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When exposed to different relative humidities (RHs), the optical properties of atmospheric aerosols will change because of changes in the aerosol particle size and complex refractive index (RI), which will affect haze formation and global climate change. The potential contributions of ultrafine particles to the atmospheric optical characteristics and to haze spreading cannot be ignored because of their high particle number concentrations and strong diffusibility; measurement of the optical properties of wet ultrafine particles is thus highly important for environmental assessment. Therefore, a surface plasmon resonance microscopy with azimuthal rotation illumination (SPRM-ARI) system is designed to determine the RIs of single particle aerosols with diameters of less than 100 nm in the hygroscopic growth process. Measurements are taken using mixed single particles with different mass ratios. The RIs of mixed single aerosols at different RHs are retrieved by measuring the scattering light intensity using the SPRM-ARI system and almost all the RIs of the bicomponent particles with different mass ratios decrease with increasing water content under high RH conditions. Finally, for each of the bicomponent particles, the maximum standard deviations for the retrieved RI values are only 2.06×10

In the current exploration an impact of 44 µm estimated B4C support expansion on the mechanical conduct of Al2618 combination has been contemplated. The Al2618 compound metal composites with 2, 4, 6 and 8 changing weight rates of B4C were created by stircast strategy. The orchestrated 44 µm estimated B4C particles built up Al2618 combination composites were exposed to microstructural considers, mechanical properties testing. Microstructural portrayals of acquired examples were completed by SEM microscopy and EDS spectrums. The B4C particles were uniformly disseminated and existences of these particles were affirmed by the EDS patterns. The hardness and tensile qualities of metal composites have been upgraded with the expansion of B4C support. There was decline in the ductility of the Al2618 compound after the joining of the support. Different crack and failure systems were seen in the Al2618-B4C composites utilizing SEM.

Electrochemiluminescence (ECL) of luminol is a luminescence process that proceeds in the presence of reactive oxygen species (e.g. hydrogen peroxide (H2O2)) at a suitable electrode potential, the reaction mechanism of which is complicated and remains ambiguous. In this work, we report a visualization approach for measuring the thickness of the ECL layer (TEL) of the luminol/H2O2 system to decipher the reaction process by combined use of the microtube electrode, ECL microscopy, and finite element simulations. With the increase of solution pH, the ECL image captured with the microtube electrode tends to vary from spot to ring, corresponding to the decrease of TEL from >9.1 μm to ca. 4.3 μm. We propose that different intermediates are involved in the course of ECL reaction. At a low pH (e.g. pH

Fluorescence nanoscopy provides imaging techniques that overcome the diffraction-limited resolution barrier in light microscopy, thereby opening up a new area of research in biomedical imaging in fields such as neuroscience. Here, we review the foremost fluorescence nanoscopy techniques, including descriptions of their applications in elucidating protein architectures and mobility, the real-time determination of synaptic parameters involved in neural processes, three-dimensional imaging, and the tracking of nanoscale neural activity. We conclude by discussing the prospects of fluorescence nanoscopy, with a particular focus on its deployment in combination with related techniques (e.g., machine learning) in neuroscience.

To digitise our old archive and evaluate the efficiency of this task, both in medical and economic terms.All slides and negatives (8254) archived in our clinic were collected, digitised with a 5-megapixel slide scanner. The images were taken from 1972 to 1999. Quality and utility of images were taken into account, as far as costs of the task (up to 2100 euros), all the work done by the same ophthalmologist.Of those identifiable, 62% of the patients had already died. Only 1.5% were archived for use; 70 images for teaching reasons and 60 for medical reasons, being incorporated into the patient's history. About 210h were spent on scanning, identifying, checking and uploading images. 84% corresponded to retinal pathology, 4% to glaucomatous pathology, 3% to anterior segment pathology and the last 9% to learning material. The quality of most images is good, and, in some cases, were important for the correct diagnosis of the pathology. If only medical reasons are taken into account, the number of images uploaded is very low when working with archives older than 50 years.Although there was a low percentage of scanned images, the task was efficient because of a low cost. Images older than 50 years must be evaluated before scanning because of their low utility.

Many institutions have cytopathology case archives for education. Unfortunately, these slides deteriorate over time and have limited accessibility. Whole slide imaging (WSI) can overcome these limitations. However, suboptimal image quality and scanning effort are barriers.We selected 123 slides from cytopathology study sets for WSI scanning at 400x magnification without z-stacking. The Ventana DP 200 scanner and Virtuoso software were used. Slides were scanned in 2 rounds: the first round of slides was prepared for scanning with light cleaning, and the second round was performed only on slides that had unacceptable WSI quality after thorough cleaning. Slides were assessed with a 4-tier grading system created by the authors. Time to scan each slide was recorded.Within the first round, 96 of 123 (78%) slides scanned were determined to be of acceptable quality. After the second round of scanning, in total, 118 of 123 (95.9%) slides were determined to be of acceptable quality. The average time needed to scan each slide was 213 seconds.The majority of slides scanned were of acceptable quality in the first round of scanning. After cleaning and rescanning, nearly every slide investigated was of acceptable quality. The primary objective is to provide other institutions that may be considering a similar project a benchmark so that they know what to expect in terms of slide scan success rate and the amount of time needed to digitize slides for educational archiving. This pilot study demonstrates the feasibility of using WSI for cytology education cases.

