Light Microscopy Research Articles

Quantifying DNA damage following light sheet and confocal imaging of the mammalian embryo

Embryo quality assessment by optical imaging is increasing in popularity. Among available optical techniques, light sheet microscopy has emerged as a superior alternative to confocal microscopy due to its geometry, enabling faster image acquisition with reduced photodamage to the sample. However, previous assessments of photodamage induced by imaging may have failed to measure more subtle impacts. In this study, we employed DNA damage as a sensitive indicator of photodamage. We use light sheet microscopy with excitation at a wavelength of 405 nm for imaging embryo autofluorescence and compare its performance to laser scanning confocal microscopy. At an equivalent signal-to-noise ratio for images acquired with both modalities, light sheet microscopy reduced image acquisition time by ten-fold, and did not induce DNA damage when compared to non-imaged embryos. In contrast, imaging with confocal microscopy led to significantly higher levels of DNA damage within embryos and had a higher photobleaching rate. Light sheet imaging is also capable of inducing DNA damage within the embryo but requires multiple cycles of volumetric imaging. Collectively, this study confirms that light sheet microscopy is faster and safer than confocal microscopy for imaging live embryos, indicating its potential as a label-free diagnostic for embryo quality.

Evaluation of Known Markers of Ferroptosis in Semen of Patients with Different Reproductive Pathologies and Fertile Men.

This study aims to investigate the role of ferroptosis, an iron-dependent form of regulated cell death, in male infertility. The motivation behind this research stems from the increasing recognition of oxidative stress and iron metabolism dysregulation as critical factors in male reproductive health. In this study, 28 infertile patients (grouped by the presence of urogenital infections or varicocele) and 19 fertile men were selected. Spermiograms were performed by light microscopy (WHO, 2021). Testosterone, ferritin, transferrin-bound iron, transferrin, and F2-isoprostanes (F2-IsoPs) were detected in seminal plasma. Glutathione peroxidase 4 (GPX4) and acyl coenzyme A synthetase long chain family member 4 (ACSL4) were also assessed in sperm cells using enzyme-linked immunosorbent assays (ELISA). All the variables were correlated (statistically significant Spearman's rank correlations) in the whole population, and then the comparison between variables of the different groups of men were carried out. Seminal ferritin and transferrin positively correlated with seminal F2-IsoPs, which had positive correlations with ACSL4 detected in sperm cells. Ferritin and ACSL4 negatively correlated with the seminal parameters. No correlation was detected for GPX4. Comparing the variables in the three examined groups, elevated levels of ACSL4 were observed in infertile patients with urogenital infections and varicocele; GPX4 levels were similar in the three groups. These results suggested a mechanism of ferroptosis, identified by increased ACSL4 levels and the occurrence of lipid peroxidation. Such events appear to be GPX4-independent in reproductive pathologies such as varicocele and urogenital infections.

Exploring the antibacterial efficacy of Satureja hortensis and Bacillus subtilis extracts in combating Xanthomonas citri

Biofilm plays a critical role in protecting the associated bacterial colonies. Citrus canker caused by Xanthomonas citri, resulted in remarkable yield reduction in citrus orchards. This study aims to evaluate the anti-biofilm properties of S. hortensis and B. subtilis extracts against X. citri. The antibacterial effects of both extracts were evaluated using the disk diffusion method and 96-well microdilution plates. Crystal violet and XTT procedures were employed to assess the inhibition of adhesion and antibiofilm effects of both extracts. The checkboard titration method was applied to determine the synergistic effects of the plant-bacterial extracts. The antibiofilm effects were confirmed by the light microscopy method. The results showed that the antibacterial and antibiofilm properties of both extracts varied. The MIC values of S. hortensis and B. subtilis extracts were 6.25 and 50 mg/ml, respectively. In these concentrations the inhibition of adhesion effects of S. hortensis and B. subtilis were 70% and 80%, whereas their antibiofilm effects were 60% and 72%. S. hortensis and B. subtilis extracts showed 63% and 76% antibiofilm activity, respectively, using the XTT test. The extracts showed synergistic effects, resulting in higher inhibition rates in combinatorial applications. Molecular docking results also confirmed that the secondary metabolites of S. hortensis could interact with different X. citri proteins differentially. Further large-scale studies on combinatorial antibiofilm effects of plant and bacterial extracts against citrus pathogens will provide new opportunities to develop safe and applicable reagents to control these destructive diseases.

