Contrast Electron Microscopy Research Articles

Genomic and morphological characterization of Knufia obscura isolated from the Mars 2020 spacecraft assembly facility

Members of the family Trichomeriaceae, belonging to the Chaetothyriales order and the Ascomycota phylum, are known for their capability to inhabit hostile environments characterized by extreme temperatures, oligotrophic conditions, drought, or presence of toxic compounds. The genus Knufia encompasses many polyextremophilic species. In this report, the genomic and morphological features of the strain FJI-L2-BK-P2 presented, which was isolated from the Mars 2020 mission spacecraft assembly facility located at the Jet Propulsion Laboratory in Pasadena, California. The identification is based on sequence alignment for marker genes, multi-locus sequence analysis, and whole genome sequence phylogeny. The morphological features were studied using a diverse range of microscopic techniques (bright field, phase contrast, differential interference contrast and scanning electron microscopy). The phylogenetic marker genes of the strain FJI-L2-BK-P2 exhibited highest similarities with type strain of Knufia obscura (CBS 148926T) that was isolated from the gas tank of a car in Italy. To validate the species identity, whole genomes of both strains (FJI-L2-BK-P2 and CBS 148926T) were sequenced, annotated, and strain FJI-L2-BK-P2 was confirmed as K. obscura. The morphological analysis and description of the genomic characteristics of K. obscura FJI-L2-BK-P2 may contribute to refining the taxonomy of Knufia species. Key morphological features are reported in this K. obscura strain, resembling microsclerotia and chlamydospore-like propagules. These features known to be characteristic features in black fungi which could potentially facilitate their adaptation to harsh environments.

Toward Bioactive Hydrogels: A Tunable Approach via Nucleic Acid-Collagen Complexation

PurposeNucleic acid-collagen complexes (NACCs) are unique biomaterials formed by binding short, monodisperse single-stranded DNA (ssDNA) with type I collagen. These complexes spontaneously generate microfibers and nanoparticles of varying sizes, offering a versatile platform with potential applications in tissue engineering and regenerative medicine. However, the detailed mechanisms behind the nucleic acid-driven assembly of collagen fibers still need to be established. We aim to understand the relationship between microscopic structure and bulk material properties and demonstrate that NACCs can be engineered as mechanically tunable systems.MethodsWe present a study to test NACCs with varying molar ratios of collagen to random ssDNA oligonucleotides. Our methods encompass the assessment of molecular interactions through infrared spectroscopy and the characterization of gelation and rheological behavior. We also include phase contrast, confocal reflectance, and transmission electron microscopy to provide complementary information on the 3D structural organization of the hydrogels.ResultsWe report that adding DNA oligonucleotides within collagen robustly reinforces and rearranges the hydrogel network and accelerates gelation by triggering rapid fiber formation and spontaneous self-assembly. The elasticity of NACC hydrogels can be tailored according to the collagen-to-DNA molar ratio, ssDNA length, and collagen species.ConclusionOur findings hold significant implications for the design of mechanically tunable DNA-based hydrogel systems. The ability to manipulate hydrogel stiffness by tailoring DNA content and collagen concentration offers new avenues for fine tuning material properties, enhancing the versatility of bioactive hydrogels in diverse biomedical applications.Lay SummaryThis work is an example of forming fibers and gels with tunable elasticity that stems from the complexation of short-length nucleic acids (on the order of size of aptamers) and collagen, which can be potentially extended to a variety of functionalized hydrogel designs and tailored biomedical applications. Incorporating DNA induces mechanical changes in NACCs.Graphical

Comparison of differential phase Contrast, Lorentz and non-Lorentz transmission electron microscopy for microstructural and magnetic imaging of alnico magnet

In this work, non-Lorentz, Lorentz transmission electron microscopy (LTEM) and differential phase contrast (DPC) methods were used to observe the phase and magnetic structure of the alnico magnet. All observations were performed on alnico-5 in transverse orientation. The results showed the magnetic contrast of the domain walls in the conventional TEM images despite the effects of the lens magnetic field, which was verified and explained using DPC. Experimental methodology and sample results are summarized with advice for further research.

