Hasil untuk "Microscopy"

Hyejin Park, Jiwon Yoon, Sumin Park et al.

Accurate focus quality assessment (FQA) in fluorescence microscopy is challenging due to stain-dependent optical variations that induce heterogeneous focus behavior across images. Existing methods, however, treat focus quality as a stain-agnostic problem, assuming a shared global ordering. We formulate stain-aware FQA for fluorescence microscopy, showing that focus-rank relationships vary substantially across stains due to stain-dependent imaging characteristics and invalidate this assumption. To support this formulation, we introduce FluoMix, the first dataset for stain-aware FQA spanning multiple tissues, fluorescent stains, and focus levels. We further propose FluoCLIP, a two-stage vision-language framework that grounds stain semantics and enables stain-conditioned ordinal reasoning for focus prediction, effectively decoupling stain representation from ordinal structure. By explicitly modeling stain-dependent focus behavior, FluoCLIP consistently outperforms both conventional FQA methods and recent vision-language baselines, demonstrating strong generalization across diverse fluorescence microscopy conditions. Code and dataset are publicly available at https://fluoclip.github.io/.

Ranjan Datta, Sneha Kobri M., Sudip Mahato

In this review article fundamentals of aberration corrected phase contrast transmission electron microscopy for the structural characterization of materials at atomic length scale is presented. The word structure entails atomic arrangement as well as electronic structure information of the materials. The article summarily covers a range of topics on the basics of aberrations, aberration correctors, direct image interpretation with negative Cs phase contrast microscopy, a discussion in comparison with the competitive atomic resolution phase contrast methods for example, off-axis electron holography, electron ptychography, differential phase contrast microscopy. Additionally, various examples of quantitative imaging of materials at atomic length scale, associated image simulation and reconstruction methods for retrieving the phase information are presented. With the tremendous advancement in instrumentation and recording devices, potential future perspective of such tools and methods in solving challenging materials science problems are outlined.

Songyuan Yan, Sara Louise Pacheco, Asa K. Laskie et al.

Maintaining favorable biological productivities in photosynthetic biomanufacturing systems, especially when the risk of contamination with competing microbes is high, remains a challenge to achieve while maintaining economic feasibility. This study presents a dielectrophoresis (DEP)-based microfluidic approach for isolating a desired strain within a co-culture. The cyanobacterium <i>Synechocystis</i> sp. PCC 6803 (a strain capable of producing the bioplastic precursor polyhydroxybutyrate, or PHB) was enriched from mixed cultures containing the competing cyanobacterium Synechococcus elongatus PCC 7942 (which does not naturally produce PHB). A DEP cascade electrode system was established to increase purification efficiency through sequential enrichment, which leveraged inherent differences in cell morphology and dielectric properties, to achieve the selective separation of these strains under physiological conditions. A substantial increase in the relative abundance of PHB-producing cells was assessed by optical microscopy and flow cytometry characterization, confirming more than five-fold reduction of the Synechococcus fraction in the refined cell mix. The presented electrokinetic platform offers a scalable and effective approach for selectively enhancing desired microbial components within microbial biomanufacturing systems, leading towards improved product yields.

Ahmed. E. Suliman, Ahmed H. Mangood, Naema S. Yehia et al.

Abstract The petroleum industry, characterized by the significant investment in costly equipment and devices utilized in the extraction, production, or processing of crude oil, can result in the loss of valuable assets or the crude itself. This research involved the synthesis of a Schiff base from substituted gallic acid derivatives through an intermediate reaction known as N-(2-{2-[2-(2-amino-ethylamino)-ethylamino]-ethylamino}-ethyl)-3,4,5-trihydroxy-benzamide (AEET). The synthesized compound was characterized using FTIR and 1HNMR spectroscopy to evaluate its effectiveness in inhibition. The performance of the inhibitors was assessed through an electrochemical process that included Tafel and EIS. This evaluation was supported by theoretical mechanisms involving density functional theory (DFT) and molecular dynamics simulations (MDS). To validate the findings from the electrochemical studies, the scanning electron microscopy (SEM) technique was employed to examine the topographic anisotropy characteristics between the treated and untreated samples of mild steel species. The bioassay diluted serial technique was utilized to assess the AEET as effective biocides for managing bacterial growth issues. This evaluation included an analysis of the AEET’s efficiency in inhibiting sulfate-reducing bacteria (SRB). Additionally, computational methods were described, demonstrating optimal scores, RMSD values, and binding interaction energies associated with the formation of hydrogen bonds with specific receptor residues to investigate the biological activity.