Single-molecule localization microscopy (SMLM) permits the visualization of cellular structures an order of magnitude smaller than the diffraction limit of visible light, and an accurate, objective evaluation of the resolution of an SMLM data set is an essential aspect of the image processing and analysis pipeline. Here, we present a simple method to estimate the localization spread function (LSF) of a static SMLM data set directly from acquired localizations, exploiting the correlated dynamics of individual emitters and properties of the pair autocorrelation function evaluated in both time and space. The method is demonstrated on simulated localizations, DNA origami rulers, and cellular structures labeled by dye-conjugated antibodies, DNA-PAINT, or fluorescent fusion proteins. We show that experimentally obtained images have LSFs that are broader than expected from the localization precision alone, due to additional uncertainty accrued when localizing molecules imaged over time.

Less than a quarter of the world population has access to microneurosurgical care within a range of 2 hours. We introduce a simplified exoscope system to achieve magnification, illumination, and video recording in low-resource settings.We combined an industrial microscope tube, a heavy-duty support arm, a wide-field c-mount digital microscope camera, and a light-emitting diode ring light. All parts were sterilized with ethylene oxide. We performed 13 spinal and 3 cranial surgeries with the help of the low-budget exoscope.The average preoperative setup time was 12.8 minutes. The exoscope provided similar magnification and illumination like a conventional binocular microscope. It allowed operating in a comfortable posture. The field of vision ranged from 30 mm-60 mm. The surgical field was captured by a 16-megapixel two-dimensional camera and projected to a 55-inch high-definition television screen in real time. Image quality was similar to that of a conventional microscope although our exoscope lacked stereoscopic view. Adjusting camera position and angle was time-consuming. Thus, the benefit of the exoscope was most notable in spine surgeries where the camera remained static for most of the time. The total cost of the exoscope was approximately U.S. $ 750.Our low-budget exoscope offers similar image quality, magnification, and illumination like a conventional binocular microscope. It may thus help expand access to neurosurgical care worldwide. Users may face difficulty adapting to the lack of depth perception in the beginning. Prospective studies are needed to assess its usability and effectiveness compared to the microscope.

Background Millions of people throughout the world suffer from parasite infections. Traditionally, technicians use manual eye inspection of microscopic specimens to perform a parasite examination. However, manual operations have limitations that hinder the ability to obtain precise egg counts and cause inefficient identification of infected parasites on co-infections. The technician requirements for handling a large number of microscopic examinations in countries that have limited medical resources are substantial. We developed the helminth egg analysis platform (HEAP) as a user-friendly microscopic helminth eggs identification and quantification platform to assist medical technicians during parasite infection examination. Methods Multiple deep learning strategies including SSD (Single Shot MultiBox Detector), U-net, and Faster R-CNN (Faster Region-based Convolutional Neural Network) are integrated to identify the same specimen allowing users to choose the best predictions. An image binning and egg-in-edge algorithm based on pixel density detection was developed to increase the performance. Computers with different operation systems can be gathered to lower the computation time using our easy-to-deploy software architecture. Results A user-friendly interface is provided to substantially increase the efficiency of manual validation. To adapt to low-cost computers, we architected a distributed computing structure with high flexibilities. Conclusions HEAP serves not only as a prediction service provider but also as a parasitic egg database of microscopic helminth egg image collection, labeling data and pretrained models. All images and labeling resources are free and accessible at http://heap.cgu.edu.tw . HEAP can also be an ideal education and training resource for helminth egg examination.

Time-lapse microscopy images generated by biological experiments have been widely used for observing target activities, such as the motion trajectories and survival states. Based on these observations, biologists can conclude experimental results or present new hypotheses for several biological applications, i.e. virus research or drug design. Many methods or tools have been proposed in the past to observe cell and particle activities, which are formed as single cell tracking and single particle tracking problems, by using algorithms and deep learning technologies. In this article, a review for these works is presented in order to summarize the past methods and research topics at first, then points out the problems raised by these works, and finally proposes future research directions. The contributions of this article will help researchers to understand past development trends and further propose innovative technologies.

Monitoring neural activity and associating neural dynamics with the anatomical connectome are required to understand how the brain works. Neural dynamics are measured by electrophysiology and optical imaging. Since the discovery of the two-photon excitation phenomenon, significant progress has been made in deep imaging for capturing neural activity from numerous neurons in vivo. The development of two-photon microscopy is aimed to image neural activity from a large and deep region with high spatial (x, y, and z) and temporal (t) resolutions at a high signal-to-noise ratio. Imaging deep regions along the optical axis (z-axis) is particularly challenging because heterogeneous biological tissues scatter and absorb light. Recent advances in the light focus modulation technology at high speeds in three dimensions (x, y, and z) have allowed multiplane two-photon imaging. z-Focus control by varifocal optical systems, such as ferroelectric liquid lenses, gradient refractive index lenses, and adaptive optical element systems, and multiplexing by time- and wavelength-division strategies have allowed to rapidly observe specimens at different focal depths. Herein, we overview the recent advances in multiplane functional imaging systems that enable four-dimensional (x, y, z, and t) analysis of neural dynamics, with a special emphasis on z-scanning mechanisms and multiplexing strategies.

Single molecule detection is necessary to find out physical, chemical properties and their mechanism involved in the normal functioning of body cells. In this way, they can provide a new direction to the healthcare system. Various techniques have been developed and employed for their successful detection. Herein, we have emphasized various traditional methods as well as biosensing technology which offer single molecule sensitivity. The various methods including plasmonic resonance, nanopores, whispering gallery mode, Simoa assay and recognition tunneling are discussed in the initial part which has been followed by a discussion about biosensor-based detection. Plasmonic, SERS, CRISPR/Cas, and other types of biosensors are focused in this review and found to be highly sensitive for single molecule detection. This review provides an overview of progression in different techniques employed for single molecule detection.

