Microscopy Images Research Articles

Combining Mesoporous Bioactive Glass Nanoparticles (MBGNs) with Essential Oils to Tackle Bacterial Infection and Oxidative Stress for Bone Regeneration Applications.

Bacterial infectious diseases remain one of the significant challenges in the field of bone regeneration applications. Despite the development of new antibiotics, their improper administration has led to the development of multiresistant bacterial strains. In this study, we proposed a novel approach to tackle this problem by loading clove oil (CLV), a natural antibacterial compound, into amino-functionalized mesoporous bioactive glass nanoparticles (MBGNs). The scanning electron microscopy images (SEM) revealed that amino-functionalization and CLV loading did not affect the shape and size of the MBGNs. The successful grafting of the amino groups on the MBGNs' surface and the presence of CLV in the material were confirmed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and zeta potential measurements. The increased CLV concentration led to a higher loading capacity, encapsulation efficiency, and antioxidant activity. The in vitro CLV release profile exhibited an initial burst release, followed by a controlled release over 14 days. The loading of CLV into MBGNs led to a stronger antibacterial effect against E. coli and S. aureus, while MG-63 osteoblast-like cell viability was enhanced with no morphological changes compared to the control group. In conclusion, the CLV-MBGNs nanocarriers showed promising properties in vitro as novel drug delivery systems, exploiting essential oils for treating bone infections and oxidative stress.

A Planar Darkfield Illuminator for Wide‐Field High‐Contrast Imaging of Specimens in Microfluidic Chips

AbstractDarkfield microscopy is an effective technique and well suited for use involving live and unstained biological samples, where a bulky condenser should be precisely aligned in the optical path to produce a cone of illumination light. Here, a compact darkfield illuminator is invented that has a planar shape and can work at broadband wavelengths. It can be easily incorporated into a conventional microscope as a substrate to produce hollow cones of light for large‐area darkfield illumination, resulting in a high‐contrast high‐sensitivity widefield microscopy images. Taking advantages of its planar and compact structure, this darkfield illuminator is successfully integrated with a microfluidic chip; thus, the actions and reactions of biological and chemical specimens flowing inside micrometer‐sized channels, can be real‐time imaged with high contrast and sensitivity in a large field‐of‐view (FoV). For the first time, the work integrates a compact darkfield illuminator with microfluidic chips, which produces a new platform for applications in biology, chemistry, and biomedical engineering.

Fuel Cell Catalyst Layers with Platinum Nanoparticles Synthesized by Sputtering onto Liquid Substrates.

Platinum (Pt) nanoparticles are widely used as catalysts in proton exchange membrane fuel cells. In recent decades, sputter deposition onto liquid substrates has emerged as a potential alternative for nanoparticle synthesis, offering a synthesis process free of contaminant oxygen, capping agents, and chemical precursors. Here, we present a method for the synthesis of supported nanoparticles based on magnetron sputtering onto liquid poly(ethylene glycol) (PEG) combined with a heat-treatment step for attachment of nanoparticles to a carbon support. Transmission electron microscopy imaging reveals Pt nanoparticle growth during the heat-treatment process, facilitated by the carbon support and the reducing properties of PEG. Following the heat treatment, a bimodal size distribution of Pt nanoparticles is observed, with sizes of 2.5 ± 0.8 and 6.7 ± 1.8 nm, compared to 1.8 ± 0.4 nm after sputtering. Synthesized Pt nanoparticles display excellent specific and mass activities for the oxygen reduction reaction, with 1.75 mA/cm2 Pt and 0.27 A/mgPt respectively, measured at 0.9 V vs the reversible hydrogen electrode. The specific activities reported herein outperform literature values of commercial Pt/C catalysts with similar loading and are on par with values of bulk Pt and mass-selected nanoparticles of comparable size. Also, the mass activities agree well with the literature values. The results provide new insights into the growth processes of SoL-synthesized carbon-supported Pt catalyst nanoparticles, and most crucially, the high performance of the synthesized catalyst layers, along with the possibility of nanoparticle growth through a straightforward heat-treatment step at relatively low temperatures, offer a scalable new approach for producing fuel cell catalysts with more efficient material utilization and new material combinations.

