A straightforward DOPE (double labeling of oligonucleotide probes)-FISH (fluorescence in situ hybridization) method for simultaneous multicolor detection of six microbial populations

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is an essential tool for the cultivation-independent identification of microbes within environmental and clinical samples. However, one of the major constraints of conventional FISH is the very limited number of different target organisms that can be detected simultaneously with standard epifluorescence or confocal laser scanning microscopy. Recently, this limitation has been overcome via an elegant approach termed combinatorial labeling and spectral imaging FISH (CLASI-FISH) (23). This technique, however, suffers compared to conventional FISH from an inherent loss in sensitivity and potential probe binding biases caused by the competition of two differentially labeled oligonucleotide probes for the same target site. Here we demonstrate that the application of multicolored, double-labeled oligonucleotide probes enables the simultaneous detection of up to six microbial target populations in a straightforward and robust manner with higher sensitivity and less bias. Thus, this newly developed technique should be an attractive option for all researchers interested in applying conventional FISH methods for the study of microbial communities.     

Single-cell systems biology by super-resolution imaging and combinatorial labeling

Fluorescence microscopy is a powerful quantitative tool for exploring regulatory networks in single cells. However, the number of molecular species that can be measured simultaneously is limited by the spectral overlap between fluorophores. Here we demonstrate a simple but general strategy to drastically increase the capacity for multiplex detection of molecules in single cells by using optical super-resolution microscopy (SRM) and combinatorial labeling. As a proof of principle, we labeled mRNAs with unique combinations of fluorophores using fluorescence in situ hybridization (FISH), and resolved the sequences and combinations of fluorophores with SRM. We measured mRNA levels of 32 genes simultaneously in single Saccharomyces cerevisiae cells. These experiments demonstrate that combinatorial labeling and super-resolution imaging of single cells is a natural approach to bring systems biology into single cells.     

MiL-FISH: Multilabeled Oligonucleotides for Fluorescence In Situ Hybridization Improve Visualization of Bacterial Cells

Fluorescence in situ hybridization (FISH) has become a vital tool for environmental and medical microbiology and is commonly used for the identification, localization, and isolation of defined microbial taxa. However, fluorescence signal strength is often a limiting factor for targeting all members in a microbial community. Here, we present the application of a multilabeled FISH approach (MiL-FISH) that (i) enables the simultaneous targeting of up to seven microbial groups using combinatorial labeling of a single oligonucleotide probe, (ii) is applicable for the isolation of unfixed environmental microorganisms via fluorescence-activated cell sorting (FACS), and (iii) improves signal and imaging quality of tissue sections in acrylic resin for precise localization of individual microbial cells. We show the ability of MiL-FISH to distinguish between seven microbial groups using a mock community of marine organisms and its applicability for the localization of bacteria associated with animal tissue and their isolation from host tissues using FACS. To further increase the number of potential target organisms, a streamlined combinatorial labeling and spectral imaging-FISH (CLASI-FISH) concept with MiL-FISH probes is presented here. Through the combination of increased probe signal, the possibility of targeting hard-to-detect taxa and isolating these from an environmental sample, the identification and precise localization of microbiota in host tissues, and the simultaneous multilabeling of up to seven microbial groups, we show here that MiL-FISH is a multifaceted alternative to standard monolabeled FISH that can be used for a wide range of biological and medical applications.     

Spectral imaging of multi-color chromogenic dyes in pathological specimens.

We have investigated the use of spectral imaging for multi-color analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier-transform spectroscopy and digital imaging. A pixel-by-pixel spectrum-based color classification is presented of single-, double-, and triple-color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki-67 and TP53 in paraffin-embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright-field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference library. We conclude that spectral imaging of multi-color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell- and tissue specimens.     

Signal to noise analysis of multiple color fluorescence imaging microscopy

BACKGROUND: Various approaches that were recently developed demonstrate the ability to simultaneously detect all human (or other species) chromosomes by using combinatorial labeling and fluorescence in situ hybridization (FISH). With the growing interest in this field, it is important to develop tools for optimizing and estimating the accuracy of different experimental methods. METHODS: We have analyzed the principles of multiple color fluorescence imaging microscopy. First, formalism based on the physical principles of fluorescence microscopy and noise analysis is introduced. Next, a signal to noise (S/N) analysis is performed and summarized in a simple accuracy criterion. The analysis assumes shot noise to be the dominant source of noise. RESULTS: The accuracy criterion was used to calculate the S/N of multicolor FISH (M-FISH), spectral karyotyping, ratio imaging, and a method based on using a set of broad band filters. Spectral karyotyping is tested on various types of samples and shows accurate classifications. We have also tested classification accuracy as a function of total measurement time. CONCLUSIONS: The accuracy criterion that we have developed can be used for optimizing and analyzing different multiple color fluorescence microscopy methods. The assumption that shot noise is dominant in these measurements is supported by our measurements.     

