Quantitative phase microscopy (QPM) has been an effective technique to examine stain-free biomedical tissues and cells. The development of simple and compact QPM technology with planar optical components enhances system integration and portability. Here, we propose a QPM technique by inserting a metasurface-assisted optical differential system to replace the bulky optical elements in the conventional microscope. The differential scheme allows for the separation of amplitude and phase information for a complex field, offering an opportunity to quantitatively analyze the phase distribution of samples. The experimental demonstrations showcase the well-executed application of the proposed method to artificial phase samples, paramecium cells, fishtail cross-cut tissue, and diatom cells. Notably, our imaging system allows switching between four imaging modes—brightfield, optical spatial differential, differential interference contrast, and QPM. At a high level, this work may drive the advancement of single-shot and multi-mode integrated phase microscopy for biomedical imaging and diagnostics.
Existing methods in nanoscale connectomics are at present too slow to map entire mammalian brains. As an emerging approach, expansion microscopy (ExM) has enormous promise, yet it still suffers from throughput limitations. Mapping the human brain and even mapping nonhuman primate brains therefore remain distant goals. While ExM increases effective resolution linearly, it enlarges tissue volume cubically, which dramatically increases imaging time. As a rapid tomographic technique, X-ray microscopy has potential for drastically speeding up large-volume connectomics. But to the best of my knowledge, no group has so far imaged cellular features within expanded tissue using X-ray microscopy. I herein present an early-stage report featuring the first demonstration of X-ray microscopy reconstruction of cell bodies within expanded tissue. This was achieved by combining a modified enzymatic Unclearing technique with a metallic gold stain and imaging using a laboratory X-ray microscope. I emphasize that a great deal of work remains to develop "expansion X-ray microscopy" (ExXRM) to the point where it can be useful for connectomics since the current iteration of ExXRM only resolves cell bodies and not neurites due to extensive off-target staining. Additionally, the current method must be modified to accommodate for the challenges of synchrotron X-ray microscopy, a vastly speedier approach than laboratory X-ray microscopy. Nonetheless, achieving X-ray contrast in expanded tissues represents a significant first step towards realizing ExXRM as a connectomics imaging modality.
Afia Yasmin, Bristy Biswas, Md. Lutfor Rahman
et al.
CoFe2O4 was synthesized at 150 °C, 180 °C, and 210 °C temperatures using hydrothermal method to find the effect on its structural, magnetic, electric, and optical properties. The saturation magnetization, coercivity and magnetic anisotropy was found using Vibrating Sample Magnetometer (VSM), ranging from 50.36 to 53.66 emu/g. XRD (X-ray Diffraction Analysis) and SEM (Scanning Electron Microscopy), FTIR (Fourier Transform Infrared Spectroscopy) was used for structural analysis verifying the spinel ferrite structure with a single phase. The crystalline size and lattice strain was found using Size-Strain Plot (SSP) and Debye-Scherrer (D-S) method which proved that as the synthesis temperature increased, the crystallite size also increased. The crystalline size ranges from 39.40 to 82.24 nm as observed by XRD. SEM analysis found the crystal size range to be from 9 to 12 nm. It was found that the optimum temperature to synthesize cobalt ferrite nanoparticles are at 180 °C for sample H2 with a crystal size of 82.24 nm and band gap energy of 2.60 eV. The Ms value was determined to be 50.36 emu/g for H2 sample with Rs value of 0.31.
Petr Koutenský, Neli Laštovičková Streshkova, Kamila Moriová
et al.
Ultrafast electron microscopy aims for imaging transient phenomena occurring on nanoscale. One of its goals is to visualize localized optical and plasmonic modes generated by coherent excitation in the vicinity of various types of nanostructures. Such imaging capability was enabled by photoninduced near-field optical microscopy, which is based on spectral filtering of electrons inelastically scattered due to the stimulated interaction with the nearfield. Here, we report on the development of ultrafast four-dimensional (4D) scanning transmission electron microscopy, which allows us to image the transverse components of the optical near-field while avoiding the need of electron spectral filtering. We demonstrate that this method is capable of imaging the integrated Lorentz force generated by optical near-fields of a tungsten nanotip and the ponderomotive potential of an optical standing wave with a spatial resolution of 21 nm.