MSIreg: an R package for unsupervised coregistration of mass spectrometry and H&E images.

Joint analysis of mass spectrometry images (MS images) and microscopy images of hematoxylin and eosin (H&E) stained tissues assists pathologists in characterizing the morphological structure of the tissues, and in performing diagnosis. Unfortunately, the analysis is undermined by substantial differences between these modalities in terms of aspect ratios, spatial resolution, number of channels in each image, as well as by large global or small local elastic spatial deformations of one image with respect to the other. Therefore, accurate coregistration of the images is a critical pre-requisite for their joint interpretation. We introduce MSIreg, an open-source R package for coregistration of MSI and H&E images. MSIreg is designed for high-dimensional MSI experiments where each spatial location is represented by thousands of mass features. Unlike most existing coregistration methods, MSIreg implements a landmark free workflow, and quantitative metrics for performance evaluation. We evaluate the performance of MSIreg on six case studies, including coregistration of contiguous tissues with large deformations, as well as simultaneous coregistration of 29 tissue microarray cores. The R package, installation instructions, and fully reproducible vignettes describing methods and Case Studies are are available open-source under the GPL-3.0 license at https://github.com/sslakkimsetty/msireg/.

Applications of deep learning-based denoising methodologies for scanning electron microscope images

Abstract In this paper, we use five types of deep-learning algorithms for denoising scanning electron microscope (SEM) measurement data. Denoising of SEM images is an important task since the images often suffer from noise, which can make it difficult to accurately interpret the data. We also investigate realistic SEM denoising characteristics using a variety of metrics to assess the quality of denoised images. Overall, we find that the trained generative models provide superior denoising performance and that it is crucial to objectively quantify the performance, just like in the scanning process itself. It is anticipated that the deep-learning based technique can accelerate image measurements, which can be utilized for very fast analytical investigations. We also demonstrate that the success of a generative model may depend on the appropriate assessment of noise characteristics in the specific image data analysis of interest. Moreover, it is addressed that denoising performance can be properly evaluated when a relevant metrics that aligns well with human visual systems.

Inhibitory effects of chlorhexidine-loaded calcium carbonate nanoparticles against dental implant infections

This study aimed to design sustained released biodegradable calcium carbonate nanoparticles loaded with chlorhexidine (CHX-loaded NPs) and to investigate the early osteogenic differentiation and antimicrobial effects on the important bacteria involved in infections of dental implants. The microemulsion method was used to prepare the calcium carbonate nanoparticles loaded with chlorhexidine. The prepared nanoparticles were characterized using conventional methods. The release pattern determination and the biodegradation test were performed for the prepared nanoparticles. For the early osteogenic differentiation test of the prepared nanoparticles, alkaline phosphatase (ALP) activity was detected in human dental pulp stem cells (HDPSCs). The antimicrobial effects of the nanoparticles were evaluated against Escherichia coli, Streptococcus mutans, Enterococcus faecalis, Staphylococcus aureus, and Pseudomonas aeruginosa. The sizes of free calcium carbonate nanoparticles and CHX-loaded NPs were 105 ± 1.63 and 118 ± 1.47 nm and their zeta potentials were − 27 and − 36, respectively. A 50% degradation of nanoparticles was achieved after 100 days. These nanoparticles showed a two-stage sustained release pattern in vitro. Microscopic images revealed that the morphology of free calcium carbonate nanoparticles primarily took on a spherical calcite form, while CHX-loaded NPs predominantly exhibited a cauliflower-like vaterite polymorph. The nanoparticles increased the activity of ALP in cells in two weeks significantly (p < 0.05). Antimicrobial and antibiofilm results showed an efficient effect of the prepared nanoparticle against the studied bacteria. Calcium carbonate nanoparticles are an efficient multifunctional vector for chlorhexidine and can be used as a bioactive antibacterial agent against various oral microorganisms to prevent implant infections.