Automatic segmentation of erythrocytes in microscopic blood smear phone images is a critical step to visualize and identify malaria using machine learning technologies. However, it still remains a challenging problem due to the scarcity of experts, low image qualities, slow manual and inefficient quality of diagnosis. To handle these issues to some extent, we proposed an effective multi-magnification deep residual neural network (MM-ResNet), where we fully automatically classify the microscopic blood smear images as either infected/ non-infected at multiple magnifications. We have experimentally evaluated our approach by using it to train more efficient variants of different compact deep convolutional neural networks (CNN), evaluated on phone datasets. The MM-ResNet end-to-end framework shows similar or superior accuracy than the baseline architectures, as measured by GPU timings on the publicly available microscopic blood smear phone images. This approach is the first application of a MM-ResNet for malaria-infected erythrocyte identification in microscopic blood smear images.

The increasing throughput of experiments in biomaterials research makes automatic techniques more and more necessary. Among all the characterization methods, microscopy makes fundamental contributions to biomaterials science where precisely focused images are the basis of related research. Although automatic focusing has been widely applied in all kinds of microscopes, defocused images can still be acquired now and then due to factors including background noises of materials and mechanical errors. Herein, we present a deep-learning-based method for the automatic sorting and reconstruction of defocused cell images. First, the defocusing problem is illustrated on a high-throughput cell microarray. Then, a comprehensive dataset of phase-contrast images captured from varied conditions containing multiple cell types, magnifications, and substrate materials is prepared to establish and test our method. We obtain high accuracy of over 0.993 on the dataset using a simple network architecture that requires less than half of the training time compared with the classical ResNetV2 architecture. Moreover, the subcellular-level reconstruction of heavily defocused cell images is achieved with another architecture. The applicability of the established workflow in practice is finally demonstrated on the high-throughput cell microarray. The intelligent workflow does not require a priori knowledge of focusing algorithms, possessing widespread application value in cell experiments concerning high-throughput or time-lapse imaging., Graphical abstract Image 1, Highlights • An automatic workflow for the sorting and reconstruction of defocused images. • Representative data containing varied cells, substrate materials, and magnifications. • Effectiveness of the workflow in high-throughput experiments. • Publicly accessible image datasets and codes.

Since the 1990s, continuous technical and scientific advances have defied the diffraction limit in microscopy and enabled three-dimensional (3D) super-resolution imaging. An important milestone in this pursuit is the coherent utilization of two opposing objectives (4Pi geometry) and its combination with super-resolution microscopy. Herein, we review the recent progress in 4Pi nanoscopy, which provides a 3D, non-invasive, diffraction-unlimited, and isotropic resolution in transparent samples. This review includes both the targeted and stochastic switching modalities of 4Pi nanoscopy. The schematics, principles, applications, and future potential of 4Pi nanoscopy are discussed in detail.

A current trend to simplify dental restorative procedures is toward using universal chromatic light-cured resin-based composites (RBCs) designed to adapt esthetically to various clinical situations. This study offers a comparative characterization of the mechanical and cytotoxic behavior of such materials that use different techniques to adjust their optical properties (e.g., structural color instead of pigment addition), have different filler systems but are based on a comparable organic matrix.The structural appearance of the filler systems was assessed by scanning electron microscopy. Various quasi-static and viscoelastic parameters were evaluated at clinically relevant frequencies (0.5-5 Hz) using an instrumented indentation test with a Dynamic Mechanical Analysis (DMA) module. Cytotoxicity on human gingival fibroblasts (HGF-1), when exposed to eluates from tested RBCs specimens (up to one month), was assessed using a WST-1 colorimetric proliferation assay. Multifactor analysis of variance was applied to compare the parameters of interest (Martens, Vickers, and indentation hardness; elastic and total indentation work; creep, indentation depth; storage, loss, and indentation moduli; loss factor; cell viability) between analyzed RBCs, loading frequencies, and eluate age.Structural particularities of the filler systems are directly reflected in the mechanical behavior of the analyzed materials. Changes in the filler system, necessary to achieve structural color, generally resulted in lower mechanical properties but a better ability to absorb shock. In contrast, the cytotoxicity was comparable.Based on the performed characterization, universal chromatic RBCs fits in the conventional RBCs class to expect comparable clinical behavior.

PURPOSE. To evaluate the thickness of the tear film lipid layer (TFLL) in meibomian gland dysfunction (MGD) using a high-resolution optical microscope. METHODS. The Ocular Surface Disease Index (OSDI) and meibum grade score (MGS) were used to classify 190 subjects into four groups: normal (OSDI

Here, all-solid scanning electrochemical cell microscopy (SECCM) is first established by filling polyacrylamide (PAM) into nanocapillaries as a solid electrolyte. A solid PAM nanoball at the tip of a nanocapillary contacts graphene and behaves as an electrochemical cell for simultaneously measuring the morphology and electrochemical activity. Compared with liquid droplet-based SECCM, this solid nanoball is stable and does not leave any electrolyte at the contact regions, which permits accurate and continuous scanning of the surface without any intervals. Accordingly, the resolutions in the lateral (x-y) and vertical (z) directions are improved to ∼10 nm. The complete scanning of the wrinkles on graphene records low currents at the two sidewalls of the wrinkles and a relatively high current at the center of the wrinkles. The heterogeneity in the electrochemical activity of the wrinkle illustrates different electron transfer features on surfaces with varied curvatures, which is hardly observed by the current electrochemical or optical methods. The successful establishment of this high spatial electrochemical microscopy overcomes the current challenges in investigating the electrochemical activity of materials at the nanoscale, which is significant for a better understanding of electron transfer in materials.