Determination of microplastics in the wetlands of specific regional area and unveiling the toxic properties of predominant HDPE particle on animal and plant models

Microplastics are emerging environmental pollutants that cause a great threat to all forms of living organisms. However, the information on MPs existence in freshwater sources and its environmental implications on freshwater associated flora and fauna remain limited. Hence, the present study was designed to identify the presence of MPs in the sediments of major wetlands of Coimbatore, Tamilnadu, India and to assess the toxicity of predominantly detected MPs in animal and plant models. In this study, six different types of MPs such as filaments (FI), fragments (F), irregular filaments (IF), pellets (P), films (FM) and colored fragments (CF) were identified from the samples through Phase contrast and Scanning Electron Microscopy. Besides, high Density Polyethylene (HDPE) was identified as predominant MPs from the wetlands by micro–Raman spectroscopy analysis and hence, HDPE has been selected for further toxicity assessment studies. In toxicity study, HDPE exposure significantly affected the phenotype, survival and hatching rate, heart shape and function of the zebrafish embryos. Moreover, HDPE toxicity significantly increased the expression pattern of major stress marker genes such as P5CS, CAT, SOD and APX1 in Sorghum bicolor plant. This study spotlighted the presence of MPs in major wetlands of Coimbatore and elucidated the toxicity nature of predominant MPs pollutant HDPE in zebrafish embryos and major agriculture crop S. bicolor as the first report.

Autophagy of Candida albicans cells after the action of earthworm Venetin-1 nanoparticle with protease inhibitor activity

The present studies show the effect of the Venetin-1 protein-polysaccharide complex obtained from the coelomic fluid of the earthworm Dendrobaena veneta on Candida albicans cells. They are a continuation of research on the mechanisms of action, cellular targets, and modes of cell death. After the action of Venetin-1, a reduced survival rate of the yeast cells was noted. The cells were observed to be enlarged compared to the controls and deformed. In addition, an increase in the number of cells with clearly enlarged vacuoles was noted. The detected autophagy process was confirmed using differential interference contrast, fluorescence microscopy, and transmission electron microscopy. Autophagic vesicles were best visible after incubation of fungus cells with the Venetin-1 complex at a concentration of 50 and 100 µg mL−1. The changes in the vacuoles were accompanied by changes in the size of mitochondria, which is probably related to the previously documented oxidative stress. The aggregation properties of Venetin-1 were characterized. Based on the results of the zeta potential at the Venetin-1/KCl interface, the pHiep = 4 point was determined, i.e. the zeta potential becomes positive above pH = 4 and is negative below this value, which may affect the electrostatic interactions with other particles surrounding Venetin-1.

Femtosecond laser preparation of resin embedded samples for correlative microscopy workflows in life sciences.

Correlative multimodal imaging is a useful approach to investigate complex structural relations in life sciences across multiple scales. For these experiments, sample preparation workflows that are compatible with multiple imaging techniques must be established. In one such implementation, a fluorescently labeled region of interest in a biological soft tissue sample can be imaged with light microscopy before staining the specimen with heavy metals, enabling follow-up higher resolution structural imaging at the targeted location, bringing context where it is required. Alternatively, or in addition to fluorescence imaging, other microscopy methods, such as synchrotron x-ray computed tomography with propagation-based phase contrast or serial blockface scanning electron microscopy, might also be applied. When combining imaging techniques across scales, it is common that a volumetric region of interest (ROI) needs to be carved from the total sample volume before high resolution imaging with a subsequent technique can be performed. In these situations, the overall success of the correlative workflow depends on the precise targeting of the ROI and the trimming of the sample down to a suitable dimension and geometry for downstream imaging. Here, we showcase the utility of a femtosecond laser (fs laser) device to prepare microscopic samples (1) of an optimized geometry for synchrotron x-ray tomography as well as (2) for volume electron microscopy applications and compatible with correlative multimodal imaging workflows that link both imaging modalities.