A highly sensitive and selective ZnO-based ammonia sensor: Fe- doping effect

Abstract This study investigates the gas sensing capabilities of ZnO and Fe-doped ZnO films prepared via a cost-effective spin coating method. The films were characterized optically, structurally, and morphologically. X-ray diffraction revealed good crystal quality with a polycrystalline nature, and wurzite structure, though crystal quality and crystallite size decreased with Fe doping. Optical measurements indicated an increased optical band gap from 3.205 ± 0.002 to 3.220 ± 0.002 eV after Fe doping. Scanning electron microscope images confirmed spherical grainy structures with reduced grain sizes after Fe doping. Gas sensing measurements at room temperature (RT) at an exposure of vapors of various toxic chemicals (acetone, ethanol, propanol, methanol, and ammonia), demonstrated a highly selective nature of ZnO and Fe-ZnO towards the ammonia. The Fe-doping into ZnO improved the ammonia sensing capability of ZnO film. The Fe-ZnO film exhibited a gas response of 388.8 ± 5.5 at 400 ppm ammonia exposure, which was nearly 10 times larger than that of ZnO film with a response/recovery time of 22/51 s, good stability, and good repeatability. The Fe-ZnO film’s higher response is attributed to its smaller grain sizes and surplus of charge carriers after Fe doping which promote the adsorption of extra oxygen ions onto the film’s surface and the subsequent interaction between the adsorbed oxygen ions ammonia molecules. It could detect up to the lower limit of 1 ppm ammonia with a response of 24.2 ± 0.9, which is better than the previous reports. These results reveal the Fe-ZnO film as a viable material for developing a cost-effective and efficient ammonia sensor at room temperature.

Stability of kangaroo rat burrows in the Sonoran Desert: Evidence of biocementation

Kangaroo rats (Dipodomys deserti) construct complex burrow systems in loose desert sand that survive temperature and relative humidity fluctuations and storms. Animals that burrow in desert sand typically burrow in compacted sand, near plant roots, or when the soil is unsaturated. However, these processes are insufficient to explain tunnel stability of kangaroo rats. Our goal is to understand how kangaroo rat burrows remain stable in loose desert sand, intending to translate this knowledge to geotechnical engineering. A kangaroo rat habitat in the dunes of The Sonoran Desert, AZ, was selected for the study. Dynamic cone penetrometer tests performed at active, abandoned, and no-burrow sites demonstrated that the animals prefer loose sand for burrow construction. Soil samples collected from the burrows' ceilings, subsurface, and surface were characterized. Brazilian tensile strength test results showed that burrow soil has approximately 3 times greater tensile strength than the rest at dry state, which indicates increased interparticle attractive stress in burrow ceilings due to biocementation. Laboratory experiments, scanning electron microscopy, and confocal microscopy images showed that fungal and microbial biofilms provided 17 kPa increase in interparticle attractive stress at less than 1% biomass concentration, indicating potential to be used in soil improvement applications.

Correlation between Broken Contact Fingers and I–V Characteristics of Partially Shaded Photovoltaic Modules

This paper reports on the correlation between broken contact fingers and the shape of the current–voltage (I–V) curve of a photovoltaic (PV) module. It was found that the broken contact fingers of a solar cell in the PV module cause a noticeable change in the I–V curve of the PV module when the solar cell was partially shaded. The broken contact fingers were inspected by microscopic imaging and electroluminescence (EL) imaging, and a further investigation was carried out using a single solar cell. The results show that the fill factor of the cell decreased from 0.75 of full contact to 0.47 after 16 contact fingers were broken, confirming the correlation between the I–V curve shape and broken contact fingers. This result reveals that the shape of the I–V curve of a PV module under individual-cell partial shading may be used as an indicator of broken contact fingers, which offers an alternative approach to EL imaging for detecting broken contact fingers in PV modules in daylight.

