Explore the vast range of titles available.

Purpose Fluorescence in situ hybridization (FISH) plays a critical role in cancer screening but faces challenges in signal clarity and manual intervention. This study aims to enhance FISH signal clarity, improve screening efficiency, and reduce false negatives through an automated image acquisition and signal enhancement framework. Methods An automated workflow was developed, integrating a dynamic signal enhancement method that optimizes global and local features. An improved Cycle-GAN network was introduced, incorporating residual connections and layer-wise supervision to accurately model and compensate for complex signal characteristics. Key metrics such as signal brightness, edge gradients, contrast improvement index (CII), and structural similarity index (SSIM) were used to evaluate performance. Results The proposed method increased weak signal brightness by 49.02%, edge gradients by 48.61%, and CII by 32.52%. The SSIM reached 0.996, indicating high fidelity to original signals. Conclusion Visual analysis demonstrated clearer, more continuous, and uniform fluorescence signals, effectively mitigating fragmentation and uneven distribution. These improvements reduced false negatives and enhanced genomic abnormality detection accuracy. The proposed method significantly improves FISH signal clarity and stability, providing reliable support for cancer screening, genomic abnormality detection, molecular typing, prognosis evaluation, and targeted treatment planning.

Fluorescence in situ hybridization (FISH) was developed more than 30 years ago and has been the most paradigm-changing technique in cytogenetic research. FISH has been used to answer questions related to structure, mutation, and evolution of not only individual chromosomes but also entire genomes. FISH has served as an important tool for chromosome identification in many plant species. This review intends to summarize and discuss key technical development and applications of FISH in plants since 2006. The most significant recent advance of FISH is the development and application of probes based on synthetic oligonucleotides (oligos). Oligos specific to a repetitive DNA sequence, to a specific chromosomal region, or to an entire chromosome can be computationally identified, synthesized in parallel, and fluorescently labeled. Oligo probes designed from conserved DNA sequences from one species can be used among genetically related species, allowing comparative cytogenetic mapping of these species. The advances with synthetic oligo probes will significantly expand the applications of FISH especially in non-model plant species. Recent achievements and future applications of FISH and oligo-FISH are discussed.

One of the biggest challenges in microbiome research in environmental and medical samples is to better understand functional properties of microbial community members at a single-cell level. Single-cell isotope probing has become a key tool for this purpose, but the current detection methods for determination of isotope incorporation into single cells do not allow high-throughput analyses. Here, we report on the development of an imaging-based approach termed stimulated Raman scattering–two-photon fluorescence in situ hybridization (SRS-FISH) for high-throughputmetabolism and identity analyses of microbial communities with single-cell resolution. SRS-FISH offers an imaging speed of 10 to 100 ms per cell, which is two to three orders ofmagnitude faster than achievable by state-of-the-art methods. Using this technique, we delineated metabolic responses of 30,000 individual cells to various mucosal sugars in the human gut microbiome via incorporation of deuterium from heavy water as an activity marker. Application of SRS-FISH to investigate the utilization of host-derived nutrients by two major human gut microbiome taxa revealed that response to mucosal sugars tends to be dominated by Bacteroidales, with an unexpected finding that Clostridia can outperform Bacteroidales at foraging fucose.With high sensitivity and speed, SRS-FISH will enable researchers to probe the fine-scale temporal, spatial, and individual activity patterns of microbial cells in complex communities with unprecedented detail.

