Light Microscopy Research Articles

The ferns of Calilegua National Park, Jujuy, Argentina: A look through their spores. Second part

This work is part of a bigger project to study the spore morphology of isosporous ferns growing in the Calilegua National Park (CNP), in the province of Jujuy, Argentina. This park belongs to the phytogeographic region of the Yungas, where climatic and terrain conditions are optimal for the growth of ferns. The aim of this work is to present the morphology of the spores of 53 taxa corresponding to 6 families of isosporate ferns that grow in this protected area. The study was carried out with herbarium material and field trips. The families studied are Hymenophyllaceae, Ophioglossaceae, Polypodiaceae, Pteridaceae, Thelypteridaceae and Woodsiaceae. According to the spore aperture type, 24 taxa are monolete and 29 trilete. The spores are yellowish, light to dark brown or brown greenish. The largest spores belong to Pleopeltis tweediana and the smallest to Polyphlebium angustatum. The ornamentation observed were echinae, folds, wings, reticles, cristae, verrucae or rugulae. The spores of 35 species are illustrated for the first time under the light microscope and five species with scanning electron microscope. An identification key of the spores is also provided. The morphological characteristics of the spores allowed the identification of 16 species, contributes to spore bank analysis, aero and palaeopalynological studies and taxonomic identifications.

Detection of TurboID fusion proteins by fluorescent streptavidin outcompetes antibody signals and visualises targets not accessible to antibodies.

Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent streptavidin offers an 'all in one' solution to these restrictions. For all proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.

Oral supplementation with probiotics, potassium citrate, and magnesium in reducing crystalluria in stone formers: A phase II study.

Crystalluria is an important indicator of renal stone recurrence. Mechanisms underlying urinary stone formation are still not fully understood and raising interests has been giving to intestinal commensal bacteria for their contribute in maintaining urinary solutes equilibrium. The aim of our phase II study was to examine the administration of potassium citrate, magnesium and probiotics in order to reduce crystalluria. Since May 2021, we enrolled 23 patients candidates for ureterorenolithotripsy for calcium oxalate kidney stones with crystalluria and a normal metabolic profile. The analysis was validated by the Institution's Ethical Committee (no. approval STS CE Lazio 1/N-823). At discharge, patients were provided with daily food supplementation for 20 days of 1 billion Lactobacillus paracasei LPC09, 1 billion Lactobacillus plantarum LP01, 1 billion Bifidobacterium breve BR03, potassium (520 mg), citrate (1400 mg), and magnesium (80 mg). Crystalluria was re-assessed at 1, 3, 6, and 12-months follow-up by polarized light microscopy. After one month from the oral supplementation, no patient reported crystalluria; at 3 months, among the 20 participants available for re-evaluation, still no patient reported crystalluria. Instead, crystalluria was reported in three patients (15%) at 6 months, and in five patients (25%) at 12 months follow-up. The oral supplementation with Lactobacillus spp. and Bifidobacterium spp. was found able to reduce the prevalence of crystalluria in a cohort of patients with diagnosis of calcium oxalate kidney stones with crystalluria candidate to ureterorenolithotripsy.

Ultrastructure and functional morphology of the mouthparts in Pterygosoma pseudotrapelus (Bochkov, Melnikov et Nazarov, 2009) (Acariformes, Pterygosomatidae)

ABSTRACT The mouthparts of the pterygosomatid mite, Pterygosoma pseudotrapelus (Bochkov, Melnikov et Nazarov, 2009) (Acariformes, Pterygosomatidae), were studied with light microscopy, scanning (SEM) and transmission (TEM) electron microscopy. The mouthparts are located within the (pseudo)tagma gnathosoma. The gnathosoma is formed of the two frontal body segments with their appendages – chelicerae and pedipalps (palps). The two-segmented chelicerae occupy a dorsal position, whereas the infracapitulum – a derivative of the fused palp coxae – occupies a ventral position. The basal cheliceral segments are widen basally, and contain protractors and retractors of the movable digits and nerve fibres. The distal cheliceral segments, the movable digits, are small and stout, bear a lateral spur, and are provided with tips looking aside. The fixed digits are long and branched. The frontal projection of the infracapitulum, the hypostome, partly envelopes laterally the distal cheliceral portions. The dorsal wall, or the roof of the infracapitulum, consists of the long labrum anteriorly possessing its own muscles and the complexly organized epistome posteriorly. The epistome and its posterior projections – the epistomal apodemes serve for origin of the pharyngeal dilators. The wide cap-shaped pharynx goes along the bottom of the infracapitulum and attaches to it. The two sigmoid pieces articulate posteriorly to the epistome and serve for origin of different sets of muscles performing retraction and protraction of the chelicerae and retraction of the gnathosoma. The peritremes are exceedingly long and protrude freely above the chelicerae. The general architecture of the mouthparts of P. pseudotrapelus corresponds more to the anystid than eleutherengonid ground pattern.