Summary: Esophageal squamous cell carcinoma (ESCC) is a common malignancy, characterized by a multistep pathogenic process regulated spatiotemporally within the esophageal epithelial microenvironment, including vessel normalization and immune infiltration. However, empirical evidence elucidating esophageal vascular remodeling and immune infiltration during ESCC tumorigenesis in situ is lacking. In this study, utilizing a mouse model recapitulating progressive human ESCC stages, we established a tissue clearing workflow for three-dimensional visualization and analysis of esophageal vessels and T cell distribution. Through this workflow, we delineated the spatial dynamics of vascular remodeling, CD3+ T cells, and characteristic T cell aggregates employing high-resolution light-sheet fluorescence microscopy across five ESCC pathogenic stages. Vessel remodeling might be coupled with T cell infiltration, and their interactions predominantly occurred at the inflammatory stage. These findings provided insights into research methodologies of esophageal cancer and spatiotemporal landscapes of vascular and T cell during ESCC initiation and progression.
Adrienn Kazsoki, Krisztina Németh, Tamás Visnovitz
et al.
Abstract Due to their small size, flexibility, and adhesive properties, extracellular vesicles (EVs) hold promises as effective drug delivery systems. However, challenges such as the variability in vesicle types and the need to maintain their integrity for medical applications exist. Curcumin, a compound found in turmeric and known for its diverse health benefits, including anti-cancer and anti-inflammatory properties, faces obstacles in clinical use due to issues like low solubility, limited absorption, and rapid breakdown in the body. This study aimed to incorporate large-sized curcumin-loaded extracellular vesicles (lEVs) into fast-dissolving nanofibers made of poly(vinyl alcohol) (PVA) by electrospinning. By using aqueous PVA-based solutions for electrospinning, the presence of curcumin-loaded lEVs in the nanofibers was confirmed by confocal laser scanning microscopy. Furthermore, the release study demonstrated high concentrations of the drug in nanofibers containing lEVs. These findings are significant for advancing the development and utilization of active ingredient-loaded EV systems within nanofibrous formulations, potentially leading to improved patient outcomes.
Samuel Antwi-Baffour, Benjamin Tetteh Mensah, Simon Aglona Ahiakonu
et al.
Abstract Background Malaria is a life-threatening parasitic disease typically transmitted through the bite of an infected Anopheles mosquito. There is ample evidence showing the potential of malaria infection to affect the counts of lymphocyte subpopulations in the peripheral blood, but the extent of alteration might not be consistent in all geographical locations, due to several local factors. Although Ghana is among the malaria-endemic countries, there is currently no available data on the level of alterations that occur in the counts of lymphocyte subpopulations during P. falciparum malaria infection among adults. Aim The study was to determine the immunophenotypic alterations in the level of peripheral blood lymphocytes and their subsets in adults with uncomplicated P. falciparum malaria infection and apparently healthy participants. Methods The study was a cross-sectional comparative study conducted in two municipalities of the Volta region of Ghana. Blood samples were collected from study participants and taken through serology (P. falciparum/Pan Rapid Diagnostic Kits), microscopy (Thick and thin blood films) and Haematological (Flow cytometric and Full blood count) analysis. Results A total of 414 participants, comprising 214 patients with malaria and 200 apparently healthy individuals (controls) were recruited into this study. Parasite density of the malaria patients ranged from 75/µL to 84,364/µL, with a mean of 3,520/µL. It was also observed that the total lymphocytes slightly decreased in the P. falciparum-infected individuals (Mean ± SD: 2.08 ± 4.93 × 109/L) compared to the control group (Mean ± SD: 2.47 ± 0.80 × 109/L). Again, there was a significant moderate positive correlation between parasite density and haematocrit levels (r = 0.321, p < 0.001). Apart from CD45 + T-cells, more people in the control group had normal values for the lymphocyte subsets measured compared to the malaria patients. Conclusions From the results obtained, there was high parasite density among the malaria patients suggestive of high intensity of infection in the case group. The malaria patients again showed considerable haematological alterations in lymphocyte sub-sets and the parasite density appeared to be strongly associated with CD4 + T-cell reduction. Also, the parasite density significantly associated with decreasing haematocrit levels. This indicates that lymphocyte subset enumeration can be used to effectively support malaria diagnosis.