Right detection of anaplastic lymphoma kinase (ALK) gene rearrangement is pivotal to selection of patients with lung adenocarcinoma for ALK-targeted therapy. We explored the potential of combination of immunohistochemistry (IHC) screening and fluorescence in situ hybridization (FISH) as an affordable practice. We analyzed 410 unselected lung adenocarcinomas by ALK IHC (D5F3 clone) and FISH. Some equivocal cases were further analyzed by RT-PCR. The EGFR mutation was detected by pyrosequencing assay. In total 368 cases which got all IHC, FISH, EGFR mutation results were eligible for analysis. Cases were evaluated as IHC score 3+ (n = 26), score 2+ (n = 9), score 1+ (n = 51), and score 0 (n = 282), respectively. 23 of 26 IHC 3+ and 5 of 9 IHC 2+ cases were FISH positive, whereas 3 of 26 IHC 3+, 4 of 9 IHC 2+ and all 333 IHC 1+/0 cases were FISH negative. If considering FISH as the standard, the sensitivity and specificity of ALK IHC 3+/2+ as ALK positive were 100% and 97.9%, respectively. Three IHC 3+ cases reported as FISH \"negative\" were actually ALK positive confirmed by ALK RT-PCR or re-detected. Based on the final classify, ALK IHC 3+/2+ was 100% sensitive and 98.8% specific. However, FISH was 90.3% sensitive and 100% specific. IHC 2+ was regarded as equivocal and need to be confirmed by FISH or RT-PCR. In the 368 cases, 8.4% cases had ALK positive, 52.2% cases had EGFR mutation, and only one case had a coexisting. Manually semiquantitative ALK IHC (primary antibody D5F3 coupled with secondary DAKO Envision system) used as the initial screening combined with auxiliary FISH confirmation is a reliable, economical approach to identify ALK positive lung adenocarcinoma. The IHC can find some ALK positive cases which would be missed by FISH only.

Candidate phylum Saccharibacteria (former TM7) are abundant and widespread in nature, but little is known about their ecophysiology and detailed phylogeny. In this study phylogeny, morphology and ecophysiology of Saccharibacteria were investigated in activated sludge from nine wastewater treatment plants (WWTPs) from Japan and Denmark using the full-cycle 16S rRNA approach in combination with microautoradiography (MAR) and fluorescence in situ hybridization (FISH). Phylogenetic analysis showed that Saccharibacteria from all WWTPs were evenly distributed within subdivision 1 and 3 and in a distinct phylogenetic clade. Three probes were designed for the distinct saccharibacterial groups, and revealed morphotypes representing thin filaments, thick filaments and rods/cocci. MAR-FISH results showed that most probe-defined Saccharibacteria utilized glucose under aerobic-, nitrate reducing- and anaerobic conditions. Some Saccharibacteria also utilized N-acetylglucosamine, oleic acid, amino acids and butyrate, which are not predicted from available genomes so far. In addition, some filamentous Saccharibacteria exhibited β-galactosidase and lipase activities determined using a combination of enzyme-labeled fluorescence and FISH (ELF-FISH). No uptake of acetate, propionate, pyruvate, glycerol and ethanol was observed. These results indicate that Saccharibacteria is a phylogenetically diverse group and play a role in the degradation of various organic compounds as well as sugar compounds under aerobic-, nitrate reducing- and anaerobic conditions. Candidatus Saccharibacteria in activated sludge are phylogenetically diverse and utilize oleic acid, amino acids,and N-acetylglucosamine as well as glucose as the carbon sources. Graphical Abstract Figure. Candidatus Saccharibacteria in activated sludge are phylogenetically diverse and utilize oleic acid, amino acids,and N-acetylglucosamine as well as glucose as the carbon sources.

The cultivated peanut Arachis hypogaea (AABB) is thought to have originated from the hybridization of Arachis duranensis (AA) and Arachis ipaënsis (BB) followed by spontaneous chromosome doubling. In this study, we cloned and analyzed chromosome markers from cultivated peanut and its wild relatives. A fluorescence in situ hybridization (FISH)-based karyotyping cocktail was developed with which to study the karyotypes and chromosome evolution of peanut and its wild relatives. Karyotypes were constructed in cultivated peanut and its two putative progenitors using our FISH-based karyotyping system. Comparative karyotyping analysis revealed that chromosome organization was highly conserved in cultivated peanut and its two putative progenitors, especially in the B genome chromosomes. However, variations existed between A. duranensis and the A genome chromosomes in cultivated peanut, especially for the distribution of the interstitial telomere repeats (ITRs). A search of additional A. duranensis varieties from different geographic regions revealed both numeric and positional variations of ITRs, which were similar to the variations in tetraploid peanut varieties. The results provide evidence for the origin of cultivated peanut from the two diploid ancestors, and also suggest that multiple hybridization events of A. ipaënsis with different varieties of A. duranensis may have occurred during the origination of peanut.