In this study, ZnO nanotubes, ZnO/ZnS core-shell nanorods, and ZnO/ZnS nanotubes were fabricated. To fabricate different types of ZnO-based nanocomposites, the central area of the ZnO nanorods was etched by a KCl solution and the outward shell was sulfurized to form ZnS/ZnO shell structures. Surface morphologies and material properties of the various nanostructures were examined by Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Transmission X-ray Microscopy (TXM). Multiple material characterizations including X-Ray Diffraction (XRD), X-Ray Photoelectron Spectroscopy (XPS), and Photoluminescence (PL) were performed. Results indicate that larger surface areas and higher defect concentrations can be obtained by the etching of ZnO nanorods. ZnO/ZnS nanotubes formed by sulfurization roughen the shell surface and mitigate defects. Results indicate that ZnO and ZnO/ZnS nanotubes show promise for future sensing device applications.

The triple cation mixed perovskites (CsFAMA) are known as one of the most efficient candidates for perovskite solar cells (PSCs). It is found that the power conversion efficiency (PCE) of triple-cation based devices would increase with the test time extending, and the maximum efficiency is normally obtained after several days aging storage. Here, the relationship between enhanced device performance, excess PbI2 and its evolution in triple cation perovskite films of initial days was systematically explored. The CsFAMA-PSCs are prepared by two-step methods under two environmental conditions, including in the glove box and the ambient air (30% humidity). After 7 days testing, the maximum PCE of PSCs under two conditions dramatically increased 12.4% and 12.2%, reached 21.68% and 21.89%, respectively. At initial days, the XRD peak intensities of perovskite phase gradually decreased and those corresponding to PbI2 increased. Along with time-resolved photoluminescence (TRPL) and kelvin probe force microscopy (KPFM), it was found that the defects were passivated with the evolution of PbI2. This work reveals the excess PbI2 and its evolution in perovskite film, which can further supplement the understanding of PbI2 defect passivation.

BackgroundThe Molecular Microscope (MMDx) system classifies heart transplant endomyocardial biopsies as No-rejection (NR), Early-injury, T cell-mediated (TCMR), antibody-mediated (ABMR), mixed, and possible rejection (possible TCMR, possible ABMR). Rejection-like gene expression patterns in NR biopsies have not been described. We extended the MMDx methodology, using a larger data set, to define a new "Minor" category characterized by low-level inflammation in non-rejecting biopsies.MethodsUsing MMDx criteria from a previous study, molecular rejection was assessed in 1,320 biopsies (645 patients) using microarray expression of rejection-associated transcripts (RATs). Of these biopsies, 819 were NR. A new archetypal analysis model in the 1,320 data set split the NRs into NR-Normal (N = 462) and NR-Minor (N = 359).ResultsCompared to NR-Normal, NR-Minor were more often histologic TCMR1R, with a higher prevalence of donor-specific antibody (DSA). DSA positivity increased in a gradient: NR-Normal 24%; NR-Minor 34%; possible ABMR 42%; ABMR 66%. The top 20 transcripts distinguishing NR-Minor from NR-Normal were all ABMR-related and/or IFNG-inducible, and also exhibited a gradient of increasing expression from NR-Normal through ABMR. In random forest analysis, TCMR and Early-injury were associated with reduced LVEF and increased graft loss, but NR-Minor and ABMR scores were not. Surprisingly, hearts with MMDx ABMR showed comparatively little graft loss.ConclusionsMany heart transplants currently diagnosed as NR by histologic or molecular assessment have minor increases in ABMR-related and IFNG-inducible transcripts, associated with DSA positivity and mild histologic inflammation. These results suggest that low-level ABMR-related molecular stress may be operating in many more hearts than previously estimated. (ClinicalTrials.gov #NCT02670408).

Van der Waals (vdWs) heterostructures based on two-dimensional (2D) materials have become a promising candidate for photoelectrochemical (PEC) catalyst not only because of the freedom in materials design that enable the band-offset construction and facilitate the charge separation. They also provide a platform for the study of various of interface effect in PEC. Here, we report a new kind of mixed-dimensional vdWs heterostructure photoelectrode and investigate the strain enhanced PEC performance at vdWs interfaces. Our heterostructures are composed of 2D n-type MoS2 nanosheets and three-dimensional (3D) p-type Cu2O nanorod arrays (NRAs), where Cu2O NRAs introduce periodically strain in the p-n junction interface. We find a promotion of the HER catalytic activities in heterostructure based PEC photoelectrodes using in-situ measurement techniques including the scanning electrochemical cell microscopy and various local spectrum probe measurements. This is attributed to the efficient charge separation at the strained heterointerface. Our results demonstrate an interesting venue for understanding the local interface effects with high spatial resolution, and shed light on design and developing high-efficiency photoelectrodes. 1L MoS2/Cu2O vdWs heterostructure photocathodes were prepared by nanoindentation technology. The effects of strain on promoting charge separation at the heterointerface were verified by the enhanced performances in PEC hydrogen evolution reaction of vdWs heterostructure through scanning electrochemical cell microscopy technique and various local spectrum probe measurements.

Conventional clinical histopathology analysis is based on bright-field microscopy of thinly sliced tissue specimens. Although bright-field microscopy is a simple and robust method for examining microscope slides, preparing the slides needed is a lengthy and labor-intensive process. Slide-free histopathology technologies address this problem by directly imaging intact, minimally processed tissue samples using advanced optical imaging systems, bypassing the extended workflow now required for preparation of tissue sections. This manuscript explains the technical basis of slide-free microscopy, reviews common slide-free optical microscopy techniques, and discusses the opportunities and challenges involved in clinical implementation.

Quantitative optical microscopy-an emerging, transformative approach to single-cell biology-has seen dramatic methodological advancements over the past few years. However, its impact has been hampered by challenges in the areas of data generation, management, and analysis. Here we outline these technical and cultural challenges and provide our perspective on the trajectory of this field, ushering in a new era of quantitative, data-driven microscopy. We also contrast it to the three decades of enormous advances in the field of genomics that have significantly enhanced the reproducibility and wider adoption of a plethora of genomic approaches.