Chaiti Saha, Shova Pandit, Md Motaher Hossain et al.

Rice blast caused by Pyricularia oryzae (syn. Magnaporthe oryzae ) is one of the most devastating diseases in rice, resulting in substantial yield loss. Currently, the control of rice blast relies significantly on chemical fungicides, which raise numerous environmental concerns. However, silver nanoparticles (AgNPs) are emerging as innovative, non-resistant substitutes for conventional fungicides. This study aimed to synthesize AgNPs from banana flowers via a green approach and assess their antifungal activity against Pyricularia oryzae strain SP2 in vitro . The pathogen Pyricularia oryzae strain SP2 was identified by morphological features and study of the internal transcribed spacer (ITS) sequence. The AgNPs were characterized via UV–Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), x-ray diffraction (XRD), transmission electron microscopy (TEM) and energy-dispersive x-ray analysis (EDX). The optimal reaction conditions for the green synthesis of the AgNPs resulted in a rapid color change from light yellow to dark brown when the floral extract (15 ml) was added. A surface plasmon resonance peak was observed in the UV-visible spectrum at approximately 433 cm ^−1 , which was correlated with the synthesis of the AgNPs. The biogenic AgNPs had a spherical form with an average particle size of 47.63 nm, according to TEM and SEM examination. The greatest mycelial growth inhibition of P. oryzae (87.25%) was observed when it was exposed to biofabricated AgNPs at 40 μg ml ^−1 . The AgNPs successfully suppressed spore germination and germ tube development of P. oryzae , with increased doses demonstrating enhanced antifungal efficacy. Additionally, the appressorium formation rates decreased from 78.34% in the control to 41.67%, 16.45%, and 4% at increasing concentrations (20, 30, 40 μg ml ^−1 ). Overall, this study demonstrated that bioinspired AgNPs have antifungal activity against the rice blast pathogen P. oryzae and can be used to control blast diseases in rice.

Chenyu Xu, Zhouyu Jin, Chengkang Shen et al.

Compared to light-field microscopy (LFM), which enables high-speed volumetric imaging but suffers from non-uniform spatial sampling, Fourier light-field microscopy (FLFM) introduces sub-aperture division at the pupil plane, thereby ensuring spatially invariant sampling and enhancing spatial resolution. Conventional FLFM reconstruction methods, such as Richardson-Lucy (RL) deconvolution, exhibit poor axial resolution and signal degradation due to the ill-posed nature of the inverse problem. While data-driven approaches enhance spatial resolution by leveraging high-quality paired datasets or imposing structural priors, Neural Radiance Fields (NeRF)-based methods employ physics-informed self-supervised learning to overcome these limitations, yet they are hindered by substantial computational costs and memory demands. Therefore, we propose 3D Gaussian Adaptive Tomography (3DGAT) for FLFM, a 3D gaussian splatting based self-supervised learning framework that significantly improves the volumetric reconstruction quality of FLFM while maintaining computational efficiency. Experimental results indicate that our approach achieves higher resolution and improved reconstruction accuracy, highlighting its potential to advance FLFM imaging and broaden its applications in 3D optical microscopy.

Jan Christian Wohlsein, Marita Meurer, Matthias Mörgelin et al.