Beyond the Scent: New Evidence about Micromorphological, Phytochemical and Biological Features of Plumeria rubra 'Tonda Palermitana' (Apocynaceae).

Plumeria rubra L. is an ornamental Caribbean plant widely known for its ethnobotanical uses and pharmacological activities. The 'Tonda Palermitana' cultivar, on which no data are to date available, is commonly cultivated in Sicily. The aim of our study was to characterize the micro-morphological features of leaves and flowers of this cultivar by light and Scanning Electron Microscopy and to investigate the phytochemical profile and the biological properties of their food-grade extracts (LE and FE, respectively) by LC-DAD-ESI-MS analysis and different in vitro assays. Numerous branched laticifers were observed, and their secretion contained alkaloids and lipophilic compounds as confirmed by histological analyses. Phytochemical analyses showed the presence of alkaloids (9%), terpenoids (13%) and fatty acids (6%), together with a very abundant presence of iridoids (28%) and polyphenols (39%). The most notable biological activity of both extracts appears to be the antioxidant one, showing half-inhibitory concentrations (IC50) about 5 times lower than those detected in anti-inflammatory assays (383.74 ± 5.65 and 232.05 ± 2.87 vs. 1981.23 ± 12.82 and 1215.13 ± 10.15, for FE and LE, respectively), with LE showing the best, and statistically significant (p < 0.001), biological activity. These results allow us to speculate promising nutraceutical and cosmeceutical applications for this old Sicilian cultivar.

Lost &amp;amp; Found - Rediscovery of H. Hauffen’s Carychium material in the Dominik Bilimek Collection, BOKU University, including a contemporary assessment within the genus Zospeum (Gastropoda, Ellobioidea, Carychiinae)

The syntypes of Carychium reticulatum Hauffen, 1856 and Carychium bidentatum Hauffen, 1856, thought to be lost to science, have been rediscovered in a recent inventory of the shell collection of Domink Bilimek (1813–1884) at the University of Natural Resources and Life Sciences in Vienna, Austria (BOKU). In this work, we present (1) the historical context of the Dominik Bilimek and Heinrich (also: Henrik) Hauffen collections and (2) provide a contemporary image dataset of Hauffen’s original material while considering today’s taxonomic understanding of the genus Zospeum. We clarify the taxonomic status of the syntypes by using light microscopy, Scanning Electron Microcopy (SEM) and 3D X-ray Micro-CT data in conjunction with Hauffen’s original illustrations and compare them to contemporary investigations of their closest congeners. Analysis of Zospeum reticulatum (Hauffen, 1856) is supported by historical documentation from Heinrich Hauffen’s original annotations and Bilimek’s diary entries, as well as from individual collection labels in Hauffen’s own script (verified by archive material from the Archive of the National Museum of Slovenia, NMS). Hauffen’s descriptions are based on apertural morphology and shell microstructure. Our re-investigation of Zospeum reticulatum provides corroborating evidence supporting its assignment to Zospeum spelaeum (Rossmaessler, 1839) while that of Carychium bidentatum corroborates its status as junior synonym of Zospeum costatum (Freyer, 1855).

Occurrence of microplastics in the gastrointestinal tracts of four most consumed fish species in Giresun, the Southeastern Black Sea.

Microplastic studies investigating concentrations in water are numerous, but the majority of microplastics settle and are retained in sediment, and higher concentrations are regularly reported in sediments. Thus, MPs accumulation may be more threatening to benthic fish living in sediments than to pelagic fish. The presence, abundance and diversity of microplastics were investigated by collecting samples from two pelagic, European anchovy, and horse mackerel and two benthic fish species, red mullet, and whiting that are popularly consumed in Giresun province of Türkiye, located on the southern coast of the Black Sea. Visual classification and chemical compositions of microplastics was performed using a light microscope and ATR-FTIR spectrophotometry, consecutively. The overall incidence and mean microplastics abundance in sampled fishes were 17 and 1.7 ± 0.18 MP fish-1, respectively. MPs were within the range of 0.026-5mm in size. In most of the cases, the MP was black in color with 41%. With the rates of 56%, polypropylene was the predominant polymer type. The most dominant MP type was identified as fiber followed by fragments and pellets. The relationship between MP amounts in fish and Fulton condition factor was not strong enough to establish a cause-effect relationship.

