Fluorescence in situ hybridization of scarce leptin receptor mRNA using the enzyme-labeled fluorescent substrate method and tyramide signal amplification.

To increase the sensitivity of fluorescence in situ hybridization (FISH) for detection of low-abundance mRNAs, we performed FISH on cryostat sections of rat hypothalamus with biotin-labeled riboprobes to leptin receptor (ObRb) and amplified the signal by combining tyramide signal amplification (TSA) and Enzyme-Labeled Fluorescent alkaline phosphatase substrate (ELF) methods. First, TSA amplification was done with biotinylated tyramide. Second, streptavidin-alkaline phosphatase was followed by the ELF substrate, producing a bright green fluorescent reaction product. FISH signal for ObRb was undetectable when TSA or ELF methods were used alone, but intense ELF FISH signal was visible in hypothalamic neurons when the ELF protocol was preceded by TSA. The TSA-ELF was combined with FISH for pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) mRNAs by hybridizing brain sections in a cocktail containing digoxigenin-labeled riboprobes to NPY or POMC mRNA and biotin-labeled riboprobes to ObRb mRNA. Dioxigenin-labeled NPY or POMC mRNA hybrids were subsequently detected first with IgG-Cy3. Then biotin-labeled leptin receptor hybrids were detected with the TSA-ELF method. Combining the ELF and TSA amplification techniques enabled FISH detection of scarce leptin receptor mRNAs and permitted the identification of leptin receptor mRNA in cells that also express NPY and POMC gene products.     

Fluorescence in situ hybridisation (FISH)--the next generation

Fluorescence in situ hybridisation (FISH) has become one of the major techniques in environmental microbiology. The original version of this technique often suffered from limited sensitivity due to low target copy number or target inaccessibility. In recent years there have been several developments to amend this problem by increasing signal intensity. This review summarises various approaches for signal amplification, focussing especially on two widely recognised varieties, tyramide signal amplification and multiply labelled polynucleotide probes. Furthermore, new applications for FISH are discussed, which arise from the increased sensitivity of the method.     

In situ detection of antibiotic-resistance elements in single Bacillus cereus spores

Rapid detection of Bacillus spores is a challenging task in food and defense industries. In situ labeling of spores would be advantageous for detection by automated systems based on single-cell analysis. Determination of antibiotic-resistance genes in bacterial spores using in situ labeling has never been developed. Most of the in situ detection schemes employ techniques such as fluorescence in situ hybridization (FISH) that target the naturally amplified ribosomal RNA (rRNA). However, the majority of antibiotic-resistance genes has a plasmidic or chromosomal origin and is present in low copy numbers in the cell. The main challenge in the development of low-target in situ detection in spores is the permeabilization procedure and the signal amplification required for detection. This study presents permeabilization and in situ signal amplification protocols, using Bacillus cereus spores as a model, in order to detect antibiotic-resistance genes. The permeabilization protocol was designed based on the different layers of the Bacillus spore. Catalyzed reporter deposition (CARD)–FISH and in situ polymerase chain reaction (PCR) were used as signal amplification techniques. B. cereus was transformed with the high copy number pC194 and low copy number pMTL500Eres plasmids in order to induce resistance to chloramphenicol and erythromycin, respectively. In addition, a rifampicin-resistant B. cereus strain, conferred by a single-nucleotide polymorphism (SNP) in the chromosome, was used. Using CARD–FISH, only the high copy number plasmid pC194 was detected. On the other hand, in situ PCR gave positive results in all tested instances. This study demonstrated that it was feasible to detect antibiotic-resistance genes in Bacillus spores using in situ techniques. In addition, in situ PCR has been shown to be more sensitive and more applicable than CARD–FISH in detecting low copy targets.     

Quantitative determination of free-DNA uptake in river bacteria at the single-cell level by in situ rolling-circle amplification

Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.     

Genomic sequencing of single microbial cells from environmental samples

Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification. Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.     

Evaluation of the redox dye 5-cyano-2,3-tolyl-tetrazolium chloride for activity studies by simultaneous use of microautoradiography and fluorescence in situ hybridization

Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge. The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in Thiothrix filaments and in single floc-forming bacteria. The signals from MAR and FISH correlated well, whereas only 65% of the active Thiothrix cells and 41% of all single cells were detectable by CTC reduction, which mainly targeted the most active cells.     

Estimation of the State of the Bacterial Cell Wall by Fluorescent In Situ Hybridization

Fluorescent in situ hybridization (FISH) is now a widely used method for identification of bacteria at the single-cell level. With gram-positive bacteria, the thick peptidoglycan layer of a cell wall presents a barrier for entry of horseradish peroxidase (HRP)-labeled probes. Therefore, such probes do not give any signal in FISH unless cells are first treated with enzymes which hydrolyze the peptidoglycan. We explored this feature of FISH to detect cells which have undergone permeabilization due to expression of autolytic enzymes. Our results indicate that FISH performed with HRP-labeled probes provides a sensitive method to estimate the states of cell walls of individual gram-positive bacteria.     