Transcription is inherently stochastic even in clonal cell populations 1. Studies at single-cell-single-molecule level enable a quantitative understanding of the underlying regulatory mechanisms 2,3. A widely used technique is single-molecule RNA fluorescence in-situ hybridization (FISH), in which fluorescent probes target the mRNA of interest and individual molecules appear as bright diffraction-limited spots (Fig. 1a,b) 3. Recent experimental progress makes FISH easy to use 4 , but a dedicated image analysis tool is currently missing. Available methods allow counting of isolated mature mRNAs but cannot reliably quantify the dense mRNA aggregates at transcription sites (TS) in three dimensions (3D), particularly of highly transcribing genes 4. We developed FISH-QUANT to close this gap (Supplementary Note 1)

Background Wheat is important for global food security. Understanding the molecular mechanisms driving spike and spikelet development can benefit the development of more productive varieties. Results Here we integrate single-molecule fluorescence in situ hybridization (smFISH) and single-cell RNA sequencing (scRNA-seq) to generate an atlas of cell clusters and expression domains during the early stages of wheat spike development. We characterize spatiotemporal expression of 99 genes by smFISH in 48,225 cells at early transition (W1.5), late double ridge (W2.5), and floret primordia stages (W3.5). These cells are grouped into 21 different expression domains, including four in the basal region of the developing spikelets and three different meristematic regions, which are consistent across spikelets and sections. Using induced mutants, we reveal functional roles associated with the specific expression patterns of LFY in intercalary meristems, SPL14 in inflorescence meristems, and FZP in glume axillae. Complementary scRNA-seq profiling of 26,009 cells from W2.5 and W3.5 stages identifies 23 distinct cell clusters. We use the scRNA-seq information to impute the expression of 74,464 genes into the spatially anchored smFISH-labelled cells and generate a public website to visualize them. We then use experimental and imputed expression profiles, together with co-expression studies and correlation matrices, to annotate the scRNA-seq clusters. From co-expression analyses, we identify genes associated with boundary genes TCP24 and FZP , as well as the meristematic genes AGL6 and ULT1 . Conclusions The smFISH and scRNA-seq studies provide complementary tools for dissecting gene networks that regulate spike development and identifying new co-expressed genes for functional characterization.

Biofilms are complex structures with an intricate relationship between the resident microorganisms, the extracellular matrix, and the surrounding environment. Interest in biofilms is growing exponentially given its ubiquity in so diverse fields such as healthcare, environmental and industry. Molecular techniques (e.g., next-generation sequencing, RNA-seq) have been used to study biofilm properties. However, these techniques disrupt the spatial structure of biofilms; therefore, they do not allow to observe the location/position of biofilm components (e.g., cells, genes, metabolites), which is particularly relevant to explore and study the interactions and functions of microorganisms. Fluorescence in situ hybridization (FISH) has been arguably the most widely used method for an in situ analysis of spatial distribution of biofilms. In this review, an overview on different FISH variants already applied on biofilm studies (e.g., CLASI-FISH, BONCAT-FISH, HiPR-FISH, seq-FISH) will be explored. In combination with confocal laser scanning microscopy, these variants emerged as a powerful approach to visualize, quantify and locate microorganisms, genes, and metabolites inside biofilms. Finally, we discuss new possible research directions for the development of robust and accurate FISH-based approaches that will allow to dig deeper into the biofilm structure and function.

Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the spatial organization of microbial communities. However, traditional FISH has several limitations, including limited phylogenetic resolution, difficulty visualizing certain lineages, and the design and optimization of new probes is time consuming and does not scale to the known diversity of microbial life. Here, we present GenomeFISH, a high-throughput, genome-based FISH approach that can differentiate strains within complex communities. Fluorescent probes are generated from the genomes of single cells, which are obtained from environmental or clinical samples through fluorescence activated single-cell sorting. GenomeFISH can distinguish between strains with up to 99% average nucleotide identity and was successfully applied to visualize strains in mock communities and human fecal samples. Given the superior sensitivity and specificity of GenomeFISH, we envisage it will become widely used for the visualization of complex microbial systems.