In steroid-responsive meningitis-arteritis (SRMA), inflammatory dysregulation is driven by neutrophilic granulocytes resulting in purulent leptomeningitis. Neutrophils can generate neutrophil extracellular traps (NET). Uncontrolled NET-formation or impaired NET-clearance evidently cause tissue and organ damage resulting in immune-mediated diseases. The aim of the study was to verify that NET-formation is detectable in ex vivo samples of acute diseased dogs with SRMA by visualizing and measuring NET-markers in serum and cerebrospinal fluid (CSF) samples. CSF-samples of dogs with acute SRMA (n = 5) and in remission (n = 4) were examined using immunofluorescence (IF)-staining of DNA-histone-1-complexes, myeloperoxidase and citrullinated Histone H3 (H3Cit). Immunogold-labeling of H3Cit and neutrophil elastase followed by transmission electron microscopy (TEM) were used to determine ultrastructural NET-formation in the CSF of one exemplary dog. H3Cit-levels and DNase-activity were measured in CSF and serum samples using an H3Cit-ELISA and a DNase-activity-assay, respectively in patients with the following diseases: acute SRMA (n = 34), SRMA in remission (n = 4), bacterial encephalitis (n = 3), meningioma with neutrophilic inflammation (n = 4), healthy dogs (n = 6). NET-formation was detectable with IF-staining in n = 3/5 CSF samples of dogs with acute SRMA but were not detectable during remission. Vesicular NET-formation was detectable in one exemplary dog using TEM. DNase-activity was significantly reduced in dogs suffering from acute SRMA compared to healthy control group (p < 0.0001). There were no statistical differences of H3Cit levels in CSF or serum samples of acute diseased dogs compared to dogs under treatment, dogs suffering from meningioma or bacterial encephalitis or the healthy control group. Our findings demonstrate that NET-formation and insufficient NET-clearance possibly drive the immunologic dysregulation and complement the pathogenesis of SRMA. The detection of NETs in SRMA offers many possibilities to explore the aetiopathogenetic influence of this defence mechanism of the innate immune system in infectious and non-infectious canine neuropathies.

Miao Li, Jiajie Gan, Xuhui Xu et al.

Baicalin, a flavonoid extracted from traditional Chinese medicine, Scutellaria baicalensis has significant anti-inflammatory effects. Microsponges are drug delivery systems that improve drug stability and slow the release rate. The combination of baicalin and the microsponges produced a new and stable system for its delivery, resulting in a novel formulation of baicalin. Baicalin microsponges (BM) were prepared using the quasi-emulsion solvent diffusion method. Effects of the mass ratio of the polymer (ethylcellulose) to baicalin, the concentration of the emulsifier polyvinyl alcohol (PVA), the stirring speed on the encapsulation efficiency (EE), and yield of the microsponges were investigated by combining the one-factor test and Box-Behnken design (BBD). The preparation process was standardised using 2.61:1 mass ratio of ethyl cellulose to baicalin, 2.17% concentration of PVA, with stirring at 794 rpm. Optimised BM formulations were evaluated for the parameters of EE (54.06 ± 3.02)% and yield of (70.37 ± 2.41)%, transmission electron microscopy (TEM), and in vitro cell evaluation. Results of the in vitro anti-inflammatory assay showed that baicalin microsponges-pretreated-lipopolysaccharide (LPS)-induced RAW264.7, mouse macrophages showed reduced inflammatory response, similar to that seen in baicalin-treated macrophages.

Silvia Bonettini, Luca Calatroni, Danilo Pezzi et al.

We propose an unfolded accelerated projected-gradient descent procedure to estimate model and algorithmic parameters for image super-resolution and molecule localization problems in image microscopy. The variational lower-level constraint enforces sparsity of the solution and encodes different noise statistics (Gaussian, Poisson), while the upper-level cost assesses optimality w.r.t.~the task considered. In more detail, a standard $\ell_2$ cost is considered for image reconstruction (e.g., deconvolution/super-resolution, semi-blind deconvolution) problems, while a smoothed $\ell_1$ is employed to assess localization precision in some exemplary fluorescence microscopy problems exploiting single-molecule activation. Several numerical experiments are reported to validate the proposed approach on synthetic and realistic ISBI data.

Daniele Ancora, Alessandro Zunino, Giuseppe Vicidomini et al.

Confocal laser scanning microscopy (CLSM) stands out as one of the most widely used microscopy techniques, thanks to its three-dimensional imaging capability and its sub-diffraction spatial resolution, achieved through the closure of a pinhole in front of a single-element detector. However, the pinhole also rejects useful photons and beating the diffraction limit comes at the price of irremediably compromising the signal-to-noise ratio (SNR) of the data. Image scanning microscopy (ISM) emerged as the natural evolution of CLSM, exploiting a small array detector in place of the pinhole and the single-element detector. Each sensitive element is small enough to achieve sub-diffraction resolution through the confocal effect, but the size of the whole detector is large enough to guarantee excellent collection efficiency and SNR. However, the raw data produced by an ISM setup consists of a 4D dataset which can be seen as a set of confocal-like images. Thus, fusing the dataset into a single super-resolved image requires a dedicated reconstruction algorithm. Conventional methods are multi-image deconvolution, which requires prior knowledge of the system point spread functions (PSF), or adaptive pixel reassignment (APR), which is effective only on a limited range of experimental conditions. In this work, we describe and validate a novel concept for ISM image reconstruction based on autocorrelation inversion. We leverage unique properties of the autocorrelation to discard low-frequency components and maximize the resolution of the reconstructed image, without any assumption on the image or any knowledge of the PSF. Our results push the quality of the ISM reconstruction beyond the level provided by APR and open new perspectives for multi-dimensional image processing.