Detection of single-copy functional genes in prokaryotic cells by two-pass TSA-FISH with polynucleotide probes

In situ detection of functional genes with single-cell resolution is currently of interest to microbiologists. Here, we developed a two-pass tyramide signal amplification (TSA)-fluorescence in situ hybridization (FISH) protocol with PCR-derived polynucleotide probes for the detection of single-copy genes in prokaryotic cells. The mcrA gene and the apsA gene in methanogens and sulfate-reducing bacteria, respectively, were targeted. The protocol showed bright fluorescence with a good signal-to-noise ratio and achieved a high efficiency of detection (> 98%). The discrimination threshold was approximately 82-89% sequence identity. Microorganisms possessing the mcrA or apsA gene in anaerobic sludge samples were successfully detected by two-pass TSA-FISH with polynucleotide probes. The developed protocol is useful for identifying single microbial cells based on functional gene sequences.     

单细胞全基因组扩增技术与应用

同一组织中的细胞往往具有类似的结构和功能,然而通过对单个细胞进行测序分析后,发现每个细胞都具有一定异质性.单细胞全基因组扩增技术是进行单细胞测序的前提,该技术可用于揭示单细胞基因组结构差异,同时在肿瘤研究、发育生物学、微生物学等研究中发挥重要作用,并成为生命科学研究技术的热点之一.单细胞全基因组扩增技术的难点在于单细胞的分离和全基因组的扩增.本文介绍了单细胞全基因组扩增技术中常用的单细胞分离技术和单细胞全基因组扩增技术,并对各技术间的优缺点进行比较,同时着重讨论该技术在肿瘤研究、发育生物学和微生物学研究中的应用.     

Quantitative Determination of Free-DNA Uptake in River Bacteria at the Single-Cell Level by In Situ Rolling-Circle Amplification

Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.     

Assessment of NMYC amplification: a comparison of FISH, quantitative PCR monoplexing and traditional blotting methods used with formalin-fixed, paraffin-embedded neuroblastomas

OBJECTIVE: To determine the degree of agreement between fluorescence in situ hybridization (FISH), Southern blot analysis and LightCycler monoplex polymerase chain reaction (PCR) analysis in the assessment of NMYC gene amplification status in neuroblastoma. STUDY DESIGN: We performed a retrospective analysis of NMYC amplification, using FISH, LightCycler monoplex PCR and Southern blot techniques to assess NMYC amplification in a series of 18 neuroblastomas and 20 histologically normal tissues (15 lymph nodes, 2 pancreas specimens, 1 section each of thyroid, prostate and uterus). RESULTS: Nine neuroblastomas were NMYC amplified, and the remaining cases were nonamplified. All cases yielded interpretable results by Southern blotting and PCR monoplexing techniques. A single case of neuroblastoma was difficult to interpret by FISH due to high background debris. A single case demonstrated low-level NMYC amplification by LightCycler PCR monoplexing but was nonamplified by the other 2 techniques. FISH analysis in 1 case showed amplification, while the other 2 techniques demonstrated nonamplified status. The case in which FISH analysis incorrectly demonstrated amplification was the same one in which there was high background debris. The Southern blot results were reported as amplified or nonamplified, while numeric amplification ratios were obtained by both FISH and PCR LightCycler monoplex analysis. Comparison of these techniques demonstrated FISH to underestimate the degree of amplification in cases in which the amplification level was high by PCR. In fact, FISH appeared to saturate at amplification ratios > 10. CONCLUSION: The study revealed a high level of concordance between the 3 techniques for assessment of NMYC amplification status. However, FISH analysis has the advantage of allowing concurrent assessment of NMYC amplification status and architecture. LightCycler PCR monoplexing appears to have the advantage of more accurately quantitating high levels of NMYC amplification, including those amplified 20-fold or higher. Both FISH and PCR LightCycler monoplexing have the advantage of being performable on formalin-fixed, paraffin-embedded tissue.     

Evaluation of the Redox Dye 5-Cyano-2,3-Tolyl-Tetrazolium Chloride for Activity Studies by Simultaneous Use of Microautoradiography and Fluorescence In Situ Hybridization

Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge. The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in Thiothrix filaments and in single floc-forming bacteria. The signals from MAR and FISH correlated well, whereas only 65% of the active Thiothrix cells and 41% of all single cells were detectable by CTC reduction, which mainly targeted the most active cells.     

Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) of Aureobasidium pullulans

Image and multifactorial statistical analyses were used to evaluate the intensity of fluorescence signal from cells of three strains of A. pullulans and one strain of Rhodosporidium toruloides, as an outgroup, hybridized with either a universal or an A. pullulans 18S rRNA oligonucleotide probe in direct or indirect FISH reactions. In general, type of fixation (paraformaldehyde or methanol-acetic acid) had no apparent effect on cell integrity and minimal impact on fluorescence. Permeabilization by enzyme treatment for various times, though needed to admit high Mw detection reagents (avidin-FITC) in indirect FISH, tended to nonspecifically degrade cells and lower the signal. Digestion was unnecessary and undesirable for the directly labelled probes. Multilabelled (five fluorescein molecules) probes enhanced fluorescence about fourfold over unilabelled probes. Overall, direct FISH was preferable to indirect FISH and is recommended especially for studies of microbes on natural substrata.     

FISH methods in phycology: Phototrophic biofilm and phytoplankton applications

Abstract

Many photosynthetic microorganisms, living attached to immersed substrates or free in the water column, lack distinct morphological details, are small in size and often unculturable. Thus, whole-cell fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has become a valuable and widely used technique to identify bacteria and protists within their natural communities. FISH methods not only allow direct, cultivation independent determination of community composition, but provide spatio-temporal quantification of microorganisms in the environment. Coupling of FISH techniques to Confocal Laser Scanning Microscopy (CLSM) has become essential for the assessment of diversity and structural integrity in three-dimensional complex biofilm samples. Combining FISH with microautoradiography (FISH-MAR) and microsensors also opens new perspectives in microbial ecology by providing new tools for revealing physiological properties of organisms with single-cell resolution. This paper briefly summarizes the application of FISH methods to phototrophic biofilm and phytoplankton research. The potential of DNA microarray technology in phycological research is highlighted, especially for the fast and accurate identification of HAB (Harmful Algal Bloom) species in marine phytoplankton. Some CLSM and FISH data from phototrophic biofilms from an Italian wastewater treatment plant are shown.     

Permeabilization method for in-situ investigation of fungal conidia on surfaces

Aims:  4',6-diamidino-2-phenylindole (DAPI) staining and fluorescent in-situ hybridization (FISH) show great potential for the detection of fungal conidia, also conserving the spatial architecture of their colonization. These investigations are often greatly hampered by the complicated wall structure of many fungal taxa. The aim of the present study was to develop an efficient permeabilization strategy for both DAPI staining and the FISH technique, applicable to various fungal species and maintaining their relationships with surfaces.
Methods and Results:  We compared different DAPI staining permeabilization strategies based on alcohol dehydration, surfactants and osmotic shock, tested with Aspergillus niger conidia. Among four permeabilization methods leading to a strong DAPI signal, only one, based on Triton X-100, EDTA and β-mercaptoethanol followed by hyperosmotic stress, appeared suitable for FISH investigation and was successfully applied to an additional 10 fungal taxa and three environmental samples.
Conclusions:  The effective permeabilization method, which employed a combination of surfactant and osmotic strategies, was successfully applied as preliminary step in both DAPI staining and the FISH protocol.
Significance and Impact of the Study:  The method described is reproducible, simple and inexpensive and might be attractive for other direct visualization techniques.     

Identification of programmed cell death in situ in individual plant cells in vivo using a chromosome preparation technique

A simple procedure, which combines a chromosome preparation technique with an in situ labelling technique modified from fluorescence in situ hybridization (FISH), has been developed for in situ detection of plant programmed cell death (PCD) at the single-cell level. After exposure of chromosomes and nuclei on slides by enzymolysis, Klenow or TdT was used to incorporate Bio-dUTP or fluorescein-dUTP at sites of DNA breaks. After Klenow-mediated labelling, the signals were amplified by a cascade of antigen-antibody reaction according to the detection system of FISH. This method enables in situ detection of plant PCD in vivo morphologically and biochemically at the chromosome, nuclear and DNA levels without cell culture and histological sectioning. This technique permits labelling of DNA breaks with high sensitivity due to increased chromosome and nucleus exposure to the labelling solutions, as well as due to the immunological amplification of the signals. Moreover, the changes in the cells were easier to be observed because the spatial obstacle of the cell wall and its autofluorescence were eliminated. It is potentially useful for in situ detection of PCD in plant root meristematic cells triggered by various environmental abiotic factors. It is proposed that the root tip is a versatile in vivo system for studying PCD induced by environmental abiotic factors.     

荧光原位杂交技术及其在微生物生态学中的应用

综述了荧光原位杂交技术 (fluorescence in situ hybridization FISH)在微生物生态学领域的各种应用 ,同时就其发展过程、原理及种类做了介绍