Single molecule dynamics in a virtual cell combining a 3-dimensional matrix model with random walks

Recent advances in light microscopy have enabled single molecules to be imaged and tracked within living cells and this approach is impacting our understanding of cell biology. Computer modeling and simulation are important adjuncts to the experimental cycle since they aid interpretation of experimental results and help refine, test and generate hypotheses. Object-oriented computer modeling is particularly well-suited for simulating random, thermal, movements of individual molecules as they interact with other molecules and subcellular structures, but current models are often limited to idealized systems consisting of unit volumes or planar surfaces. Here, a simulation tool is described that combines a 3-dimensional, voxelated, representation of the cell consisting of subcellular structures (e.g. nucleus, endoplasmic reticulum, cytoskeleton, vesicles, and filopodia) combined with numerical floating-point precision simulation of thousands of individual molecules moving and interacting within the 3-dimensional space. Simulations produce realistic time-series video sequences comprising single fluorophore intensities and realistic background noise which can be directly compared to experimental fluorescence video microscopy data sets.

Fascicular injury is rare following needle transfixion: a study on median and ulnar isolated human nerves

BackgroundNeedle trauma has been associated with peripheral nerve injury and neurological dysfunction. However, inadvertent needle puncture is frequent while postblock dysfunction is rare. We conducted a cadaveric study to evaluate...

Molluscicidal property of symbiotic bacteria associated with entomopathogenic nematodes against Indoplanorbis exustus and Radix rubiginosa, the intermediate hosts of trematode parasites

Indoplanorbis exustus and Radix rubiginosa act as intermediate hosts for veterinary and medical trematode parasites. Snail control is a strategy used to decrease the number of snails and interrupt the life cycle of parasites. The objective of this study was to evaluate the efficacy of Xenorhabdus and Photorhabdus extracts against I. exustus and R. rubiginosa in the laboratory. Ethyl acetate extracts of selected symbiotic bacteria were tested for their molluscicidal activities according to World Health Organization guidelines. Additionally, pathological changes in the snails were observed after treatment with the LC50 values under a light microscope. Indoplanorbis exustus and R. rubiginosa were susceptible to all ethyl acetate extracts of symbiotic bacteria. The lowest LC50 and LC90 at 24 h for I. exustus after exposure to Photorhabdus laumondii subsp. laumondii (bALN18.2_TH) extracts were 81.66 and 151.02 ppm, respectively. Similarly, the lowest LC50 and LC90 at 24 h for R. rubiginosa after exposure to Photorhabdus luminescence subsp. akhurstii (bAPY3.5_TH) extracts were 49.21 and 147.66 ppm, respectively. Photorhabdus species had more substantial molluscicidal effects than Xenorhabdus on these snails. The ethyl acetate extracts of these bacteria are effective when contacting the epithelial cells and foot muscle of the snails. To our knowledge, this is the first report on using Xenorhabdus and Photorhabdus extracts to evaluate molluscicidal activities. These symbiotic bacteria, Xenorhabdus and Photorhabdus, may be useful for controlling snail intermediate hosts.

Melatonin prevents histopathologies stem from cadmium chloride in pregnant mice lungs.

Heavy metals may cause structural and functional changes in organs. Cadmium, taken into the body through oral and respiratory routes, can lead to lesions. Cadmium may lead to lesions by accumulating in organs. The lungs are significantly affected by cadmium. Melatonin, an antioxidant hormone with therapeutic effects, is secreted by the pineal gland. The aim of the study is to treat cadmium-induced lesions in the lungs of pregnant mice with Melatonin. Four groups were created with 24 pregnant mice, named Control, Cadmium Chloride, Melatonin, and Melatonin + Cadmium Chloride groups (n: 6) Cadmium Chloride (2mg/kg/bw) and Melatonin (3mg/kg/bw) were given orally through gavage during pregnancy (21 days) After routine histological procedures, the lung tissues were stained with Hematoxylin-Eosin and evaluated under a light and electron microscope. ANOVA tests were applied for one-way analysis of variance, and LSD tests were applied for pairwise comparisons (p < 0.05) The average lung weight decreased in the Cadmium Chloride group (p: 0.03) The average lung weight in the Cadmium Chloride + Melatonin group was found to be close to the control group (p: 0.06) Cadmium Chloride caused thickening of the lung alveolar wall, inflammatory cell infiltration, and fibrin deposition. Because the lesions were not observed in the Melatonin group, lesions may be prevented by melatonin. Additional studies may be useful to determine the protective effect of Melatonin at different doses of Cadmium Chloride.