Electronic Speckle Pattern Interference (ESPI) is a non-contact optical technique generally used for the measurement of surface deformations of various kinds. With the help of phase retrieval algorithms, phase of the deformed object is calculated in this method which can be then converted into displacement information. This paper demonstrates the use of ESPI method to measure the thickness of some nano-sized metallic thin films. Calculated thickness values are in good agreement with the values measured via Profilometer. The least thickness (z-axis) measured by this method is ~ 52 ± 3.1 nm. The z-axis resolution of the used method is at par with other expensive microscopy techniques like atomic force microscopy. Therefore, a low-cost alternative to expensive microscopy techniques is presented in this paper as far as z-axis is concern. Surface roughness of thin films can also be calculated using retrieved surface profiles and they are also found to be in the nanometer range.

Highly porous SiOC ceramic foams with gradient or uniform macrostructures were obtained through polymer derived ceramic routes. Precuring of preceramic polymers and introduction of SiO2 powders were used to tailor precursor viscosity and hence SiOC foam macrostructure. Effects of polymer viscosity on porosity, pore size, pore distribution were investigated by light microscopy and micro-computed tomography techniques. SiOC ceramic foams. Foams from one unmodified precursor, showed pore size gradient with small pores located at bottom and large pores at the top. To address this non-uniformity, the viscosity of the precursor was increased by pre-curing the preceramic polymer, which resulted in decrease of the average pore size and improvement in pore size uniformity. For a different system with a self-foaming preceramic polymer, because of the simultaneous release of foaming gases and rapid increase in viscosity during crosslinking, the foam had non-uniform macrostructure with large pores and thick struts at the bottom. By addition of SiO2 fillers, the crosslinking reaction rate was reduced leading to homogeneous pore nucleation and uniform small pore size foams.

This task is intended to perform impact tests with a low-speed test on the specimen. The tests are carried out to improve their tolerance of impact so that they can be used for realistic applications in the real world. The specimen is treated with acid and a base for one day. By the hand-laying method, the specimen shall be prepared. Microscopy of the electron scanning is carried out in order to receive data on the surface topography of the affected specimen. Various low speed experiments have been evaluated in this paper.

Since its initial demonstration in 2000, far-field super-resolution light microscopy has undergone tremendous technological developments. In parallel, these developments have opened a new window into visualizing the inner life of cells at unprecedented levels of detail. Here, we review the technical details behind the most commonly used implementations of super-resolution microscopy and highlight some of the recent, promising advances in this field.

Light Sheet Fluorescence Microscopy (LSFM) has revolutionized how optical imaging of biological specimens can be performed as this technique allows to produce 3D fluorescence images of entire samples with a high spatiotemporal resolution. In this manuscript, we aim to provide readers with an overview of the field of LSFM on ex vivo samples. Recent advances in LSFM architectures have made the technique widely accessible and have improved its acquisition speed and resolution, among other features. These developments are strongly supported by quantitative analysis of the huge image volumes produced thanks to the boost in computational capacities, the advent of Deep Learning techniques, and by the combination of LSFM with other imaging modalities. Namely, LSFM allows for the characterization of biological structures, disease manifestations and drug effectivity studies. This information can ultimately serve to develop novel diagnostic procedures, treatments and even to model the organs physiology in healthy and pathological conditions. This work was produced with the support of the Spanish Ministry of Science, Innovation and Universities (TEC2016-78052-R, RTC-2017-6600-1, PID2019-109820RB-100, FPU19/02854).

Over the last ten years, developments in whole-brain microscopy now allow for high-resolution imaging of intact brains of small animals such as mice. These complex images contain a wealth of information, but many neuroscience laboratories do not have all of the computational knowledge and tools needed to process these data. We review recent open source tools for registration of images to atlases, and the segmentation, visualisation and analysis of brain regions and labelled structures such as neurons. Since the field lacks fully integrated analysis pipelines for all types of whole-brain microscopy analysis, we propose a pathway for tool developers to work together to meet this challenge.

As an essential part in the toolbox of super-resolution microscopy, stimulated emission depletion (STED) nanoscopy has been widely explored in revealing the substructure and bioactivities in fluorescence imaging. Among the applied STED fluorophores, silicon-substituted rhodamines (SiRs) belong to one of the most extensively employed fluorophores. The carboxy-SiR was favored in STED bioimaging with many advantages, including reliable photostability, cell permeability, tunable fluorogenicity, feasible structural decoration and so on. We reviewed the research of carboxy-SiR in the STED nanoscopy and hopefully this can inspire more efforts in the design and application of STED fluorophores.

Scanning probe microscopy is a group of measurements that provides 3D visualization of viruses in different environmental conditions including liquids and air. Besides 3D topography it is possible to measure the properties like mechanical rigidity and stability, adhesion, tendency to crystallization, surface charge, etc. Choosing the right substrate and scanning parameters makes it much easier to obtain reliable data. Rational interpretation of experimental results should take into account possible artifacts, proper filtering and data presentation using specially designed software packages. Animal and human virus characterization is in the focus of many intensive studies because of their potential harm to higher organisms. The article focuses on high-resolution visualization of plant viruses. Tobacco mosaic virus, potato viruses X and B and others are not dangerous for the human being and are widely used in different applications such as vaccine preparation, construction of building units in nanotechnology and material science applications, nanoparticle production and delivery, and even metrology. The methods of virus's deposition, visualization, and consequent image processing and interpretation are described in details. Specific examples of viruses imaging are illustrated using the FemtoScan Online software, which has typical and all the necessary built-in functions for constructing three-dimensional images, their processing and analysis. Despite visible progress in visualizing the viruses using probe microscopy, many unresolved problems still remain. At present time the probe microscopy data on viruses is not systemized. There is no descriptive atlas of the images and morphology as revealed by this type of high resolution microscopy. It is worth emphasizing that new virus investigation methods will appear due to the progress of science.