Structured Illumination Microscopy (SIM) overcomes the optical diffraction limit by folding high-frequency components into the baseband of the optical system, where they can be extracted and then repositioned to their original location in the Fourier domain. Although SIM is considered superior to other super-resolution (SR) methods in terms of compatibility with live cell imaging and optical setup simplicity, its reliance on image reconstruction restricts its temporal resolution and may introduce distortions in the super-resolved image. These inherent drawbacks are exacerbated in extended-SIM im-plementations, where spatial resolution surpasses the diffraction limit by more than 2-fold. Here, we present and demon-strate the Tessellation Structured Illumination Microscopy (TSIM) framework, which introduces a revived image recon-struction paradigm. With TSIM both the temporal resolution limit and the reconstruction artifacts that impact extended-SIM, are alleviated, without compromising the achievable spatial resolution.
Malcolm Bogroff, Gabriel Cowley, Ariel Nicastro
et al.
Cathodoluminescence microscopy is now a well-established and powerful tool for probing the photonic properties of nanoscale materials, but in many cases, nanophotonic materials are easily damaged by the electron-beam doses necessary to achieve reasonable cathodoluminescence signal-to-noise ratios. Two-dimensional materials have proven particularly susceptible to beam-induced modifications, yielding both obstacles to high spatial-resolution measurement and opportunities for beam-induced patterning of quantum photonic systems. Here pan-sharpening techniques are applied to cathodoluminescence microscopy in order to address these challenges and experimentally demonstrate the promise of pan-sharpening for minimally-perturbative high-spatial-resolution spectrum imaging of beam-sensitive materials.
Heidi V. Kastenholz, Michael I. Topper, Warren S. Warren
et al.
Carbon-based black pigments, a widely used class of pigments, are difficult to differentiate with the noninvasive techniques currently used in cultural heritage science. We utilize pump-probe microscopy to distinguish four common carbon-based black pigments as pure pigments, as two-component black pigment mixtures, and as a mixture of a black and a colorful pigment. This work also demonstrates that even nominally homogeneous pigments present remarkable, and useful, heterogeneity in pump-probe microscopy.
Chen Liu, Danqi Liao, Alejandro Parada-Mayorga
et al.
The proliferation of digital microscopy images, driven by advances in automated whole slide scanning, presents significant opportunities for biomedical research and clinical diagnostics. However, accurately annotating densely packed information in these images remains a major challenge. To address this, we introduce DiffKillR, a novel framework that reframes cell annotation as the combination of archetype matching and image registration tasks. DiffKillR employs two complementary neural networks: one that learns a diffeomorphism-invariant feature space for robust cell matching and another that computes the precise warping field between cells for annotation mapping. Using a small set of annotated archetypes, DiffKillR efficiently propagates annotations across large microscopy images, reducing the need for extensive manual labeling. More importantly, it is suitable for any type of pixel-level annotation. We will discuss the theoretical properties of DiffKillR and validate it on three microscopy tasks, demonstrating its advantages over existing supervised, semi-supervised, and unsupervised methods. The code is available at https://github.com/KrishnaswamyLab/DiffKillR.
Optic deconvolution in light microscopy (LM) refers to recovering the object details from images, revealing the ground truth of samples. Traditional explicit methods in LM rely on the point spread function (PSF) during image acquisition. Yet, these approaches often fall short due to inaccurate PSF models and noise artifacts, hampering the overall restoration quality. In this paper, we approached the optic deconvolution as an inverse problem. Motivated by the nonstandard-form compression scheme introduced by Beylkin, Coifman, and Rokhlin (BCR), we proposed an innovative physics-informed neural network Multi-Stage Residual-BCR Net (m-rBCR) to approximate the optic deconvolution. We validated the m-rBCR model on four microscopy datasets - two simulated microscopy datasets from ImageNet and BioSR, real dSTORM microscopy images, and real widefield microscopy images. In contrast to the explicit deconvolution methods (e.g. Richardson-Lucy) and other state-of-the-art NN models (U-Net, DDPM, CARE, DnCNN, ESRGAN, RCAN, Noise2Noise, MPRNet, and MIMO-U-Net), the m-rBCR model demonstrates superior performance to other candidates by PSNR and SSIM in two real microscopy datasets and the simulated BioSR dataset. In the simulated ImageNet dataset, m-rBCR ranks the second-best place (right after MIMO-U-Net). With the backbone from the optical physics, m-rBCR exploits the trainable parameters with better performances (from ~30 times fewer than the benchmark MIMO-U-Net to ~210 times than ESRGAN). This enables m-rBCR to achieve a shorter runtime (from ~3 times faster than MIMO-U-Net to ~300 times faster than DDPM). To summarize, by leveraging physics constraints our model reduced potentially redundant parameters significantly in expertise-oriented NN candidates and achieved high efficiency with superior performance.