Gynoecium structure and pollen tube pathway in the cactus family with emphasis on tribe Trichocereeae (Cactaceae: Cactoideae)

Abstract The structural details of the gynoecium are key to understanding the reproductive systems and successful diversification of flowering plants. However, the gynoecium morpho-anatomy in South American species of Cactaceae that evolved in the Andean region remains largely unknown. Here we selected 18 species, most of them of evolutionarily related genera of Cactaceae tribe Trichocereeae, to conduct a detailed comparative study. Observations were made using light, fluorescence, differential interference contrast and scanning electron microscopy. Most of the characters of the ovary and ovule were typical of the family, except for the nucellar beak in Echinopsis aurea and E. haematantha, here reported for the first time in cacti. We found evidence suggesting that the stigmatic surface covered with multiseriate trichomes, the semi-closed style type and the pollen tube transmitting tract are conserved characters among species of Trichocereeae; this finding may be explained by a phylogenetic conservatism of the investigated genera of the tribe. We integrated the available information about structural and histological characters of the gynoecium in the family, taking into account the current phylogenetic context of the examined genera. Our results reinforce the significance of floral anatomical traits for the systematics of Cactaceae.

Characterization, antibacterial and anticancer study of silk fibroin hydrogel

Purpose: Protein-based hydrogels such as silk fibroin hydrogel, are used in tissue engineering and regenerative medicine applications as they showed striking characteristics like biocompatibility and offered various benefits as biomaterials. The current study sought to prepare silk fibroin hydrogel and characterise it in order to assess its antibacterial and anticancer activity.
 Methodology: Silk fibroin hydrogel was prepared and characterized by using different microscopy methods, namely Scanning Electron Microscopy (SEM), Phase Contrast Electron (PCM) microscopy, and foldscope analysis. Further, it was characterized through 1H-NMR analysis, Fourier Transform Infrared spectroscopy (FTIR) analysis, and swelling properties. A Current study also covers an antimicrobial and anticancer analysis of silk fibroin hydrogel by disk diffusion method and SRB (Sulforhodamine B) assay respectively.
 Results: The antibacterial study confirmed that SF hydrogel has a moderate antibacterial activity for Streptococcus mutans, and Salmonella typhi. Additionally, the SRB assay test showed that silk fibroin hydrogels had moderate anticancer activity against the human lung cancer cell line (A549).
 Conclusion: The current study unequivocally demonstrates that silk fibroin hydrogel has antibacterial and anti-cancerous properties, making it a suitable scaffold for future studies that seek to target a specific drug delivery site.
 Keywords: Silk fibroin; Swelling behaviour; Anticancer; Drug carrier; Wound healing

Mechanical Properties of Aluminium/Copper Bimetallic Sheets Subjected to Cyclic Bending

The aim of this study is to evaluate the influence of the roll bonding (RB) process on the mechanical properties of Al/Cu bimetallic strip specimens. A specially designed test instrument for the cyclic bending test of strip specimens produced by RB has been proposed. The test consists of repeated pulling of a strip of sheet metal through a system of cylindrical countersamples. Samples cut along and across the sheet rolling direction were tested. The specimens were subjected to preliminary microstructural tests using an optical microscope with Nomarski contrast and scanning electron microscopy. The tests showed a slight influence of the orientation of the samples on changes to the mechanical properties of the bimetallic sheets in the cyclic bending process. For samples oriented along the sheet rolling direction, discontinuities in the transition layer were found. The samples oriented perpendicularly to the rolling direction were free of this defect.

Liquid to solid transition of elastin condensates

Liquid-liquid phase separation of tropoelastin has long been considered to be an important early step in the complex process of elastin fiber assembly in the body and has inspired the development of elastin-like peptides with a wide range of industrial and biomedical applications. Despite decades of study, the material state of the condensed liquid phase of elastin and its subsequent maturation remain poorly understood. Here, using a model minielastin that mimics the alternating domain structure of full-length tropoelastin, we examine the elastin liquid phase. We combine differential interference contrast (DIC), fluorescence, and scanning electron microscopy with particle-tracking microrheology to resolve the material transition occurring within elastin liquids over time in the absence of exogenous cross-linking. We find that this transition is accompanied by an intermediate stage marked by the coexistence of insoluble solid and dynamic liquid phases giving rise to significant spatial heterogeneities in material properties. We further demonstrate that varying the length of the terminal hydrophobic domains of minielastins can tune the maturation process. This work not only resolves an important step in the hierarchical assembly process of elastogenesis but further contributes mechanistic insight into the diverse repertoire of protein condensate maturation pathways with emerging importance across biology.