Generation of empty cell envelopes of Streptococcus pyogenes using biosurfactants

BackgroundBacterial ghost cells (BGCs) are cell envelopes that devoid of cytoplasmic and genetic contents in purpose of variable applications, including their great potential as vaccine candidates and their effectiveness as delivery systems for drugs and proteins. To our knowledge, this is the first study to produce Gram-positive BGCs by treating Streptococcus pyogenes (S. pyogenes) ATCC 19615 with Tween80 (TW80) or TritonX-100 (TX100), followed by preliminary testing of their antigenicity and safety in NIH/Ola-Hsd mice. The produced BGCs were confirmed by the presence of intact cells under a light microscope, the absence of growth signs upon re-cultivation. The transmembrane tunnels were visualized using a scanning electron microscope, and subsequently, considerable quantities of released DNA and protein were detected in the culture supernatant of the BGCs. The antigenicity of the produced BGCs was tested through three intra-nasal immunization doses followed by infection. Afterward, the opsonic activity and the IgG levels were measured, followed by a comprehensive histopathological examination for selected tissues and organs.ResultsThe sera of immunized mice exhibited a significant rise in both opsonic activity (TW80 produced BGC = 68% and TX100 produced BGC = 75%) and IgG levels (TW80 produced BGC = a threefold increase and TX100 produced BGC = a fourfold increase) when compared to the positive control group "non-immunized challenged with ATCC 19615." Histopathological analysis revealed that the BGCs produced by TW80 are relatively safer and have a less severe impact than those produced by TX100.ConclusionThe study's findings suggest that Sp-BGC/TW80 is initially effective and safe in vivo. However, further pre-clinical studies are necessary to confirm its effectiveness and ensure complete safety, specifically in terms of the absence of autoimmunity and antibody cross-reactivity with myosin proteins in human cardiac tissues.

Craft Beer Produced by Immobilized Yeast Cells with the Addition of Grape Pomace Seed Powder: Physico-Chemical Characterization and Antioxidant Properties.

The aim of this study was to produce and to characterize craft beer fermented by immobilized yeast cells with the addition of Prokupac grape pomace seed powder (2.5% and 5%), to obtain a beer enriched with phenolic compounds and improved sensory characteristics. The immobilization of the yeast cells was performed by electrostatic extrusion, while the obtained calcium alginate beads were characterized by light and scanning electron microscopy. Phenolic and hop-derived bitter compounds in beer with or without grape pomace seed powder (GS) phenolics were identified using UHPLC Q-ToF MS. The results indicated that GS adjunct significantly shortened the fermentation process of wort and increased the content of phenolic compounds, especially ellagic acid, flavan-3-ols and pro(antho)cyanidins in the final products compared to the control beer. A total of twenty (iso)-α-acids and one prenylflavonoid were identified, although their levels were significantly lower in beers with GS phenolics compared to the control beer. Beers with GS phenolics showed good antioxidant properties as measured by the reduction of ferric ions (FRP) and the scavenging of ABTS•+ and DPPH• radicals. The concentration of immobilized viable yeast cells was higher than 1 × 108 CFU/g wet mass after each fermentation without destroying the beads, indicating that they can be reused for the repeated fermentation of wort. Beers produced with 5% GS added to the wort exhibited the best sensory properties (acidity, astringency, bitterness intensity, mouthfeel, aftertaste and taste), and highest overall acceptability by the panelists. The results showed that grape pomace seed powder present a promising adjunct for the production of innovative craft beer with good sensory properties and improved functionality.

Red Bull Energy Drink Impact on Salivary Glands in Wistar Rats: Can Blueberry Extract Reverse the Damage?