BackgroundUrinary extracellular vesicles (uEVs) are derived from epithelia facing the renal tubule lumen in the kidney and urogenital tract; they may carry protein biomarkers of renal dysfunction and structural injury. However, there are scarce studies focusing on uEVs in diabetes with kidney injury.Materials and methodsA community-based epidemiological survey was performed, and the participants were randomly selected for our study. uEVs were enriched by dehydrated dialysis method, quantified by Coomassie Bradford protein assay, and adjusted by urinary creatinine (UCr). Then, they identified by transmission electron microscopy (TEM), nanoparticle track analysis (NTA), and western blot of tumor susceptibility gene 101.ResultsDecent uEVs with a homogeneous distribution were finally obtained, presenting a membrane-encapsulated structure like cup-shaped or roundish under TEM, having active Brownian motion, and presenting the main peak between 55 and 110 nm under NTA. The Bradford protein assay showed that the protein concentrations of uEVs were 0.02 ± 0.02, 0.04 ± 0.05, 0.05 ± 0.04, 0.07 ± 0.08, and 0.11 ± 0.15 μg/mg UCr, respectively, in normal controls and in prediabetes, diabetes with normal proteinuria, diabetes with microalbuminuria, and diabetes with macroproteinuria groups after adjusting the protein concentration with UCr by calculating the vesicles-to-creatinine ratio.ConclusionThe protein concentration of uEVs in diabetes with kidney injury increased significantly than the normal controls before and after adjusting the UCr. Therefore, diabetes with kidney injury may change the abundance and cargo of uEVs, which may be involved in the physiological and pathological changes of diabetes.
Diseases of the endocrine glands. Clinical endocrinology
Jairo Alberto Muñoz, Egor Dolgach, Vanina Tartalini
et al.
This research presents the microstructural and mechanical evolution throughout the welded seam of an austenitic stainless steel (ASS) tube. It was found that the main hardness decrement occurred in the fusion zone (FZ), followed by the heat-affected zone (HAZ) and the base material (BM). Optical microscopy indicated a dendritic structure in FZ and heterogeneous austenitic grain size from the HAZ towards the BM, ranging from 100 µm to 10 µm. The welding process generated an intense texture around the FZ and the HAZ, while the BM still showed an extrusion-like texture. In terms of mechanical behavior, the largest austenite grain size in the FZ led to the lowest strength and ductility of all zones due to the earliest strain localization manifested by heterogeneous strain distribution. However, the strain localization in all zones appeared after 0.4 true strain, indicating an overall good ductility of the seam. These high values were related to two microstructure characteristics: (1) the 10% δ-ferrite after solidification in the FZ favored by the <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mi>C</mi><msub><mi>r</mi><mrow><mi>e</mi><mi>q</mi></mrow></msub><mo>/</mo><mi>N</mi><msub><mi>i</mi><mrow><mi>e</mi><mi>q</mi></mrow></msub><mo>=</mo><mn>1.67</mn></mrow></semantics></math></inline-formula> relationship that delayed the crack propagation along the austenite grains and (2) the heterogeneous microstructure made up of soft austenite and hard martensite in the HAZ and BM producing multiple strain concentrations. Kernel Average Misorientation (KAM) maps obtained by Electron Back-Scattering Diffraction (EBSD) allowed observing higher internal misorientations in the FZ than in the HAZ due to interconnected walls between the δ-ferrite grains. However, the largest KAM values were observed in the BM between γ-austenite and the deformation-induced α’-martensite phases. X-ray diffraction revealed that the residual stresses in the cross-section of the welded seam were compression-type and then switched to tension-type in the outer surface.
Xiaosheng Wu, Michelle K. Manske, Gordon J. Ruan
et al.