Genome-wide analysis of the biophysical properties of chromatin and nuclear proteins in living cells with Hi-D.

To understand the dynamic nature of the genome, the localization and rearrangement of DNA and DNA-binding proteins must be analyzed across the entire nucleus of single living cells. Recently, we developed a computational light microscopy technique, called high-resolution diffusion (Hi-D) mapping, which can accurately detect, classify and map diffusion dynamics and biophysical parameters such as the diffusion constant, the anomalous exponent, drift velocity and model physical diffusion from the data at a high spatial resolution across the genome in living cells. Hi-D combines dense optical flow to detect and track local chromatin and nuclear protein motion genome-wide and Bayesian inference to characterize this local movement at nanoscale resolution. Here we present the Python implementation of Hi-D, with an option for parallelizing the calculations to run on multicore central processing units (CPUs). The functionality of Hi-D is presented to the users via user-friendly documented Python notebooks. Hi-D reduces the analysis time to less than 1 h using a multicore CPU with a single compute node. We also present different applications of Hi-D for live-imaging of DNA, histone H2B and RNA polymerase II sequences acquired with spinning disk confocal and super-resolution structured illumination microscopy.

Loss-of-function variants in JPH1 cause congenital myopathy with prominent facial and ocular involvement

BackgroundWeakness of facial, ocular and axial muscles is a common clinical presentation in congenital myopathies caused by pathogenic variants in genes encoding triad proteins. Abnormalities in triad structure and function...

Teaching medical students hematopathology: a randomized crossover study comparing direct inspection by light microscope versus projected images.

Students' ability to diagnose various blood disorders could be substantially improved by continuously reviewing approaches toward teaching hematology. This study aims to compare the effectiveness of light microscopes and projected images on students' learning and determine medical students' perception of these teaching methods. A randomized trial was conducted using a crossover design. Two groups, each with 30 students, were subjected to teaching methods based on light microscopes and projected images alternatively. No differences were found in the two study groups' baseline characteristics, such as median age, sex, and prior academic performance, as well as in the pre-test scores. Post-test scores were significantly higher among students subjected to the projection method than in the control group (Mean ± SD = 9.8 ± 1.7 vs. 5.1 ± 1.3, p < 0.001). In the post-cross-over assessment, 85% (n = 51) of students reported their satisfaction for the projected images, and 78% (n = 47) of students were willing to be taught by projection. Students perceived that the projection method facilitated participation and better involvement in discussions, improved learning, provided greater motivation, and eventually increased comprehension and efficiency. The projection-based teaching method is more effective in improving knowledge and achieving intended learning outcomes. Students tend to prefer the projection method over the laboratory-based method and perceive it as an effective method to enhance their learning of hematology.

Comparing Different Methodologies to Quantify Particulate Matter Accumulation on Plant Leaves

Urban air pollution poses a significant threat to human health, with metropolitan areas particularly affected due to high emissions from human activities. Particulate matter (PMx) is among the most harmful pollutants to human health, being composed of a complex mixture of substances related to severe pulmonary conditions. Urban green spaces play a vital role in mitigating air pollution by capturing PMx, and it is essential to select plant species with a high capacity for PMx accumulation to effectively enhance air quality. This study aimed to evaluate and compare the accuracy of two PMx quantification methods—light microscopy and filtration—which demonstrated a high correlation (R2 = 0.72), suggesting that both methods are reliable for assessing PMx accumulation on leaves. Light microscopy allowed for the visualization of PMx deposition, revealing the species warranting further analysis using the filtration method. Among the species analyzed, Euonymus japonicus, Ligustrum lucidum, Alnus glutinosa, Rubus ulmifolius, and Laurus nobilis demonstrated the highest total PMx accumulation, exceeding 50 µg cm−2, making them particularly valuable for air pollution mitigation. This study examined the correlation between leaf traits such as specific leaf area (SLA), leaf area (LA), leaf dissection index (LDI), and leaf roundness and PMx accumulation across the 30 different plant species. A multiple linear regression analysis indicated that these leaf traits significantly influenced PMx accumulation, with SLA and LA showing negative correlations and leaf roundness exhibiting a positive correlation with PMx deposition. In conclusion, this study highlights the importance of selecting plant species with specific leaf traits for effective air quality improvement in urban environments particularly in highly polluted areas, to enhance air quality and public health.