Energy drink (ED) consumption has become increasingly popular. Due to a lack of evidence, it was crucial to assess the effects of Red Bull (RB) consumption on the rat submandibular salivary gland and the potential therapeutic impact of blueberry (BB). Thirty rats were randomly assigned to five groups. Group 1 (Control) received distilled water. Group 2 (RB) received RB (10 mL/100 g/day) for 8 weeks. Group 3 (BB) rats were administered BB (500 mg/day for 8 weeks). Group 4 (RB + BB (L)) received RB for 8 weeks, and from the 5th week, were concurrently given BB (250 mg/day) for 4 weeks. Group 5 (RB + BB (H)) received RB for 8 weeks, and from the 5th week, were concurrently given BB (500 mg/day) for 4 weeks. At the end of the experiment, blood samples were collected, the animals were euthanized, and their submandibular salivary glands were harvested. Oxidative stress markers (MDA, GPx, CAT, and SOD) were assessed in both serum and tissue. Inflammatory markers (TNF-α, IL-6, and IL-10) were quantified in tissue. Submandibular gland specimens were prepared for light microscopy, and immunohistochemical staining was performed using anti-α-SMA. RB consumption resulted in a significant increase in MDA, TNF-α, IL-6, and IL-10, while GPx, CAT, and SOD levels decreased significantly. Degenerative changes in the gland's structure were observed in the RB group. A significant increase in α-SMA immunoreaction was detected in myoepithelial cells. Administration of BB, particularly at a high dose, ameliorated the aforementioned findings. In conclusion, blueberry administration exhibited therapeutic effects due to its antioxidative and anti-inflammatory properties.

From tin(II) to tin(IV): Following solid‐liquid reactions by microscopy

In order to study the formation of complexes independently from stoichiometry we developed a microscale experiment that allows us to monitor the reaction progress by light microscopy In the present study, the complex formation between SnF2 and 1,10‐phenanthroline (phen) with N,N‐dimethylformamide as solvent was investigated over a period of some weeks. By use of this setup, the formation of three different crystals has been observed, whose compositions and structures were determined by single crystal X‐ray diffraction: 3SnF2 · phen, 1, 2SnF2 · SnF4 · phen, 2, SnF4 · phen, 3. While the octahedral fluorine environment of the tin(IV) atoms of 2 and 3 is very regular, the coordinations of the tin(II) atoms in 1 and 2 is irregular. Assuming a non‐bonding 5s electron pair, classical 2c‐2e‐ and symmetrical and asymmetrical, hypervalent 3c‐4e‐bonds ‐ all based on the orthogonal p atomic orbitals of Sn – an approach to analyse and describe these coordinations in a simple comprehensive way is presented.

Leaf micromorphology and anatomical traits of leaves as potential taxonomic markers for infrageneric classification of&lt;em&gt; Cinnamomum&lt;/em&gt; (Lauraceae)

This study aimed to examine the leaf anatomy of the eight species of Cinnamomum (family Lauraceae) that occur in Sri Lanka to evaluate its potential utility in taxonomic delimitation. Leaf surface micromorphology and internal anatomy were studied using standard light and scanning electron microscopy. Infrageneric and interspecific variation in micromorphology of cuticular materials was observed in both abaxial and adaxial surfaces. Both abaxial and adaxial surfaces had undulating, thick cuticles with different patterns of wax deposition. Leaves were hypostomatic with no special arrangement for epidermal pavement cells. Simple, unicellular, unbranched, solitary and non-glandular trichomes were observed in different species on both surfaces of the leaves. Both adaxial and abaxial surfaces in C. dubium and C. capparu-coronde were densely covered with simple, unicellular/ falcate, long and thin trichomes. Transverse sections (TS) of the leaves were different in shape (symmetrical, asymmetrical, irregular, saucer). The midrib contained one open arch central vascular bundle that was different in shape (oval, elongated, irregular, ‘V’shaped, partially dissected into 2 or 3 segments) in different species. The number of collateral vessels was dissimilar between the species and varied in number (±10 - ±17). Isolated or aggregated crystals were observed in different parts of the leaf and oval-to-round schizogenous secretory cells were present in C. capparu-coronde, C. citriodorum and C. dubium. Leaf cuticular features together with the presence/absence of secretory cavities, trichome shape and density, midrib cross-section outline and shape of vascular bundles are taxonomically informative characteristics that can be used to differentiate the eight species of Cinnamomum that occur in Sri Lanka.

Amoebicidal activity of essential oils and essential oil-based microemulsions of Aloysia citrodora Ortega ex Pers., Cymbopogon winterianus Jowitt ex Bor, and Ocimum gratissimum L. against Acanthamoeba polyphaga trophozoites.