Additive manufacturing (AM) is a powerful and promising manufacturing technology. Advances in AM have led to increasing demands for its applications; however, controlling the geometry of deposited objects during the AM process remains a major challenge. Geometry control and compliance to a target geometry are some of the key concerns for AM, especially for a prototype requiring high precision in its geometry. In this study, an online geometry estimation methodology for continuous monitoring during the directed energy deposition (DED) process was developed using a laser line scanner. Our proposed methodology comprises (1) real-time scanning of each deposited track's profile, (2) online extraction of the track's geometry, and (3) online plotting and comparison of the as-designed and as-built models. In the methodology, data analysis following real-time scanning, such as geometry discrepancy estimation and online plotting of the as-built model, is attained for both single- and multi-layer objects. The effectiveness of our developed methodology is examined by comparing the profiles of the single- and multi-layer objects estimated during the DED process with the reference profiles obtained via laser line scanning and microscopy after the completion of the DED process.

Recent developments in clearing and microscopy enable 3D imaging with cellular resolution up to the whole organ level. These methods have been used extensively in neurobiology, but their uptake in other fields has been much more limited. Application of this approach to the human heart and effective use of the data acquired present challenges of scale and complexity. Four interlinked issues need to be addressed: 1) efficient clearing and labelling of heart tissue, 2) fast microscopic imaging of human-scale samples, 3) handling and processing of multi-terabyte 3D images, and 4) extraction of structural information in computationally tractable structure-based models of cardiac function. Preliminary studies show that each of these requirements can be achieved with the appropriate application and development of existing technologies.

Prevention of healthcare-associated infections (HAI) like ventilator associated pneumonia (VAP) is particularly challenging especially in resource limited settings. Complex microbial interactions between patients and health care workers (HCWs) further complicate the situation, requiring a holistic approach for successful management. To bridge the gap between laboratory and intensive care unit (ICU) this study was conducted to find the role of hand-held microscope 'Foldscope' in restricting empirical therapy in intubated patients.A total of 75 endotracheal aspirates (ETA) were collected from intubated patients in the ICU with (group 1) and without (group 2) VAP. For group 2, those with less than 48 ​h ventilation and with endotracheal tube (ETT) in situ were considered. Presence of biomass was detected through foldscope and ETA samples were processed for quantitative gram staining (QGS), semi-quantitative and quantitative culture. Phenotypic and genotypic characterization of Acinetobacter baumannii, the commonest isolate, was done and findings were statistically analysed.Biomass was present as seen through a foldscope in 45 cases (90%) in group 1 and 17 cases (68%) in group 2. In both the groups, A. baumannii was the most common isolate. Biomass production, significant QGS and culture was significantly more in group 1 (p ​ ​0.05). However, carbapenem resistant A. baumannii (CRAB) was comparably present in both the groups thus showing limited role of empirical carbapenem therapy.Early assessment of biomass in mechanically ventilated patients could provide guidance for empirical antibiotic therapy. Foldscope proved to be an excellent tool for restricting empirical therapy and driving antimicrobial stewardship in low resource settings.

The chemical synapse is one type of cell-adhesion system that transmits information from a neuron to another neuron in the complex neuronal network in the brain. Synaptic transmission is the rate-limiting step during the information processing in the neuronal network and its plasticity is involved in cognitive functions. Thus, morphological and electrophysiological analyses of synapses are of particular importance in neuroscience research. In the current study, we applied super-resolved three-dimensional stimulated emission depletion (3D-STED) microscopy for the morphological analyses of synapses. This approach allowed us to estimate the precise number of excitatory and inhibitory synapses in the mouse hippocampal tissue. We discovered a region-specific increase in excitatory synapses in a model mouse of autism spectrum disorder, Neuroligin-3 KO, with this method. This type of analysis will open a new field in developmental neuroscience in the future.

Due to the atomic thickness and planar characteristics, two-dimensional (2D) materials such as graphene and transition metal dichalcogenides (TMDs) are considered to be excellent electronic materials, which endow them with great potential for future device applications. The robust and reliable application of their functional devices requires an in-depth understanding of their mechanical properties and deformation behavior, which is also of fundamental importance in nanomechanics. Considering their exceedingly small sizes and thicknesses, this is a very challenge task. In situ microscopy techniques show great superiority in this respect. This review focuses on the progress in in situ microscopy techniques (including atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM)) in characterizing the mechanical properties and deformation behavior of 2D materials. The technical characteristics, advantages, disadvantages, and main research fields of various in situ AFM, SEM, and TEM techniques are analyzed in detail, and the corresponding mechanical scenarios from point to plane are realized, including local indentation, planar stretching, friction sliding between atomic layers and atomic movement mechanisms. By virtue of their complementary advantages, in situ integrated microscopy techniques enable the simultaneous study of various mechanical properties, nanomechanical behavior, and inherent atomic mechanisms of 2D materials. Based on the present research, we look forward to further optimized in situ integrated microscopy techniques with high spatiotemporal atomic resolution that can reveal the dynamic structure-performance correlations and corresponding atomic mechanisms between the physical properties, such as mechanical, electrical, optical, thermal, and magnetic properties of 2D materials and their crystal structures, electronic structures, atomic layers, defect densities and other influencing factors under multifield coupling conditions. This will provide beneficial predictions and guidance for the design, construction and application of 2D material-based mechanoelectronic, piezoelectric, photoelectric, thermoelectric, etc. nanoelectronic devices.