Illia Tsiporenko, Pavel Chizhov, Dmytro Fishman

Segmentation is a crucial step in microscopy image analysis. Numerous approaches have been developed over the past years, ranging from classical segmentation algorithms to advanced deep learning models. While U-Net remains one of the most popular and well-established models for biomedical segmentation tasks, recently developed transformer-based models promise to enhance the segmentation process of microscopy images. In this work, we assess the efficacy of transformers, including UNETR, the Segment Anything Model, and Swin-UPerNet, and compare them with the well-established U-Net model across various image modalities such as electron microscopy, brightfield, histopathology, and phase-contrast. Our evaluation identifies several limitations in the original Swin Transformer model, which we address through architectural modifications to optimise its performance. The results demonstrate that these modifications improve segmentation performance compared to the classical U-Net model and the unmodified Swin-UPerNet. This comparative analysis highlights the promise of transformer models for advancing biomedical image segmentation. It demonstrates that their efficiency and applicability can be improved with careful modifications, facilitating their future use in microscopy image analysis tools.

A. Vetráková, R. Kalianková Chovanová, R. Rechtoríková et al.

Bacillus subtilis spores are considered to be efficient and useful vehicles for the surface display and delivery of heterologous proteins. In this study, we prepared recombinant spores with the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein displayed on their surface in fusion with the CotZ or CotY spore coat proteins as a possible tool for the development of an oral vaccine against the SARS-CoV-2 virus. The RBD was attached to the N-terminus or C-terminus of the coat proteins. We also directly adsorbed non-recombinantly produced RBD to the spore surface. SDS-PAGE, western blot and fluorescence microscopy were used to analyze RBD surface expression on purified spores. Results obtained from both display systems, recombinant and non-recombinant, demonstrated that RBD was present on the spore surfaces.

Ana M. Leiva, Juan M. Pardo, Warren Arinaitwe et al.

Abstract Cassava witches' broom disease (CWBD) is a devastating disease of cassava in Southeast Asia (SEA), of unknown etiology. Affected plants show reduced internodal length, proliferation of leaves and weakening of stems. This results in poor germination of infected stem cuttings (i.e., planting material) and significant reductions in fresh root yields and starch content, causing economic losses for farmers and processors. Using a metagenomic approach, we identified a fungus belonging to the Ceratobasidium genus, sharing more than 98.3–99.7% nucleotide identity at the Internal Transcribed Spacer (ITS), with Ceratobasidium theobromae a pathogen causing similar symptoms in cacao. Microscopy analysis confirmed the identity of the fungus and specific designed PCR tests readily showed (1) Ceratobasidium sp. of cassava is strongly associated with CWBD symptoms, (2) the fungus is present in diseased samples collected since the first recorded CWBD outbreaks in SEA and (3) the fungus is transmissible by grafting. No phytoplasma sequences were detected in diseased plants. Current disease management efforts include adjustment of quarantine protocols and guarantee the production and distribution of Ceratobasidium-free planting material. Implications of related Ceratobasidium fungi, infecting cassava, and cacao in SEA and in other potential risk areas are discussed.

Hongyuan Li, Ziyu Xiang, Mit H. Naik et al.

