalent probe in which the FITC was linked directly to the 5'-amino group of the DNA.     

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.     

Confocal multilaser focusing and single-laser characterization of ultraviolet excitable stains of cellular preparations

BACKGROUND: The aims of this study were (1) to realign cellular preparations when spots and structures are excited by different lasers of a confocal laser scanning microscope (multilaser studies); (2) to avoid the use of realigment methods by selecting fluorochromes that can be excited by only one laser (single-laser experiments). METHODS: In multilaser studies, we used propidium iodide fluorescent beads, as well as tetramethyl rhodamine isothiocyanate (TRITC), fluorescein isothiocyanate (FITC), and 4'-6 diamidino-2-phenylindole (DAPI)-stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser lines of a confocal laser scanning microscope. Single-laser experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines and tissue were counterstained by DAPI and cytoplasms were labeled with ELF-97 substrates. Factor analysis of medical images (FAMIS) and correlation methods were used to realign shifted images, focus images, and characterize each fluorochrome when necessary. RESULTS: In multilaser studies, superimposition of factor images corrected Z shifts and correlation methods provided X, Y correction values. In single-laser experiments, each fluorochrome was clearly distinguished in the group of fluorochromes. Estimated images in both studies showed colocalizations of structures. CONCLUSIONS: It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is also possible to study colocalization of UV excitable fluorochromes (DAPI, ELF-97, europium) in cellular and tissular preparations via multilaser or single-laser experiments.     

Improved indirect fluorescence immunocytochemical method using counterstains.

Immunocytochemistry in recent years has provided powerful tools for research in neurobiology. One of the more popular techniques is the indirect fluorescence technique in which fluorescein isothiocyanate (FITC) or tetrahodamine isothiocyanate (TRITC) is used. Although widely used, this technique has two disadvantages: (1) localization may be difficult in relation to background morphology, and (2) the fluorescence fades. The study reported here describes a modification of an indirect immunocytochemical technique using FITC, TRITC and 7-amino-4-methyl-coumarin-3-acetic acid (AMCA) which enhances localization and significantly prolongs fluorescence. Evans blue was used as a counterstain. The results show that FITC and AMCA stained cells are seen against a background of clearly distinguishable cells after counterstaining with Evans blue. However, Evans blue is not compatible with TRITC. In addition, the fluorescence life of the FITC is extended from several days to several weeks with Evans blue. These results clearly indicate that Evans blue can be used to improve indirect fluorescence immunocytochemical techniques.     

High-resolution in situ hybridization to whole-mount zebrafish embryos

The in situ hybridization (ISH) technique allows the sites of expression of particular genes to be detected. This protocol describes ISH of digoxigenin-labeled antisense RNA probes to whole-mount zebrafish embryos. In our method, PCR-amplified sequence of a gene of interest is used as a template for the synthesis of an antisense RNA probe, which is labeled with digoxigenin-linked nucleotides. Embryos are fixed and permeabilized before being soaked in the digoxigenin-labeled probe. We use conditions that favor specific hybridization to complementary mRNA sequences in the tissue(s) expressing the corresponding gene. After washing away excess probe, hybrids are detected by immunohistochemistry using an alkaline phosphatase-conjugated antibody against digoxigenin and a chromogenic substrate. The whole procedure takes only 3 days and, because ISH conditions are the same for each probe tested, allows high throughput analysis of zebrafish gene expression during embryogenesis.     

Conjugation of lectins with fluorochromes: an approach to histochemical double labeling of carbohydrate components.

Methodical investigations on the coupling of lectins (Con A, LcL, WGA, RcA) to tetramethylrhodamine isothiocyan ate (TRITC) are reported. 20-microgram of TRITC per mg of lectin were found to be the optimal amount of TRITC for the conjugation. With this fluorochrome: protein ratio conjugates were produced which resulted in a specific and brilliant fluorescence in tissue staining. The optimally conjugated lectins were separated on DEAE-Sephadex-A 50. Using two different lectins which were conjugated with TRITC or FITC, respectively, a double labeling of different lectin-binding sites in tissue sections was achieved.