Berberine-Loaded Bovine Serum Albumin Nanoparticles: Fabrication, Characterization, and Drug Release Evaluation

Abstract Introduction/Objective Introduction: Numerous studies have highlighted the diverse biochemical and pharmacological properties of berberine (BER), including its anti-inflammatory, antioxidant, anticancer, and hypolipidemic effects. Nanoparticles play a crucial role in enhancing drug delivery by providing a larger surface area, facilitating quicker dissolution in the bloodstream. Methods/Case Report Methods: Blank bovine serum albumin nanoparticles (BSA NPs) were prepared using a desolvation process, followed by the fabrication of berberine-loaded BSA nanoparticles (BER-NP). Physicochemical characterization, including particle size and zeta potential, was conducted using a Malvern Zetasizer. The mean particle size and distribution were determined using photon correlation spectroscopy (PCS), and transmission electron microscope (TEM) images were obtained for visualization. Fourier transform infrared (FTIR) spectra and differential scanning calorimetry (DSC) thermograms were recorded to assess drug encapsulation efficiency (EE). Berberine release from BER-NPs was evaluated using dialysis membranes. Results (if a Case Study enter NA) Results: FTIR spectra and DSC thermograms confirmed successful drug loading into the nanoparticles. Encapsulation efficiency was determined by UV absorption spectroscopy, revealing high efficiency in the encapsulation process. Dialysis membranes demonstrated controlled release kinetics of berberine from BER-NPs, indicating their potential for targeted drug delivery. Conclusion Conclusion: Berberine-loaded bovine serum albumin nanoparticles exhibit favorable characteristics for drug delivery applications, including enhanced permeation across biological barriers. These nanoparticles hold promise for targeted therapy and combination drug regimens, offering potential benefits for various medical interventions.

Correlative microscopy - illuminating the endomembrane system of plant seeds.

The endomembrane system of cereal seed endosperm is a highly plastic and dynamic system reflecting the high degree of specialization of this tissue. It is capable of coping with high levels of storage protein synthesis and undergoes rapid changes to accommodate these storage proteins in newly formed storage organelles such as endoplasmic reticulum-derived protein bodies or protein storage vacuoles. The study of endomembrane morphology in cereal endosperm is challenging due to the amount of starch that cereal seeds accumulate and the progressive desiccation of the tissue. Here, we present a comprehensive study of the endomembrane system of developing barley endosperm cells, complemented by correlative light and electron microscopy (CLEM) imaging. The use of genetically fused fluorescent protein tags in combination with the high resolution of electron microscopy brings ultrastructural research to a new level and can be used to generate novel insights in cell biology in general and in cereal seed research in particular.

Back to the future - 20 years of progress and developments in photonic microscopy and biological imaging.

In 2023, the ImaBio consortium (imabio-cnrs.fr), an interdisciplinary life microscopy research group at the Centre National de la Recherche Scientifique, celebrated its 20th anniversary. ImaBio contributes to the biological imaging community through organization of MiFoBio conferences, which are interdisciplinary conferences featuring lectures and hands-on workshops that attract specialists from around the world. MiFoBio conferences provide the community with an opportunity to reflect on the evolution of the field, and the 2023 event offered retrospective talks discussing the past 20 years of topics in microscopy, including imaging of multicellular assemblies, image analysis, quantification of molecular motions and interactions within cells, advancements in fluorescent labels, and laser technology for multiphoton and label-free imaging of thick biological samples. In this Perspective, we compile summaries of these presentations overviewing 20 years of advancements in a specific area of microscopy, each of which concludes with a brief look towards the future. The full presentations are available on the ImaBio YouTube channel (youtube.com/@gdrimabio5724).

Enhanced wound healing with biogenic zinc oxide nanoparticle-incorporated carboxymethyl cellulose/polyvinylpyrrolidone nanocomposite hydrogels.

Contemporary wound dressings lack antibacterial properties, exhibit a low water vapour transmission rate, and demonstrate inadequate porosity. In order to overcome these limitations, scientists have employed water hyacinth to produce carboxymethyl cellulose (CMC). CMC/PVP nanocomposite films containing biogenic zinc oxide nanoparticles (nZnOs) were synthesised using cost effective solution-casting technique. As the proportion of nZnOs in the film increased, swelling and water permeability decreased, whereas mechanical stability improved. Dynamic light scattering testing and transmission electron microscopy confirmed that the particle size was around 50.7 nm. Field emission scanning electron microscopy (FESEM) images showed that nZnOs were distributed uniformly in the polymer matrix. Cell viability against Vero cells was greater than 94%, and a substantial zone of inhibition against S. aureus and E. coli bacteria was observed. Wounds of albino mice were treated with CMC/PVP and CMC/PVP/nZnO (6%) nanocomposite hydrogels and healed in 20 and 12 days, respectively, as demonstrated by wound healing assay and histological staining. In vitro and in vivo studies revealed that the novel nanocomposite hydrogels exhibit improved cell viability and wound healing features. Therefore, they could be exploited as promising skin wound dressing materials.