Near-field optical imaging of abasic sites on a single DNA molecule

Scanning near-field optical microscopy (SNOM) imaging was performed to allow for the direct visualization of damaged sites on individual DNA molecules to a scale of a few tens of nanometers. Fluorescence in situ hybridization on extended DNA molecules was modified to detect a single abasic site. Abasic sites were specifically labelled with a biotinlylated aldehyde-reactive probe and fluorochrome-conjugated streptavidin. By optimizing the performance of the SNOM technique, we could obtain high contrast near-field optical images that enabled high-resolution near-field fluorescence imaging using optical fiber probes with small aperture sizes. High-resolution near-field fluorescence imaging demonstrated that two abasic sites within a distance of 120 nm are clearly obtainable, something which is not possible using conventional fluorescence in situ hybridization combined with far-field fluorescence microscopy.     

Double and triple in situ hybridization for coexpression studies: combined fluorescent and chromogenic detection of neuropeptide Y (NPY) and serotonin receptor subtype mRNAs expressed at different abundance levels

Multiple fluorescence in situ hybridization is the method of choice for studies aimed at determining simultaneous production of signal transduction molecules and neuromodulators in neurons. In our analyses of the monoamine receptor mRNA expression of peptidergic neurons in the rat telencephalon, double tyramide-signal-amplified fluorescence in situ hybridization delivered satisfactory results for coexpression analysis of neuropeptide Y (NPY) and serotonin receptor 2C (5-HT2C) mRNA, a receptor subtype expressed at high-to-moderate abundance in the regions analyzed. However, expression of 5-HT1A mRNA, which is expressed at comparatively low abundance in many telencephalic areas, could not be unequivocally identified in NPY mRNA-reactive neurons due to high background and poor signal-to-noise ratio in fluorescent receptor mRNA detections. Parallel chromogenic in situ hybridization provided clear labeling for 5-HT1A mRNA and additionally offered the possibility to monitor the chromogen deposition at regular time intervals to determine the optimal signal-to-noise ratio. We first developed a double labeling protocol combining fluorescence and chromogenic in situ hybridization and subsequently expanded this variation to combine double fluorescence and chromogenic in situ hybridization for triple labelings. With this method, we documented expression of 5-HT2C and/or 5-HT1A in subpopulations of telencephalic NPY-producing neurons. The method developed in the present study appears suitable for conventional light and fluorescence microscopy, combines advantages of fluorescence and chromogenic in situ hybridization protocols and thus provides a reliable non-radioactive alternative to previously published multiple labeling methods for coexpression analyses in which one mRNA species requires highly sensitive detection.     

Nonradioactive in Situ Hybridization with Digoxigenin Labeled DNA Probes

Nonradioactive in situ hybridization techniques are becoming increasingly important tools for rapid analysis of the topological organization of DNA and RNA sequences within cells. Prerequisite for further advances with these techniques are multiple labeling and detection systems for different probes. Here we summarize our results with a recently developed labeling and detection system. The DNA probe for in situ hybridization is modified with digoxigenin-labeled deoxyuridine-triphosphate. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[11]-dUTP). Labeled DNA probes were hybridized in situ to chromosome preparations. The hybridization signal was detected using digoxigenin-specific antibodies covalently coupled to enzyme markers (alkaline phosphatase or peroxidase) or to fluorescent dyes. Color reactions catalyzed by the enzymes resulted in precipitates located on the chromosomes at the site of probe hybridization. This was verified by hybridizing DNA probes of known chromosomal origin. The signals were analyzed by bright field, reflection contrast and fluorescence microscopy. The results indicate that the new technique gives strong signals and can also be used in combination with other systems (e.g., biotin) to detect differently labeled DNA probes on the same metaphase plate.     

Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS

To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with (13)C-carbon and (15)N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.     

The labeling efficiency of human telomeres is increased by double-strand PRINS

Telomeres are composed of tandem repeated sequences, TTAGGG, that can be detected either by fluorescence in situ hybridization (FISH), more efficiently by using a peptide nucleic acid (PNA) probe, or by the primed in situ (PRINS) technique. However, the efficiency of human telomere labeling using PRINS is somewhat lower than the efficiency using PNA-FISH. To solve this problem, we developed a double-strand PRINS technique, which uses two primers, (TTAGGG)₇ and (CCCTAA)₇, to label both forward and reverse telomeric DNA strands. A total of 120 lymphocyte metaphases obtained from three normal adults were scored to evaluate the labeling efficiency based upon the telomere signal frequency present in chromatid ends and chromosome arms. As a comparison, 30 metaphases from the same three individuals were evaluated using PNA-FISH. The average labeling efficiency of PRINS was increased to a level very close to that obtained with PNA-FISH. Therefore, we demonstrated that the low labeling efficiency of human telomeres with regular PRINS was likely caused by uneven annealing of primers at the relatively short human telomere sequences, resulting in some telomere sites with very weak or absent labeling. We suggest that the present double-strand labeling protocol is critical to maximize the labeling efficiency of the human telomere sequence when using the PRINS technique.     

GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data

Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers.     

荧光原位杂交探针应用于平衡易位t（1，6）（q42；p23）胚胎植入前诊断的研究

背景：染色体相互易位在人群中比较常见,下一代常常产生相同或不同的易位,易导致容易流产,而植入前诊断方法之一的CGH难以检测到相互易位,因此原位杂交（FISH）依然是解决诊断相互易位的有力手段。目的：通过设计个体化的FISH探针,制备探针,并在卵裂球单细胞水平进一步验证探针的准确性,为筛选正常核型的囊胚进行植入奠定技术基础,为个体化的FISH探针植入前诊断提供应用研究基础。方法：通过设计1 q和6p平衡易位探针,进行探针标记,再采用患者和正常人核型验证探针质量,通过荧光原位杂交技术进一步检测正常人受精后的卵裂球中1q 和6p平衡易位对易位染色体状态。结果：3个卵接球裂均呈现单个完整细胞核,荧光原位杂交中各细胞核均有清晰明亮的杂交信号。信号数分别为2。均为正常胚胎,可以考虑进一步对该易位患者进行卵裂球进行诊断,上述研究对个体化的易位探针的应用研究提供了研究基础。     

Chromosomal in situ hybridization using yeast artificial chromosomes

Large DNA fragment cloning methods using yeast artificial chromosomes (YACs) have vastly improved the strategies for constructing physical maps of regions of complex genomes, as well as for isolating and cloning genes important for human disease. We present here a simple and rapid method for carrying out in situ hybridization to metaphase chromosomes using isolated YAC clones by labeling DNA directly in agarose gel slices. Nonisotopic labeling and chromosomal in situ hybridization can be used to determine the chromosomal localization of individual YAC clones on human metaphase chromosomes. This method can also be used to characterize YAC clones consisting of single fragments from those that contain concatamerized, and thus artifactual, inserts. This technique also offers a valuable tool to study consistent translocations in neoplastic diseases by identifying YACs that span a specific chromosomal breakpoint.     

Toward a multicolor chromosome bar code for the entire human karyotype by fluorescence in situ hybridization

A colored banding pattern for human chromosomes is described that distinguishes each chromosome in a single fluorescence in situ hybridization with a set of subregional DNA probes. Alu/polymerase chain reaction products of various human/rodent somatic cell hybrids (fragment hybrids) were pooled into two probe sets that were labeled differentially and detected by red and green fluorescence. Chromosome regions hybridized by DNA present in both pools appeared yellow. The result was a multi-color set of 110 distinct signals per haploid chromosome set for the human karyotype. Each individual chromosome showed a unique sequence of signals, a result termed the “chromosome bar code”. The reproducibility of the hybridization pattern in various labeling and hybridization experiments was analyzed by computer densitometry. We have applied the chromosome bar code both in diagnostic cytogenetics and in genome studies. The approach allows the rapid identification of chromosomes and chromosome rearrangements. Although not yet showing the resolution of classical banding patterns, the present experiments demonstrate various applications in which the present multi-color bar code can significantly add to the spectrum of cytogenetic techniques. Received: 18 December 1996 / Accepted: 10 February 1997     

Unlabeled Helper Oligonucleotides Increase the In Situ Accessibility to 16S rRNA of Fluorescently Labeled Oligonucleotide Probes

Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in Escherichia coli DSM 30083^T. The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for E. coli. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.     

Quencher-free multiplexed monitoring of DNA reaction circuits

We present a simple yet efficient technique to monitor the dynamics of DNA-based reaction circuits. This technique relies on the labeling of DNA oligonucleotides with a single fluorescent modification. In this quencher-free setup, the signal is modulated by the interaction of the 3'-terminus fluorophore with the nucleobases themselves. Depending on the nature of the fluorophore's nearest base pair, fluorescence intensity is decreased or increased upon hybridization. By tuning the 3'-terminal nucleotides, it is possible to obtain opposite changes in fluorescence intensity for oligonucleotides whose hybridization site is shifted by a single base. Quenching by nucleobases provides a highly sequence-specific monitoring technique, which presents a high sensitivity even for small oligonucleotides. Compared with other sequence-specific detection methods, it is relatively non-invasive and compatible with the complex dynamics of DNA reaction circuits. As an application, we show the implementation of nucleobase quenching to monitor a DNA-based chemical oscillator, allowing us to follow in real time and quantitatively the dephased oscillations of the components of the network. This cost-effective monitoring technique should be widely implementable to other DNA-based reaction systems.     