Molecular Characterization of Native Bacillus thuringiensis Strains from Root Nodules with Toxicity Against the Fall Armyworm (FAW, Spodoptera frugiperda) and Brinjal Ash Weevil (Myllocerus subfasciatus).

The fall armyworm is an exotic pest which destroys a wide variety of crops Querywhereas the brinjal ash weevil is a serious pest of eggplant and other solanaceous vegetables. The goal of this research is to find a sustainable and ecologically friendly bio-control agent for managing FAW and brinjal ash weevils. Twelve natural Bacillus thuringiensis strains were isolated from cowpea root nodules, and the Gram-positive cells with characteristic Bt crystal structures were discovered using phase contrast and scanning electron microscopy. There were bipyramidal, cuboidal, rhombus, and spherical crystals. The Bt cry gene content was characterized by PCR analysis, which revealed the presence of cry1, cry1I, cry3, cry7, cry7,8, cry14, cry26, and cry55 genes. The identity of Bt was confirmed by cloning and sequencing the cry genes. In the nucleotide sequences, no pseudo genes or indels were found in cry sequences. SDS-PAGE examination indicated the presence of bands ranging in size from 13 to 130kDa, with 50-60kDa being the most common. When compared to the control, the new native Bt strains were lethal, with pathogenicity ranging from 93 to 100% against S. frugiperda larvae and M. subfasciatus adults. The studies revealed that the native strains with conserved regions of 16S rRNA genes were compared to NCBI database sequences and classified as native Bt strains with 99-100% similarity to known Bt strains. In conclusion, native Bt strains from cowpea root nodules were shown to have bio-insecticidal activity against fall armyworm larvae and brinjal ash weevil adults.

Methuosis Contributes to Jaspine-B-Induced Cell Death.

Methuosis is a type of programmed cell death in which the cytoplasm is occupied by fluid-filled vacuoles that originate from macropinosomes (cytoplasmic vacuolation). A few molecules have been reported to behave as methuosis inducers in cancer cell lines. Jaspine B (JB) is a natural anhydrous sphingolipid (SL) derivative reported to induce cytoplasmic vacuolation and cytotoxicity in several cancer cell lines. Here, we have investigated the mechanism and signalling pathways involved in the cytotoxicity induced by the natural sphingolipid Jaspine B (JB) in lung adenocarcinoma A549 cells, which harbor the G12S K-Ras mutant. The effect of JB on inducing cytoplasmic vacuolation and modifying cell viability was determined in A549 cells, as well as in mouse embryonic fibroblasts (MEF) lacking either the autophagy-related gene ATG5 or BAX/BAK genes. Apoptosis was analyzed by flow cytometry after annexin V/propidium iodide staining, in the presence and absence of z-VAD. Autophagy was monitored by LC3-II/GFP-LC3-II analysis, and autophagic flux experiments using protease inhibitors. Phase contrast, confocal, and transmission electron microscopy were used to monitor cytoplasmic vacuolation and the uptake of Lucifer yellow to assess macropinocyosis. We present evidence that cytoplasmic vacuolation and methuosis are involved in Jaspine B cytotoxicity over A549 cells and that activation of 5′ AMP-activated protein kinase (AMPK) could be involved in Jaspine-B-induced vacuolation, independently of the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin complex 1 (PI3K/Akt/mTORC1) axis.

Sample Preparation and Warping Accuracy for Correlative Multimodal Imaging in the Mouse Olfactory Bulb Using 2-Photon, Synchrotron X-Ray and Volume Electron Microscopy.