Moiré superlattices provide a highly tunable and versatile platform to explore novel quantum phases and exotic excited states ranging from correlated insulators1-17 to moiré excitons7-10,18. Scanning tunneling microscopy has played a key role in probing microscopic behaviors of the moiré correlated ground states at the atomic scale1,11-15,19. Atomic-resolution imaging of quantum excited state in moiré heterostructures, however, has been an outstanding experimental challenge. Here we develop a novel photocurrent tunneling microscopy by combining laser excitation and scanning tunneling spectroscopy (laser-STM) to directly visualize the electron and hole distribution within the photoexcited moiré exciton in a twisted bilayer WS2 (t-WS2). We observe that the tunneling photocurrent alternates between positive and negative polarities at different locations within a single moiré unit cell. This alternating photocurrent originates from the exotic in-plane charge-transfer (ICT) moiré exciton in the t-WS2 that emerges from the competition between the electron-hole Coulomb interaction and the moiré potential landscape. Our photocurrent maps are in excellent agreement with our GW-BSE calculations for excitonic states in t-WS2. The photocurrent tunneling microscopy creates new opportunities for exploring photoexcited non-equilibrium moiré phenomena at the atomic scale.

Suprim Nakarmi, Sanam Pudasaini, Safal Thapaliya et al.

The consumption of microbial-contaminated food and water is responsible for the deaths of millions of people annually. Smartphone-based microscopy systems are portable, low-cost, and more accessible alternatives for the detection of Giardia and Cryptosporidium than traditional brightfield microscopes. However, the images from smartphone microscopes are noisier and require manual cyst identification by trained technicians, usually unavailable in resource-limited settings. Automatic detection of (oo)cysts using deep-learning-based object detection could offer a solution for this limitation. We evaluate the performance of four state-of-the-art object detectors to detect (oo)cysts of Giardia and Cryptosporidium on a custom dataset that includes both smartphone and brightfield microscopic images from vegetable samples. Faster RCNN, RetinaNet, You Only Look Once (YOLOv8s), and Deformable Detection Transformer (Deformable DETR) deep-learning models were employed to explore their efficacy and limitations. Our results show that while the deep-learning models perform better with the brightfield microscopy image dataset than the smartphone microscopy image dataset, the smartphone microscopy predictions are still comparable to the prediction performance of non-experts. Also, we publicly release brightfield and smartphone microscopy datasets with the benchmark results for the detection of Giardia and Cryptosporidium, independently captured on reference (or standard lab setting) and vegetable samples. Our code and dataset are available at https://github.com/naamiinepal/smartphone_microscopy and https://doi.org/10.5281/zenodo.7813183, respectively.

Zoi Terzopoulou, Eleftheria Xanthopoulou, Nikolaos Pardalis et al.

Lignin, being one of the main structural components of lignocellulosic biomass, is considered the most abundant natural source of phenolics and aromatics. Efforts for its valorisation were recently explored as it is mostly treated as waste from heat/energy production via combustion. Among them, polymer-based lignin composites are a promising approach to both valorise lignin and to fine tune the properties of polymers. In this work, organosolv lignin, from beech wood, was used as fillers in a poly (lactic acid) (PLA) matrix. The PLA/lignin composites were prepared using melt mixing of masterbatches with neat PLA in three different lignin contents: 0.5, 1.0 and 2.5 wt%. Lignin was used as-isolated, via the organosolv biomass pretreatment/fractionation process and after 8 h of ball milling. The composites were characterised with Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR) spectroscopy, X-ray Diffraction (XRD), and Differential Scanning Calorimetry (DSC). Additionally, their antioxidant activity was assessed with the 2,2-Diphenyil-1-picrylhydrazyl (DPPH) method, the colour was measured with a colorimeter and the mechanical properties were evaluated with tensile testing. Ball milling, at least under the conditions applied in this study, did not induce a further substantial decrease in the already relatively small organosolv lignin primary particles of ~1 μm. All the produced PLA/lignin composites had a uniform dispersion of lignin. Compression-moulded films were successfully prepared, and they were coloured brown, with ball-milled lignin, giving a slightly lighter colour in comparison with the as-received lignin. Hydrogen bonding was detected between the components of the composites, and crystallization of the PLA was suppressed by both lignin, with the suppression being less pronounced by the ball-milled lignin. All composites showed a significantly improved antioxidant activity, and their mechanical properties were maintained for filler content 1 wt%.

Luís Marangoni Júnior, Ewelina Jamróz, Sayeny de Ávila Gonçalves et al.