Evaluate the in vitro efficacy of the essential oils derived from Aloysia citrodora (Verbenaceae), Cymbopogon winterianus (Poaceae), and Ocimum gratissimum (Lamiaceae) against Acanthamoeba polyphaga trophozoites. Additionally, microemulsions formulated with these essential oils, along with their major components, were analyzed. The prepared microemulsions were characterized using polarized light microscopy and rheological techniques. The amoebicidal activity was determined by measuring the inhibitory concentration (IC50). Flow cytometry was employed to detect membrane damage and alterations in trophozoites size. The results revealed transparent and thermodynamically stable microemulsions. The essential oil from O. gratissimum exhibited a lower IC50, with values of 280.66 and 47.28µg ml-1 after 24 and 48 h, respectively. When microemulsions containing essential oils were tested, the IC50 values exhibited a reduction of over 80% after 24 h. Particularly, eugenol, a constituent of the O. gratissimum essential oil, displayed higher amoebicidal activity. The essential oils also caused damage to the cell membrane, resulting in the subsequent death of the trophozoites. The EOs of A. citrodora, C. winterianus, and O. gratissimum and their microemulsions showed antiparasitic effect against A. polyphaga trophozoites, representing promising alternatives for the treatment of diseases caused by this protozoan.

Seasonal Variances in General Urine Examination Findings among Al-Yarmouk Teaching Hospital Patients

Background: Urinalysis is a common test done for several reasons; for instance, in UTI looking for, hematuria or pseudo-hematuria, pyuria, crystalluria and also looking for different microorganisms (etc...), many laboratories perform either a regular test light microscopy, or examine varying dilutions of urine sediment and fields of view with or without the use of stains or polarization, while other laboratories used analysis of urine using automated system. As being a human; the biological sex(female or male) and broadly influences diverse immune phenotypes, including immune responses to diseases at mucosal surfaces, all contribute to how an organism will respond to diseases of the urinary tract, on the other hand, a close relationship between four seasons and the incidence of UTI has been detected in various areas of the world, where the seasons alternate in a year, and the temperature fluctuates widely among these seasons. Methods: present study was conducted on midstream sample was collected for acquiring accurate results from (68899) individuals (40800 females and 28099 males), these subjects were clinically suspected to have UTI, who were referred to the Department of Teaching Laboratories in AL-Yarmouk Teaching Hospital at Baghdad / Iraq, between January 2020 and December 2023, which examined by; Urilyzer 500 PRO (Analyticon) with Urine strips (dipstick) and Optical Microscope BA 210 (Motic). Results: A total of 68899 urine specimens were tested, of which 28099 male (40.8%) and 40800 female (59.2%) with the highest percentage of urinalysis in 2022 (33.2%), furthermore, the higher percentage of urinalysis parameters was scored in 2021 as follows; pus cells 9012 (49.3%), red blood corpuscles 5284 (48.9%) and crystalluria 3956 (44.2%) with the most common type of crystal was amorphous urate 5032 (56.2%), while highest percentages of microorganisms 1050 (40.3%) in year 2022 , and Bacteria was exhibited the highest percentages 2036 (78.1%). Current study recorded significantly differences between males and females in all urine parameters which was higher percentage in female than male and in the Seasons of (Summer and Autumn) , during the four years of this study. Conclusion: This study registered more accurate diagnostic results of UTI according to the urinary parameters in the microscopic routine urinalysis(centrifugation at 300xg at 5 min) when compared to machines (Urilyzer 500 PRO test/Urine strips) which frequently miss that , in addition, hot season in Iraq could affect to increase the seasonally variable incidence of UTI , as well, increased percentages and frequency of urinary parameters which observed in 2021-2022 out of four study years may indicates, high likelihood side effects of Covid-19 pandemic disease in study persons involved.

Investigation of the spatial distribution of sodium in bread microstructure using X-ray, light and electron microscopy

The sodium consumption in many countries is too high, which results in increased risk for hypertension, cardiovascular diseases, stroke and premature death. Inhomogeneous sodium distribution using layering is a viable way to reduce sodium in bread that normally contains a lot of sodium. Prevention of sodium migration during production and storage is important for the function of this approach. Furthermore, the distribution of sodium between starch and gluten influences their properties. The spatial distribution of sodium was investigated at high resolution using combinations of X-ray fluorescence microscopy (XFM), scanning transmission X-ray microscopy (STXM), light microscopy (LM), scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDX) and image analysis. Reference breads and layered bread samples were baked with one salt-free layer and one layer containing 3.6 wt% sodium chloride salt. The obtained results showed that the concentration of sodium is higher in the starch phase than in the gluten phase and that sodium migrates across the layer interface from the salt-containing to the salt-free layer. The ratios between the sodium concentration in the starch and gluten phases were dependent on the sodium concentration across the interfaces. Furthermore, magnesium and phosphor signals in bread yeast cells were observed using XFM.