Integrating physiology with structural insights of the same neuronal circuit provides a unique approach to understanding how the mammalian brain computes information. However, combining the techniques that provide both streams of data represents an experimental challenge. When studying glomerular column circuits in the mouse olfactory bulb, this approach involves e.g., recording the neuronal activity with in vivo 2-photon (2P) calcium imaging, retrieving the circuit structure with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT) and/or serial block-face scanning electron microscopy (SBEM) and correlating these datasets. Sample preparation and dataset correlation are two key bottlenecks in this correlative workflow. Here, we first quantify the occurrence of different artefacts when staining tissue slices with heavy metals to generate X-ray or electron contrast. We report improvements in the staining procedure, ultimately achieving perfect staining in ∼67% of the 0.6 mm thick olfactory bulb slices that were previously imaged in vivo with 2P. Secondly, we characterise the accuracy of the spatial correlation between functional and structural datasets. We demonstrate that direct, single-cell precise correlation between in vivo 2P and SXRT tissue volumes is possible and as reliable as correlating between 2P and SBEM. Altogether, these results pave the way for experiments that require retrieving physiology, circuit structure and synaptic signatures in targeted regions. These correlative function-structure studies will bring a more complete understanding of mammalian olfactory processing across spatial scales and time.

Characterization of the Growth and Morphology of a BSL-2 Coccidioides posadasii Strain That Persists in the Parasitic Life Cycle at Ambient CO2.

Coccidioides is a dimorphic fungus responsible for Valley Fever and is the cause of severe morbidity and mortality in the infected population. Although there is some insight into the genes, pathways, and growth media involved in the parasitic to saprophytic growth transition, the exact determinants that govern this switch are largely unknown. In this work, we examined the growth and morphology of a Coccidioides posadasii strain (C. posadasii S/E) that efficiently produces spherules and endospores and persists in the parasitic life cycle at ambient CO2. We demonstrated that C. posadasii S/E remains virulent in an insect infection model. Surprisingly, under spherule-inducing conditions, the C. posadasii S/E culture was found to be completely hyphal. Differential interference contrast (DIC) and transmission electron microscopy (TEM) revealed unexpected cellular changes in this strain including cell wall remodeling and formation of septal pores with Woronin bodies. Our study suggests that the C. posadasii S/E strain is a useful BSL-2 model for studying mechanisms underlying the parasitic to saprophytic growth transition—a morphological switch that can impact the pathogenicity of the organism in the host.

Interfacial ferroelectricity in marginally twisted 2D semiconductors

Twisted heterostructures of two-dimensional crystals offer almost unlimited scope for the design of new metamaterials. Here we demonstrate a room temperature ferroelectric semiconductor that is assembled using mono- or few-layer MoS2. These van der Waals heterostructures feature broken inversion symmetry, which, together with the asymmetry of atomic arrangement at the interface of two 2D crystals, enables ferroelectric domains with alternating out-of-plane polarization arranged into a twist-controlled network. The last can be moved by applying out-of-plane electrical fields, as visualized in situ using channelling contrast electron microscopy. The observed interfacial charge transfer, movement of domain walls and their bending rigidity agree well with theoretical calculations. Furthermore, we demonstrate proof-of-principle field-effect transistors, where the channel resistance exhibits a pronounced hysteresis governed by pinning of ferroelectric domain walls. Our results show a potential avenue towards room temperature electronic and optoelectronic semiconductor devices with built-in ferroelectric memory functions.

Detection and molecular characterization of astro and bocaviruses in dogs in Minnesota

Canine astrovirus (CAstV) and canine bocavirus (CBoV) are involved in cases of mild, and sometimes severe, gastroenteritis in dogs. Fecal samples from two dead dogs with gastroenteritis were received at the University of Minnesota Veterinary Diagnostic Laboratory to determine the cause of death. Small round viruses of 20–35 nm diameter were observed by negative contrast electron microscopy. The samples were subjected to Illumina MiSeq sequencing. Both samples were strongly positive for CAstV; all viral reads were related to CAstV. In addition, sample number 1 had a few reads of CBoV. Two complete sequences of CAstV were identified (6625 and 6627 nt in length) with 95% nt identity. RT-PCR and PCR were used to confirm CAstV and CBoV infections in successive samples of canine gastroenteritis. Sanger sequencing was done on nucleic acids from positive samples. Of a total of ten samples, CAstV and CBoV infections were confirmed in six and eight animals, respectively. Four animals had mixed infection with both viruses. All sequences of ORF1b gene of CAstVs showed closest clusters in phylogenetic tree with 96–100% nucleotide and amino acids identity. On the other hand, identity between VP2 gene of different CBoV strains in this study ranged from 93%− 100%. All strains were located close to each other except the divergent MT078234 strain, which was arranged in a separate branch and was closer to reference strain JN648103/USA/2010. This study highlights the importance of electron microscopy and next generation sequencing for early detection and characterization of viruses associated with dog gastroenteritis.