Refining delivery and positioning: A modified approach to FIL SSF intraocular lens implantation using the "full reverse" technique.

To present a modified surgical technique for implantation of the sutureless scleral-fixated hydrophilic intraocular lens (FIL SSF). Single surgeon retrospective case series and review of surgical videos with step-by-step technique analysis. Uncorrected and best corrected visual acuity (UCVA and BCVA), refractive error (spherical equivalent), full clinical examination with intraocular pressure (IOP) measurement, endothelial cell density on corneal specular microscopy and macular optical coherence tomography (OCT) were recorded at baseline, 1, 4 and 8 weeks postoperatively. The FIL SSF IOLs were successfully implanted using the so-called "full reverse" technique, having the lens loaded in the injector in an upside-down fashion, as opposed to IOL technical specifications. In all cases, the FIL SSF IOL was properly placed in the ciliary sulcus, well-centered and without signs of tilt. Follow up figures at 2 months are consistent with published data, confirming the potential benefits of the new implantation technique. In our preliminary experience, the "full reverse" technique of the FIL SSF IOL has proven effective in preventing incorrect IOL orientation in 100% of cases. However, larger prospective controlled studies and longer follow up are required to either support or disprove our results.

Procedure for Fabrication and Characterization of Van-der-Waals Heterostructures

In this work we provide a step-by-step description of the technique for manufacturing various van der Waals heterostructures. First, we discuss the procedure to obtain monolayer and few-layer flakes from layered materials, in particular from graphite and hexagonal boron nitride. Next, we consider different approaches to the assembly depending on the required final device. Further, we describe in detail the procedure for making ohmic contacts and give the parameters for plasma chemistry and metal deposition. We observe the field effect in transport measurements but a number of features – a strong shift of the charge neutral point from the zero-gate voltage, a large resistance away from the charge neutral point, and low mobility – indicate a problem with the quality of the resulting devices. Nevertheless, one of the fabricated devices demonstrates reasonable quality – the maximum mobility is estimated at 15000 cm2V–1s–1, the magnetic field dependences demonstrate the quantum Hall effect, which is standard for high-quality graphene. Unexpectedly, scanning electron microscope images of the resulting devices reveal a large amount of contamination on the surface of the flakes, which may explain the corresponding quality of our devices. Preliminary results of flakes cleaning with chemical compounds and thermal treatment are given.

From two segments and beyond: Investigating the onset of regeneration in Syllis malaquini.

Annelids feature a diverse range of regenerative abilities, but complete whole-body regeneration is less common, particularly in the context of the head and anterior body regeneration. This study provides a detailed morphological description of Syllis malaquini regenerative abilities. By replicating previous experiments and performing diverse surgical procedures, we explored the capacity of this species for whole-body regeneration. We detailed the precise timing of regeneration of particular structures such as the eyes, proventricle, pharyngeal tooth, nuchal organs, and body pigmentation after amputation. Our high-resolution scanning electron microscopy and confocal laser-scanning microscopy images provide details of the blastema region, revealing that while anal opening remains in connection to the exterior environment, oral opening is formed "de novo" during blastema differentiation. Additionally, we performed amputations to isolate fragments consisting of one, two, and three segments from the intestinal trunk region. We found that S. malaquini requires at least two to three segments to successfully regenerate the whole body. In addition, we verified a variable capacity to regenerate depending upon the gut region, with structures of the foregut greatly impairing some steps of the regenerative process. Our work notably addresses the gap in knowledge concerning gut formation and its impact on regenerative capabilities. Ongoing research is crucial to unravel the role of gut tissue specificity and plasticity during regeneration in annelids, and particularly in syllids.

Structural Repetition Detector: multi-scale quantitative mapping of molecular complexes through microscopy.