A method for simultaneously delineating multiple targets in 3D-FISH using limited channels, lasers, and fluorochromes

Many studies have suggested a link between the spatial organization of genomes and fundamental biological processes such as genome reprogramming, gene expression, and differentiation. Multicolor fluorescence in situ hybridization on three-dimensionally preserved nuclei (3D-FISH), in combination with confocal microscopy, has become an effective technique for analyzing 3D genome structure and spatial patterns of defined nucleus targets including entire chromosome territories and single gene loci. This technique usually requires the simultaneous visualization of numerous targets labeled with different colored fluorochromes. Thus, the number of channels and lasers must be sufficient for the commonly used labeling scheme of 3D-FISH, “one probe-one target”. However, these channels and lasers are usually restricted by a given microscope system. This paper presents a method for simultaneously delineating multiple targets in 3D-FISH using limited channels, lasers, and fluorochromes. In contrast to other labeling schemes, this method is convenient and simple for multicolor 3D-FISH studies, which may result in widespread adoption of the technique. Lastly, as an application of the method, the nucleus locations of chromosome territory 18/21 and centromere 18/21/13 in normal human lymphocytes were analyzed, which might present evidence of a radial higher order chromatin arrangement.     

An improved method for chromosome-specific labeling of alpha satellite DNA in situ by using denatured double-stranded DNA probes as primers in a primed in situ labeling (PRINS) procedure.

An improved primed in situ labeling (PRINS) procedure that provides fast, highly sensitive, and nonradioactive cytogenetic localization of chromosome-specific tandem repeat sequences is presented. The PRINS technique is based on the sequence-specific annealing in situ of unlabeled DNA. This DNA then serves as primer for chain elongation in situ catalyzed by a DNA polymerase. If biotin-labeled nucleotides are used as substrate for the chain elongation, the hybridization site becomes labeled with biotin. The biotin is subsequently made visible through the binding of FITC-labeled avidin. Tandem repeat sequences may be detected in a few hours with synthetic oligonucleotides as primers, but specific labeling of single chromosomes is not easily obtained. This may be achieved, however, if denatured double-stranded DNA fragments from polymerase-chain-reaction products or cloned probes are used as primers. In the latter case, single chromosome pairs are stained with a speed and ease (1 h reaction and no probe labeling) that are superior to traditional in situ hybridization. Subsequent high-quality Q banding of the chromosomes is also possible. The developments described here extends the range of applications of the PRINS technique, so that it now can operate with any type of probe that is available for traditional in situ hybridization.     

COBRA: combined binary ratio labeling of nucleic-acid probes for multi-color fluorescence in situ hybridization karyotyping

Combined binary ratio labeling (COBRA) is designed to increase the multiplicity of fluorescence in situ hybridization (FISH)--i.e., the number of targets that can be distinguished simultaneously. In principle, chemical (ULS), enzymatic (nick translation or random priming) or PCR-based labeling procedures of probes can be used. The method was originally designed to label chromosome-painting probes, but has also been used for probe sets specific for subtelomeric regions. COBRA imaging requires a digital fluorescence microscope equipped for sequential excitation and recording of color images. Staining of all 24 human chromosomes is accomplished with only four fluorochromes, compared with five for methods based on combinatorial labeling. The COBRA procedure takes approximately 6 h laboratory work, 2-3 d incubation and a few hours imaging. The method is routinely applied in research (cultured cells from human or mouse origin) or to support clinical diagnosis, such as postnatal and perinatal genetic testing and in solid tumors.     

PRINS detection of 18S rDNA in pig, red fox and Chinese raccoon dog, and centromere DNA in horse

The fluorescence in situ hybridization (FISH) technique is widely used in animal cytogenetics. Contrary to FISH procedure, primed in situ DNA synthesis (PRINS) does not require the DNA probe preparation (design, synthesis, gel purification of PCR products and labeling). The PRINS method with primers used as 'DNA probes' is both PCR-sensitive and allows for chromosomal localization of DNA sequences. Here, we show the application of PRINS reaction with one unlabeled oligonucleotide pair to identify 18S rDNA loci in three different animal species: domestic pig (Sus scrofa), red fox (Vulpes vulpes) and Chinese raccoon dog (Nyctereutes procyonoides procyonoides). We present the data of indirect labeling with the digoxigenin-PRINS using two different pairs of primers complementary to centromeric region of horse (Equus caballus) chromosomes. Our new PRINS application may be considered as a useful tool for chromosome investigation in the field of domestic and wild animal genetics and evolution.