The objective of this work was to evaluate the effect of green propolis extract (PE) and silica nanoparticles (SiO2) on the structure of sodium alginate-based films (SA), as well as their physical and antioxidant properties. Control samples (only SA), films with SA and PE (SA/PE3%), as well as films simultaneously containing SA, PE and SiO2 (SA/PE3%/SiO25% and SA/PE3%/SiO210%) were systematically evaluated. Scanning electron microscopy revealed homogenous distribution of PE and SiO2 within the film matrix. The addition of both compounds increased tensile strength from 12.9 ± 1.2 (control) to 19.6 ± 1.4 MPa (SA/PE3%/SiO210%), without noting any extreme changes in elongation at break or modulus of elasticity. Additionally, the incorporation of PE provided an outstanding UV light blocking effect, and strong DPPH radical-scavenging activity for all samples, therefore, suggesting their potential for future application in active food packaging.

Caitlin P. Casar, Brittany R. Kruger, Magdalena R. Osburn

The continental deep subsurface is likely the largest reservoir of biofilm-based microbial biomass on Earth, but the role of mineral selectivity in regulating its distribution and diversity is unclear. Minerals can produce hotspots for intraterrestrial life by locally enhancing biofilm biomass. Metabolic transformations of minerals by subsurface biofilms may occur widely with the potential to significantly impact subsurface biogeochemical cycles. However, the degree of impact depends upon the amount of biofilm biomass and its relationship to host rock mineralogy, estimates that are currently loosely constrained to non-existent. Here, we use in situ cultivation of biofilms on native rocks and coupled microscopy/spectroscopy to constrain mineral selectivity by biofilms in a deep continental subsurface setting: the Deep Mine Microbial Observatory (DeMMO). Through hotspot analysis and spatial modeling approaches we find that mineral distributions, particularly those putatively metabolized by microbes, indeed drive biofilm distribution at DeMMO, and that bioleaching of pyrite may be a volumetrically important process influencing fluid geochemistry at this site when considered at the kilometer scale. Given the ubiquity of iron-bearing minerals at this site and globally, and the amount of biomass they can support, we posit that rock-hosted biofilms likely contribute significantly to subsurface biogeochemical cycles. As more data becomes available, future efforts to estimate biomass in the continental subsurface should incorporate host rock mineralogy.

Hangwen Guo, Mohammad Saghayezhian, Zhen Wang et al.

Complex oxide interfaces have been one of the central focuses in condensed matter physics and material science. Over the past decade, aberration corrected scanning transmission electron microscopy and spectroscopy has proven to be invaluable to visualize and understand the emerging quantum phenomena at an interface. In this paper, we briefly review some recent progress in the utilization of electron microscopy to probe interfaces. Specifically, we discuss several important challenges for electron microscopy to advance our understanding on interface phenomena, from the perspective of variable temperature, magnetism, electron energy loss spectroscopy analysis, electronic symmetry, and defects probing.

Alex Gileles-Hillel, Hagar Mor-Shaked, David Shoseyov et al.

The diagnosis of primary ciliary dyskinesia (PCD) relies on clinical features and sophisticated studies. The detection of bi-allelic disease-causing variants confirms the diagnosis. However, a standardised genetic panel is not widely available and new disease-causing genes are continuously identified. To assess the accuracy of untargeted whole-exome sequencing (WES) as a diagnostic tool for PCD, patients with symptoms highly suggestive of PCD were consecutively included. Patients underwent measurement of nasal nitric oxide (nNO) levels, ciliary transmission electron microscopy analysis (TEM) and WES. A confirmed PCD diagnosis in symptomatic patients was defined as a recognised ciliary ultrastructural defect on TEM and/or two pathogenic variants in a known PCD-causing gene. Forty-eight patients (46% male) were enrolled, with a median age of 10.0 years (range 1.0–37 years). In 36 patients (75%) a diagnosis of PCD was confirmed, of which 14 (39%) patients had normal TEM. A standalone untargeted WES had a diagnostic yield of 94%, identifying bi-allelic variants in 11 known PCD-causing genes in 34 subjects. A nNO<77 nL·min was nonspecific when including patients younger than 5 years (area under the receiver operating characteristic curve (AUC) 0.75, 95% CI 0.60–0.90). Consecutive WES considerably improved the diagnostic accuracy of nNO in young children (AUC 0.97, 95% CI 0.93–1). Finally, WES established an alternative diagnosis in four patients. In patients with clinically suspected PCD and low nNO levels, WES is a simple, beneficial and accurate next step to confirm the diagnosis of PCD or suggest an alternative diagnosis, especially in preschool-aged children in whom nNO is less specific.