Comparative fruit morphology of nine Psittacanthus Mart. (Santalales: Loranthaceae) mistletoe species occurring in Mexico

The genus Psittacanthus is distributed from Mexico to Argentina and contains c. 110 species. The large, lipid-rich, one-seeded fruits of Psittacanthus species frequently depend on frugivorous birds for seed dispersal; however, fruit morphology of Psittacanthus (Loranthaceae) mistletoes remain poorly studied. In this study we describe the fruit morphology of nine Psittacanthus species. Fruits were collected from the study sites in which mistletoe plants were inhabiting contrasting habitats and host tree species. The morphological characteristics of fruits by species were described and compared with the use of inclusion and staining techniques and light microscopy. Our results show that fruit size varies among species, with P. macrantherus having the largest fruits. Most fruits have an ellipsoid to ovoid shape, color of fruits transits from green when immature to red or purple before ripening to blackish or purplish black, with color variations observed in different species, and the cupular pedicel length varying among species. Fruit sections indicate the following parts: exocarp, viscin layer, seed coat and a dicotylar to polycotylar embryo, with P. schiedeanus having the highest cotyledon count. No endosperm is present in the studied species. Our results provide valuable information for further species comparisons regarding the lack of endosperm and polycotylar embryo. Additionally, interspecific variation in cotyledon number and seed coat highlights distinct processes, including the potential effects of environmental differences.

Three new species of bright-spored myxomycetes from Asia and the second world record of Perichaena maculosa

Three new species of myxomycetes are described in the genera Arcyria, Metatrichia and Tubifera. Each species is given a full description, along with details of ecology and distribution. They are compared to morphologically similar species, using both light and scanning electron microscopy. DNA extracted from the 18S nrDNA regions (SSU), confirmed the three new species.

Widefield quantitative polarized light microscopy using spectrally encoded null polarimetry.

Quantitative polarized light microscopy enables determination of optical retardation and azimuth of birefringent specimens and is a powerful tool for label-free imaging in the fields of biology and pathology. We have recently proposed a device for fast laser-scanning birefringence microscopy based on a near-infrared wavelength-swept laser and spectral encoding of polarization, resulting in a channeled spectrum generated during the wavelength-sweep of the laser and highly sensitive to optical retardation [Opt. Lett.49, 387 (2024)10.1364/OL.507576]. In this Letter, we propose its transposition to visible widefield imaging using a white light source and a high-order retarder for spectral encoding and a hyperspectral camera to record the channeled spectrum at each point of the image in parallel. The method proposed here allows for straightforward conversion of any widefield microscope into a highly sensitive and quantitative polarized light microscope.

Enzymatically enhanced ultrastructure expansion microscopy unlocks expansion of in vitro Toxoplasma gondii cysts.

Toxoplasma gondii is an intracellular parasite capable of establishing long-term chronic infection in nearly all warm-blooded animals. During the chronic stage, parasites encapsulate to form cysts predominantly in neurons and skeletal muscle. Current anti-Toxoplasma drugs do not eradicate chronic parasites, leaving a reservoir of infection. The cyst is critical for disease transmission and pathology, yet it is harder to study, with the function of many chronic-stage proteins still unknown. Ultrastructure expansion microscopy, a new method to overcome the light microscopy's diffraction limit by physically expanding the sample, allowed in-depth studies of acute Toxoplasma infection. We show that Toxoplasma cysts resist expansion using standard protocol, but an additional enzymatic digestion with the mucinase StcE allows full expansion. This protocol offers new avenues for examining the chronic stage, including precise spatial organization of cyst-specific proteins, linking these locations to morphological structures, and detailed investigations of components of the durable cyst wall.

Non-redundant role of leishmanolysin-like (Lmln) zinc-metallopeptidase in retinal homeostasis

To determine if Lmln, a Zn-metallopeptidase, is important for retinal homeostasis. Combining an unbiased N-ethyl-N-nitrosourea mutagenesis pipeline in mice with optical coherence tomography (OCT) screening and automated meiotic mapping, we identified an allele (nemeth) that seemed to be associated with outer nuclear layer (ONL) thinning. Since nemeth was predicted to lead to a nonsense mutation of the Lmln gene, we targeted Lmln using CRISPR/Cas-9 technology and characterized the impact on retinal anatomy and function. OCT imaging demonstrated an outer retinal degeneration in Lmln-/- mice (P = 7.3 × 10-9 for ONL at 2 m) that progressed over the first 6 months of life and then stabilized. Light microscopy showed loss of ONL nuclei (P ranged between .00033 and .0097 for posterior measurements), and a TUNEL assay revealed a small but significant increase in apoptosis (P = .034). Lmln-/- mice accumulated fundus spots (P = .0030 by 2 m of age) and activated subretinal microglia (p ranged from .0007 to 8 × 10-13 for Gal3+ cells). Scotopic electroretinography demonstrated a decrease in retinal function in Lmln-/- mice both at 6 m (only a-wave, P < .01 for all stimuli) and at 10 m of age (P < .01 for both a-wave and b-wave with all stimuli). Our work revealed a previously unknown essential requirement for Lmln in maintaining retinal anatomy and function. Further studies using this new model will be aimed at determining the cellular expression of Lmln and its mechanisms of action within the retina.