From molecules to organelles, cells exhibit recurring structural motifs across multiple scales. Understanding these structures provides insights into their functional roles. While super-resolution microscopy can visualise such patterns, manual detection in large datasets is challenging and biased. We present the Structural Repetition Detector (SReD), an unsupervised computational framework that identifies repetitive biological structures by exploiting local texture repetition. SReD formulates structure detection as a similarity-matching problem between local image regions. It detects recurring patterns without prior knowledge or constraints on the imaging modality. We demonstrate SReD's capabilities on various fluorescence microscopy images. Quantitative analyses of three datasets highlight SReD's utility: estimating the periodicity of spectrin rings in neurons, detecting HIV-1 viral assembly, and evaluating microtubule dynamics modulated by EB3. Our open-source ImageJ and Fiji plugin enables unbiased analysis of repetitive structures across imaging modalities in diverse biological contexts.

Image Analysis in Histopathology and Cytopathology: From Early Days to Current Perspectives.

Both pathology and cytopathology still rely on recognizing microscopical morphologic features, and image analysis plays a crucial role, enabling the identification, categorization, and characterization of different tissue types, cell populations, and disease states within microscopic images. Historically, manual methods have been the primary approach, relying on expert knowledge and experience of pathologists to interpret microscopic tissue samples. Early image analysis methods were often constrained by computational power and the complexity of biological samples. The advent of computers and digital imaging technologies challenged the exclusivity of human eye vision and brain computational skills, transforming the diagnostic process in these fields. The increasing digitization of pathological images has led to the application of more objective and efficient computer-aided analysis techniques. Significant advancements were brought about by the integration of digital pathology, machine learning, and advanced imaging technologies. The continuous progress in machine learning and the increasing availability of digital pathology data offer exciting opportunities for the future. Furthermore, artificial intelligence has revolutionized this field, enabling predictive models that assist in diagnostic decision making. The future of pathology and cytopathology is predicted to be marked by advancements in computer-aided image analysis. The future of image analysis is promising, and the increasing availability of digital pathology data will invariably lead to enhanced diagnostic accuracy and improved prognostic predictions that shape personalized treatment strategies, ultimately leading to better patient outcomes.

3D alignment of distant patterns with deep-subwavelength precision using metasurfaces

Measurement of the relative positions of two objects in three dimensions with sub-nanometer precision is essential to fundamental physics experiments and applications such as aligning multi-layer patterns of semiconductor chips. Existing methods, which rely on microscopic imaging and registration of distant patterns, lack the required accuracy and precision for the next generation of three-dimensional (3D) chips. Here we show that 3D misalignment between two distant objects can be measured using metasurface alignment marks, a laser, and a camera with sub-nanometer precision. Through simulations, we demonstrate that the shot noise-limited precisions of the lateral and axial misalignments between the marks are λ0/50, 000 and λ0/6, 300 (λ0: laser’s wavelength), respectively. With its high precision and simplicity, the technique enables the next generation of 3D-integrated optical and electronic chips and paves the way for developing cost-effective and compact sensors relying on sub-nanometer displacement measurements.

This Microtubule Does Not Exist: Super-Resolution Microscopy Image Generation by a Diffusion Model.

Generative models, such as diffusion models, have made significant advancements in recent years, enabling the synthesis of high-quality realistic data across various domains. Here, the adaptation and training of a diffusion model on super-resolution microscopy images are explored. It is shown that the generated images resemble experimental images, and that the generation process does not exhibit a large degree of memorization from existing images in the training set. To demonstrate the usefulness of the generative model for data augmentation, the performance of a deep learning-based single-image super-resolution (SISR) method trained using generated high-resolution data is compared against training using experimental images alone, or images generated by mathematical modeling. Using a few experimental images, the reconstruction quality and the spatial resolution of the reconstructed images are improved, showcasing the potential of diffusion model image generation for overcoming the limitations accompanying the collection and annotation of microscopy images. Finally, the pipeline is made publicly available, runnable online, and user-friendly to enable researchers to generate their own synthetic microscopy data. This work demonstrates the potential contribution of generative diffusion models for microscopy tasks and paves the way for their future application in this field.