Summary Halide perovskites are enabling a variety of high-performance devices, yet these materials exhibit heterogeneity in their chemical, structural, morphological, and optoelectronic properties on different length scales. A global understanding of the relationship between these properties, and how they evolve under operation, is essential for commercialization. This perspective covers efforts to attain such relationships through multimodal and correlative microscopy. A variety of ex situ, in situ, and operando measurements have led to key conclusions that the length scales that are extremely important—yet least understood—are on the grain, sub-grain, and atomic scales. The multifaceted disorder of these materials and their ionic nature make complete characterization challenging, but exploitation of the latest experimental probes is stretching our knowledge, allowing rational approaches to improve performance and stability. Future adaptions will employ machine-learning-driven algorithms to process large hyperspectral datasets and operando measurements on the manufacturing line, in turn refining processing and device architectures.

Background To evaluate the quality of in vitro fenestrations during in situ fenestration (ISF) and investigate the differences between needle and laser puncture in current stent-grafts. Methods An in vitro study evaluated the damage created by needle ISF on stent-graft fabrics versus laser ISF. Fenestrations were made in 5 different commercially available stent-grafts, including polyester stent-grafts (Relay, Valiant and Hercules) and expanded polytetrafluoroethylene (ePTFE) stent-grafts (TAG and Ankura). Each stent-graft received fenestration by needle and laser separately, followed by gradual dilation (4 mm, 6 mm, 8 mm, and 10 mm sequentially) of noncompliant balloons. Quantitative and qualitative evaluations including fenestration diameter, area, shape and margins were conducted using light microscopy and scanning electron microscope. Results The primary fenestrations created by needle were slit-like with visible cut-off fibers in polyester stent-grafts and were almost circular with clear margins in ePTFE stent-grafts; those created by laser were squared or elliptical with ragged edges and burned fibers in all the stent-grafts. Fabric debris and toxic particles due to burning of the material were generated during laser-assisted fenestration. The Primary holes in polyester stent-grafts (Relay, Valiant and Hercules) by needle showed smaller area (0.05 mm2 vs. 0.22 mm2, 0.52 mm2 vs. 0.70 mm2 and 0.28 mm2 vs. 0.46 mm2; P 0.05). Ankura stent-graft showed the largest holes with best quality than the others. Conclusions The primary fenestrations were different between needle and laser puncture, laser induced fabric debris and toxic particles release should be cautiously considered. The final fenestrations were similar after gradual balloon dilation.

Degradation of therapeutic monoclonal antibodies (mAb) due to interfacial agitation through air bubbling was investigated. Samples containing mAb in phosphate buffered saline were subjected to rapid bubbling by using a peristaltic pump at an air flow rate of 11.5 mL/min. Samples were analyzed by visual observation, UV–Vis, fluorescence, circular dichroism and infrared spectroscopy, size-exclusion chromatography (SEC), dynamic light scattering, microscopy, and cell-based activity assays. The stressed samples showed increasing turbidity with bubbling time, with mAb1 showing a protein loss of 53% in the supernatant at the latest time point (240 min), indicating formation of sub-visible and visible aggregates. Aggregate rich samples exhibited altered secondary structure and higher hydrophobicity with 40% reduction in activity. The supernatants of the stressed samples showed unchanged secondary and tertiary structure without the presence of any oligomers in SEC. Furthermore, the impact of various factors that could affect aggregation was investigated and it was found that the extent of aggregation was affected by protein concentration, sample volume, presence of surfactants, temperature, air flow rate, and presence of silicone oil. In conclusion, exposure to air/liquid interfacial stress through bubbling into liquid mAb samples effectively generated sub-visible and visible aggregates, making air bubbling an attractive approach for interfacial stress degradation studies of mAbs.

Summary Cases of Leber congenital amaurosis caused by mutations in CRX (LCA7) exhibit an early form of the disease and show signs of significant photoreceptor dysfunction and eventual loss. To establish a translational in vitro model system to study gene-editing-based therapies, we generated LCA7 retinal organoids harboring a dominant disease-causing mutation in CRX. Our LCA7 retinal organoids develop signs of immature and dysfunctional photoreceptor cells, providing us with a reliable in vitro model to recapitulate LCA7. Furthermore, we performed a proof-of-concept study in which we utilize allele-specific CRISPR/Cas9-based gene editing to knock out mutant CRX and saw moderate rescue of photoreceptor phenotypes in our organoids. This work provides early evidence for an effective approach to treat LCA7, which can be applied more broadly to other dominant genetic diseases., Graphical abstract, Highlights • A retinal organoid model of CRX-associated Leber congenital amaurosis was generated • LCA7 retinal organoids show impaired photoreceptor maturation and gene expression • scRNA-seq profiling revealed both rod and cone photoreceptor dysfunction • Allele-specific editing of mutant CRX promotes disease rescue in retinal organoids, LCA7 currently has no effective treatments; therefore, Dr. Lamba and colleagues generated LCA7 retinal organoids from patient-derived stem cells. The retinal organoids recapitulated LCA7 disease phenotypes, allowing for testing of an allele-specific gene editing approach. Early signs of disease rescue were achieved, providing evidence for a promising new strategy to treat LCA7 and other dominant retinal diseases.