Summary: Despite extensive research, the specific factor associated with SARS-CoV-2 infection that mediates the life-threatening inflammatory cytokine response in patients with severe COVID-19 remains unidentified. Herein we demonstrate that the virus-encoded Open Reading Frame 8 (ORF8) protein is abundantly secreted as a glycoprotein in vitro and in symptomatic patients with COVID-19. ORF8 specifically binds to the NOD-like receptor family pyrin domain-containing 3 (NLRP3) in CD14+ monocytes to induce inflammasomal cytokine/chemokine responses including IL1β, IL8, and CCL2. Levels of ORF8 protein in the blood correlate with severity and disease-specific mortality in patients with acute SARS-CoV-2 infection. Furthermore, the ORF8-induced inflammasome response was readily inhibited by the NLRP3 inhibitor MCC950 in vitro. Our study identifies a dominant cause of pathogenesis, its underlying mechanism, and a potential new treatment strategy for severe COVID-19.
Sarlota Birnsteinova, Danilo E. Ferreira de Lima, Egor Sobolev
et al.
The X-ray microscopy technique at the European X-ray free-electron laser (EuXFEL), operating at a MHz repetition rate, provides superior contrast and spatial-temporal resolution compared to typical microscopy techniques at other X-ray sources. In both online visualization and offline data analysis for microscopy experiments, baseline normalization is essential for further processing steps such as phase retrieval and modal decomposition. In addition, access to normalized projections during data acquisition can play an important role in decision-making and improve the quality of the data. However, the stochastic nature of XFEL sources hinders the use of existing flat-flied normalization methods during MHz X-ray microscopy experiments. Here, we present an online dynamic flat-field correction method based on principal component analysis of dynamically evolving flat-field images. The method is used for the normalization of individual X-ray projections and has been implemented as an online analysis tool at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of EuXFEL.
Sergei V. Kalinin, Debangshu Mukherjee, Kevin M. Roccapriore
et al.
Machine learning (ML) has become critical for post-acquisition data analysis in (scanning) transmission electron microscopy, (S)TEM, imaging and spectroscopy. An emerging trend is the transition to real-time analysis and closed-loop microscope operation. The effective use of ML in electron microscopy now requires the development of strategies for microscopy-centered experiment workflow design and optimization. Here, we discuss the associated challenges with the transition to active ML, including sequential data analysis and out-of-distribution drift effects, the requirements for the edge operation, local and cloud data storage, and theory in the loop operations. Specifically, we discuss the relative contributions of human scientists and ML agents in the ideation, orchestration, and execution of experimental workflows and the need to develop universal hyper languages that can apply across multiple platforms. These considerations will collectively inform the operationalization of ML in next-generation experimentation.
The edible film with barrier property can restrain food oxidation rancidity and improve the effect of storage and preservation when applied to food. In this study, sodium carboxymethyl cellulose, sodium alginate and guar gum were used as film forming materials, oxygen resistance and water vapor permeability were used as indicators, and the process optimization of sodium carboxymethyl cellulose-sodium alginate-guar gum ternary mixed membrane was carried out through single factor experiment and D-optimal mixture design. The light transmittance, rheological properties, fourier transform infrared spectroscopy, scanning electron microscopy and thermodynamic properties of the edible films were analyzed and compared. The results showed that when 1.25% sodium carboxymethyl cellulose:2% sodium alginate:0.75% guar gum was 35:49:16, the reciprocal of the water vapor permeability was 505224 and the oxygen resistance was 0.52 of the edible film, indicating the best barrier. Compared with the single membrane, the optimized mixed membrane significantly improved its properties, the components had good coordination, and the structure of the mixed membrane had good compatibility and integrity. The study of the edible ternary mixed membrane can provide a reference for the development of new edible film.
Differential dynamic microscopy (DDM) is increasingly used in the fields of soft matter physics and biophysics to extract the dynamics of microscopic objects across a range of wavevectors using optical microscopy. Standard DDM is limited to detecting dynamics no faster than the camera frame rate. We report on an extension to DDM where we sequentially illuminate the sample with spectrally-distinct light and image with a color camera. By pulsing blue and then red light separated by a lag time much smaller than the camera's exposure time we are able to use this two-color DDM method to measure dynamics occurring much faster than the camera frame rate.