Instance segmentation of cells and nuclei from multi-organ cross-protocol microscopic images.

Light microscopy is a widely used technique in cell biology due to its satisfactory resolution for cellular structure analysis, prevalent availability of fluorescent probes for staining, and compatibility for the dynamic analysis of live cells. However, the segmentation of cells and nuclei from microscopic images is not a straightforward process because it has several challenges such as high variation in morphology and shape, the presence of noise and diverse contrast in backgrounds, clustering or overlapping nature of cells. Dealing with these challenges and facilitating more reliable analysis necessitates the implementation of computer-aided methods that leverage image processing techniques and deep learning algorithms. The major goal of this study is to propose a model, for instance segmentation of cells and nuclei, applying the most cutting-edge deep learning techniques. A fine-tuned You Only Look at Once version 9 extended (YOLOv9-E) model is initially applied as a prompt generator to generate bounding box prompts. Using the generated prompts, a pre-trained segment anything model (SAM) is subsequently applied through zero-short inferencing to produce raw segmentation masks. These segmentation masks are then refined using non-max suppression and simple image processing methods such as image addition and morphological processing. The proposed method is developed and evaluated using an open-sourced dataset called Expert Visual Cell Annotation (EVICAN), which is relatively large and contains 4,738 microscopy images extracted from cross organs using different protocols. Based on the evaluation results on three different levels of EVICAN test sets, the proposed method demonstrates noticeable performances showing average mAP50 [mean average precision at intersection over union (IoU) =0.50] scores of 96.25, 95.05, and 94.18 for cell segmentation, and 68.04, 54.66, and 38.29 for nucleus segmentation on easy, medium, and difficult test sets, respectively. Our proposed method for instance segmentation of cells and nuclei provided favorable performance compared to the existing methods in literature, indicating its potential utility as an assistive tool for cell culture experts, facilitating prompt and reliable analysis.

Metformin dampens the progression of cholangiofibrosis induced by thioacetamide using deep learning

PurposeThe consistent use of metformin has been linked to a reduced incidence of neoplastic diseases among diabetic populations. As a preventive intervention, metformin may offer a more favorable risk-benefit profile. Here, we explored the efficacy of metformin in the primary prevention of cholangiofibrosis, which can precede the carcinogen-induced development of cholangiocarcinoma (CCA). Our objective was to assess the potential of metformin to act as an intervention prior to the onset of these conditions. MethodsA rat model of thioacetamide (TAA)-induced cholangiofibrosis was utilized to assess the impact of metformin on the induction process of cholangiocarcinoma (CCA). Liver tissues were harvested and analyzed histologically using light microscopy, complemented by a deep-learning convolutional neural network for enhanced evaluation. Additionally, RNA sequencing (RNA-seq) was performed to investigate the genetic alterations associated with metformin treatment in this TAA-induced cholangiofibrosis model. ResultsIn the rat model, the TAA control group exhibited an increased incidence and average count of cholangiofibrosis cases in the liver, with rates of 100 % and an average of 12.0, compared to the metformin-treated group, which showed an incidence of 70 % and an average of 3.3. Notably, the progression from normal cholangioles to cholangiofibrosis was associated with the upregulation of several proteins critical for metabolic processes and the tumor microenvironment. These alterations were significantly mitigated by metformin treatment. ConclusionsLong-term metformin use may offer protective benefits against cholangiofibrosis, partially by regulating metabolic processes and improving the tumor microenvironment.

Pollen atlas and morphological analysis of the selected types from southeastern Tibet, China

The accuracy of fossil pollen identification is crucial for understanding past plant diversity, vegetation landscape and associated climate change, while dispersed/fossil pollen assignments heavily rely on how robust modern pollen reference is. There is high species richness in southeastern Tibet, China, however, pollen morphological studies in this area are still poorly documented. Here, we present pollen atlas of 57 species (40 genera, 22 families) collected from the Ranwu Lake Basin. Pollen grains were imaged with light microscope (LM, 100X oil-immersion objective lens) and scanning electron microscope (SEM). All pollen types are ordered and presented according to different aperture types and sculpture patterns, facilitating comparison with fossil pollen. Moreover, we selected some representative species mainly based on potential palaeoecological importance, including Sibiraea (Rosaceae), Koenigia cathayana (Polygonaceae), Stellera chamaejasme (Thymelaeaceae), Hordeum vulgare subsp. vulgare and Poa annua (Poaceae), to discuss their pollen identification characteristics for the first time and assess potential applications in Quaternary palaeoecological interpretation. This study enriches modern pollen atlas for Quaternary pollen analysis in the study area and the adjacent regions. Our results provide robust modern reference in pollen-based reconstruction of vegetation history, palaeoclimate and palaeoenvironment, and tracing pollen signal of local human activities as well.