Integrative Descriptions of Two New Mesobiotus Species (Tardigrada, Eutardigrada, Macrobiotidae) from Vietnam

To date, 34 tardigrade taxa have been recorded from Vietnam and this includes only two macrobiotid species belonging to the genus Mesobiotus. In this paper, two additional species of this genus, one of the M. harmsworthi group and one of the M. furciger group, are reported and described as new for science (Mesobiotus imperialis sp. nov., Mesobiotus marmoreus sp. nov.). Both descriptions have an integrative character providing detailed morphological and morphometric data collected by phase contrast and scanning electron microscopy that are linked to genetic data. The latter constitute DNA sequences of molecular markers that are commonly used in tardigrade taxonomy. The genus phylogeny is also provided, elucidating the phylogenetic position of the newly discovered taxa.

High-power near-concentric Fabry-Perot cavity for phase contrast electron microscopy.

Transmission electron microscopy (TEM) of vitrified biological macromolecules (cryo-EM) is limited by the weak phase contrast signal that is available from such samples. Using a phase plate would thus substantially improve the signal-to-noise ratio. We have previously demonstrated the use of a high-power Fabry–Perot cavity as a phase plate for TEM. We now report improvements to our laser cavity that allow us to achieve record continuous wave intensities of over 450 GW/cm2, sufficient to produce the optimal 90° phase shift for 300 keV electrons. In addition, we have performed the first cryo-EM reconstruction using a laser phase plate, demonstrating that the stability of this laser phase plate is sufficient for use during standard cryo-EM data collection.

Exploration of insecticidal potential of Cry protein purified from Bacillus thuringiensis VIID1

Insect pests are a threat to agriculture as they cause a loss of 15–22% to economically important crops every year. Bacillus thuringiensis produces parasporal crystal inclusions that have insecticidal ‘Cry’ proteins which are toxic to insect larvae of the order Lepidoptera, Coleoptera and Diptera, etc. In the present study, 40 different soil samples from Amritsar and its surrounding areas were selected for isolation of B. thuringiensis. The rod shaped, gram-positive bacterial isolates were further analyzed for characteristic crystal formation using phase contrast and scanning electron microscopy. 6 Bacillus samples containing cry genes were identified using the universal primers for cry genes, of which one isolate exhibited a protein band of ~95 kDa. This protein was purified using a Sephadex G-75 column. The insecticidal assays conducted with purified Cry protein on insect larvae of lepidopteran and dipteran orders viz. Spodoptera litura, Galleria malonella, Bactrocera cucurbitae and Culex pipens revealed considerable detrimental effects. A significant increase in larval mortality was observed for the larvae of all insects in a concentration dependent manner when treated with Cry protein purified from B. thuringenisis VIID1. The purified Cry protein did not have any significant effect on honey bee larvae.

Control and hygienic regulation of air pollution by fibrous particles

Introduction. The correct assessment of air pollution by fibrous particles allows controlling the risks of their exposure for workers and the general population. The indices for control of air pollution by fibrous particles differs from established for other dusts. The main control methods are optical and electron microscopy. Each method has its own advantages and disadvantages, which should be reflected in the national regulatory framework. The aim of study - to test the modern methods for the control of air pollution by fibrous particles. Materials and methods. A comparative analysis of modern methods for controlling air pollution by fibres with practical testing of determining their numerical concentrations by the method of membrane filters using optical phase contrast (PCOM) and scanning electron microscopy (SEM) was performed. Results. Based on field studies performed in various industries, the advantages and disadvantages of the methods used in the world to control air pollution by fibrous particles were evaluated. Conclusions. The main methods for determining fibers in the air are PCM and SEM/EDXA. The development of regulatory documents for calculating the numerical concentrations in the work area air and in the ambient air will help to ensure the correct risk assessments for occupational and non-occupational exposure of fibrous particles.