This study aimed to explore the evolution of flow lines and microstructures of M50-steel bearing ring and the anisotropy of its tensile mechanical properties after Multi-Stage Hot Forging (MSHF) and subsequent spheroidizing annealing (MSHFA). To this end, the present study mainly employed stereo microscopy, Optical Metallurgical Microscopy (OMM), Scanning Electron Microscopy (SEM), and Electron Backscatter Diffraction (EBSD) to characterize and analyze the workpiece at each processing stage of MSHF while performing microhardness measurement and uniaxial tensile experiment to test and analyze the mechanical properties of the workpiece. Macro-structure observation showed that the simulation results of flow lines at each stage were consistent with the experimental results. Microscopic observation showed that, after MSHF, deformation gradually became less significant along the outward radial direction of the bearing ring. After MSHFA, the microstructures of the bearing ring became uniform, whereas primary carbides did not dissolve. The mechanical properties were better in the axial direction (AD) than in the radial (RD) and circumferential directions (CD) after MSHF due to the smaller grain width. After MSHFA, the mechanical properties in the ADs and CDs were better than those in the RDs, which was due to the large cross-sectional area of carbides along the flow-line direction.

For 140 years, microscopy has repeatedly revolutionized the study of nucleus biology, but despite this our understanding of the evolutionarily divergent nucleus biology of Plasmodium remains limited. Here, we discuss how microscopy advances have enabled two groundbreaking studies by Simon et al. and Klaus et al. into Plasmodium nucleus biology.

This study aimed to investigate the changes in water properties and protein stability of large yellow croaker during freezing. The changes of ice crystals and muscle tissue were assessed using X-ray computed tomography (CT) and light microscopy, respectively. The results of water-holding capacity (WHC), low-field nuclear magnetic resonance, Fourier transform infrared, intrinsic fluorescence, differential scanning calorimetry (DSC), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that compared to the control, freezing treatment resulted in the reduction of WHC and protein stability of the samples. Large ice crystals had a greater effect on muscle water loss, protein secondary and tertiary structure, and protein thermal stability than small ice crystals. The results of Pearson analysis confirmed significant correlation between water properties and protein stability at different ice crystal size. Therefore, these results suggested the beneficial effects of small ice crystals on the water properties and protein stability of croaker, but the effect of low-temperature fracture on the product need to be concerned.

Background Downregulation of claudin-5 in the heart is associated with the end-stage heart failure. However, the underlying mechanism ofclaudin-5 is unclear. Here we investigated the molecular actions of claudin-5 in perspective of mitochondria in cardiomyocytes to better understand the role of claudin-5 in cardioprotection during ischemia. Methods Myocardial ischemia/reperfusion (I/R; 30 min/24 h) and hypoxia/reoxygenation (H/R; 24 h/4 h) were used in this study. Confocal microscopy and transmission electron microscope (TEM) were used to observe mitochondrial morphology. Results Claudin-5 was detected in murine heart tissue and neonatal rat cardiomyocytes (NRCM). Its protein level was severely decreased after myocardial I/R or H/R. Confocal microscopy showedclaudin-5 presented in the mitochondria of NRCM. H/R-induced claudin-5 downregulation was accompanied by mitochondrial fragmentation. The mitofusin 2 (Mfn2) expressionwas dramatically decreased while the dynamin-related protein (Drp) 1 expression was significantly increased after H/R. The TEM indicatedH/R-induced mitochondrial swelling and fission. Adenoviral claudin-5 overexpression reversed these structural disintegration of mitochondria. The mitochondria-centered intrinsic pathway of apoptosis triggered by H/R and indicated by the cytochrome c and cleaved caspase 3 in the cytoplasm of NRCMs was also reduced by overexpressing claudin-5. Claudin-5 overexpression in mouse heart also significantly decreased cleaved caspase 3 and the infarct size in ischemic heart with improved systolic function. Conclusion We demonstrated for the first time the presence of claudin-5 in the mitochondria in cardiomyocytes and provided the firm evidence for the cardioprotective role of claudin-5 in the preservation of mitochondrial dynamics and cell fate against hypoxia- or ischemia-induced stress.

The Abutilon mosaic virus (AbMV) encodes a nuclear shuttle protein (NSP), and a movement protein (MP) which cooperatively accomplish viral DNA transport through the plant. Subcellular distribution patterns of fluorescent protein-tagged NSP and MP were tracked in Nicotiana benthamiana leaves in presence or absence of an AbMV infection using light microscopy. NSP was located within the nucleus and associated with early endosomes in the presence of MP. MP appeared at the plasma membrane, plasmodesmata and in motile vesicles, trafficking along the endoplasmic reticulum in an actin-dependent manner. MP and NSP did not co-localize and employed separate cellular pathways. Correspondingly, Forster resonance energy transfer analysis did not support physical interaction between NSP and MP. Time lapse movies illustrate the cellular dynamics of both proteins on their way around the nucleus and to the cell periphery and provide a first hint for the nuclear egress of NSP complexes.

Advances in microscopy have increased output data volumes, and powerful image analysis methods are required to match. In particular, finding and characterizing nuclei from microscopy images, a core cytometry task, remains difficult to automate. While deep learning models have given encouraging results on this problem, the most powerful approaches have not yet been tested for attacking it. Here, we review and evaluate state-of-the-art very deep convolutional neural network architectures and training strategies for segmenting nuclei from brightfield cell images. We tested U-Net as a baseline model; considered U-Net++, Tiramisu, and DeepLabv3+ as latest instances of advanced families of segmentation models; and propose PPU-Net, a novel light-weight alternative. The deeper architectures outperformed standard U-Net and results from previous studies on the challenging brightfield images, with balanced pixel-wise accuracies of up to 86%. PPU-Net achieved this performance with 20-fold fewer parameters than the comparably accurate methods. All models perform better on larger nuclei and in sparser images. We further confirmed that in the absence of plentiful training data, augmentation and pretraining on other data improve performance. In particular, using only 16 images with data augmentation is enough to achieve a pixel-wise F1 score that is within 5% of the one achieved with a full data set for all models. The remaining segmentation errors are mainly due to missed nuclei in dense regions, overlapping cells, and imaging artifacts, indicating the major outstanding challenges.