Protective Role of Melatonin Against Abamectin-Induced Biochemical, Immunohistochemical, and Ultrastructural Alterations in the Testicular Tissues of Rats.

Abamectin is one of the most widely used pesticides due to its strong insecticidal and anthelmintic activities. Melatonin is a neurohormone with potent antioxidant, anti-apoptotic, and anti-inflammatory effects. This study aimed to investigate the potential ameliorative effects of melatonin against abamectin-induced testicular toxicity in rats. Twenty-four rats were divided into four groups: control group (1 mL/kg/day corn oil), melatonin-treated group (10 mg/kg/day), abamectin-treated group (0.5 mg/kg/day), and melatonin plus abamectin-treated group. Test substances were administered via oral gavage once daily for 28 days. While MDA and 8-OHdG levels increased in the testicular tissue of rats treated with abamectin, SOD, CAT, GPx, and GST enzyme activities decreased significantly. While interleukin-17 levels, TNF-α, and caspase3 expression increased in the testicular tissue, acetylcholinesterase activity decreased. At the same time, serum gonadotropins (luteinizing and follicle-stimulating hormones) and testosterone levels decreased. Light microscope examinations of testicular tissues revealed severe histopathological changes, such as atrophic hyalinized seminiferous tubules, basement membrane irregularity, degeneration, spermatogenic cell loss, and necrosis. Electron microscopy examinations revealed large vacuoles in Sertoli and spermatogenic cells, swelling and vacuolization in mitochondria, lysosomal structures, and increased pyknotic nuclei. In contrast, melatonin supplementation significantly ameliorated abamectin-induced testicular toxicity in rats through antioxidant, antiapoptotic, and anti-inflammatory mechanisms.

New morphological structures of Priapulus caudatus, Lamarck 1816 (Priapulida) and analysis of homologous characters across macroscopic priapulids

The macroscopic Priapulus caudatus Lamarck, 1816 is one of the most investigated priapulids, yet, the adult morphology warrants detailed characterization and analyses of potential priapulid homologies. Using light microscopy, scanning electron microscopy and histology, we examine adult specimens of P. caudatus from three localities in the Northeast Atlantic and the Arctic Oceans (Gullmarsfjord, North Sea, White Sea). Our examinations show ring papillae on the posterior trunk of P. caudatus for the first time in detail and describe previously overlooked structures on the circumoral field (hemispherical papillae, ring 0 scalids and circular folds). In addition, we add new information on pharyngeal teeth, scalids, trunk papillae, posterior warts, and on spinulets of the caudal appendage. The new collected data of P. caudatus allows us to compare structures across species of the macroscopic Priapulidae (Acanthopriapulus, Priapulopsis, Priapulus, Halicryptus) in detail and to build hypotheses on their homology within this clade.

Charcoal in Anaerobic Digestion: Part 1—Characterisation of Charcoal

Biochar (BC) is often used as an additive in anaerobic digestion (AD) to increase yield and/or to stabilise the process when the manure content is increased. Unfortunately, BC is rarely described in detail in terms of its raw material sources, production processes, and structural, physical and chemical properties to allow correlation with its effects on AD. It is an open question whether microorganisms from AD can penetrate into different biochar pore types, depending on their wood origin. In this paper, we describe the preparation (temperatures, treatment times, yields) and characterisation shrinkage, density, pore sizes, pore size distribution, specific surface area, ash, volatiles, fixed carbon, elemental composition, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), benzene, toluene, ethyl-toluene, xylene (BTEX) and volatile halogenated hydrocarbons (VHH) of BC cubes of Scots pine (Pinus sylvestris) and common beech (Fagus sylvatica) powder made from this BC in addition to commercial charcoal powder. The pore size distribution determined by mercury porosimetry differs from that determined by 3D-reflected light microscopy. After incubating BC cubes in AD, the cubes were mechanically cleaned and cut into two pieces. Microorganisms were detected inside the cubes by fluorescence microscopy. Particle size and wood source determine the influence of BC on AD.