Experimental review and accuracy of etchants used for phase analysis of SAF 2507 Super Duplex Stainless Steel

The mechanical and corrosion properties of duplex stainless steels (DSS) depend on the distribution of ferrite and austenite as well as the presence of secondary phases. It is therefore necessary to accurately determine both the distribution of the phases and their morphology. The aim of this research is to define a standard method for the determination of the volume fraction of the phases present in a SAF2507 SDSS by analysing images acquired by light microscope. To do this, the material is heat-treated at 850°C for 5 min, 15 min, 30 min, 1h, 5h to stimulate the formation of the σ-phase. The metallographic etchant capable of maximising phase contrast is then identified. Micrographic analyses are performed using different reagents at different holding times and temperatures, in order to find the best combination. A standard procedure is then defined for the image analysis. The data obtained were are processed to calculate the phase distribution. The results are compared with the quantitative analysis performed using X-ray diffraction as reference.The research evidence that Kalling's reagent is best etchant for fully solubilised materials, as it accurately estimates ferrite and austenite content. On the other hand, Marble's reagent should be preferred for the detection of σ-phase. For comprehensive analysis of all phases, Murakami's reagent is recommended.The approach defined in this paper not only improves the understanding of the microstructure of DSS, but also provides an important tool for industrial quality control and material characterisation.

A new heteropolar Staurosirella species (Staurosiraceae, Bacillariophyceae), widely distributed in Europe

ABSTRACT An unknown heteropolar Staurosirella taxon was observed in a survey of Lake Pavin (Massif Central, France) with peculiar marginal spines that could not be identified using the currently available literature. Additional populations of the same taxon were observed in samples from Iceland and Flanders, Belgium. The unknown taxon is described as a new species, named Staurosirella pavinensis, based on detailed light and scanning electron microscopy observations of all populations. This new species is characterised by heteropolar, slightly clavate valves, with a narrow foot pole and a more broadly rounded head pole. The marginal spines are individually located in a small depression. Only at the foot pole, a small apical pore field is present, whereas the headpole is typically depressed. A detailed comparison with morphologically similar species is provided together with notes on its ecology, based on the analysis of the associated diatom flora and physico-chemical data.

Live-cell imaging and CLEM reveal the existence of ACTN4-dependent ruffle-edge lamellipodia acting as a novel mode of cell migration

α-Actinin-4 (ACTN4) expression levels are correlated with the invasive and metastatic potential of cancer cells; however, the underlying mechanism remains unclear. Here, we identified ACTN4-localized ruffle-edge lamellipodia using live-cell imaging and correlative light and electron microscopy (CLEM). BSC-1 cells expressing EGFP-ACTN4 showed that ACTN4 was most abundant in the leading edges of lamellipodia, although it was also present in stress fibers and focal adhesions. ACTN4 localization in lamellipodia was markedly diminished by phosphoinositide 3-kinase inhibition, whereas its localization in stress fibers and focal adhesions remained. Furthermore, overexpression of ACTN4, but not ACTN1, promoted lamellipodial formation. Live-cell analysis demonstrated that ACTN4-enriched lamellipodia are highly dynamic and associated with cell migration. CLEM revealed that ACTN4-enriched lamellipodia exhibit a characteristic morphology of multilayered ruffle-edges that differs from canonical flat lamellipodia. Similar ruffle-edge lamellipodia were observed in A549 and MBA-MD-231 invasive cancer cells. ACTN4 knockdown suppressed the formation of ruffle-edge lamellipodia and cell migration during wound healing in A549 monolayer cultures. Additionally, membrane-type 1 matrix metalloproteinase was observed in the membrane ruffles, suggesting that ruffle-edge lamellipodia have the ability to degrade the extracellular matrix and may contribute to active cell migration/invasion in certain cancer cell types.