Light Microscopy Research Articles

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.

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.

Effect of Different Liquids and Thermal Aging Procedures on the Shear Bond Strength of APC II, APC Flash-Free, and Conventional Ceramic Brackets: An In Vitro Study.

The purpose of this study was to compare the effects of cherry juice, coffee, coke, gastric acid, and the thermo-aging procedure (TAP) on the shear bond strength (SBS) of APC II, APC flash-free, and conventional ceramic brackets. A total of 180 human premolar teeth were randomly divided into three major groups according to the type of ceramic bracket. Then, six subgroups (n=10) were established from each major group: Group 1: control; Group 2: only TAP; Group 3: 72 hours of cherry juice exposure + TAP; Group 4: 72 hours of coffee exposure + TAP; Group 5: 72 hours of coke exposure + TAP; and Group 6: 24 hours gastric acid exposure + TAP. SBS was assessed for each specimen using a universal test device, and the adhesive remnant index (ARI) was scored under a light microscope. Kruskal-Wallis and post-hoc Tamhane tests were used to analyze the data. Among the control groups, the highest SBS value belonged to conventional ceramic brackets (p<0.01). SBS values for all groups decreased as a result of each liquid and TAP. Gastric acid and coke had the greatest detrimental effects on SBS, while TAP had the least negative effects. The SBS values of APC II, APC flash-free, and conventional brackets were found to be statistically insignificant after different liquid exposures and TAP. TAP and various fluids had a negative impact on the SBS value of ceramic brackets. SBS values, however, were still higher than clinically acceptable (8-9 MPa) values, even after exposure to gastric acid and coke.

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.

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.

Tissues response and bone-forming capacity of zinc oxide–eugenol sealer following the addition of nanohydroxyapatite-tyrosine amino acid: An in vivo animal study

Abstract Introduction: The zinc oxide–eugenol (ZOE) sealer is used till now in many private dental clinics although many sealers have been developed. The current study aimed to compare the biocompatibility and remineralizing capacity of ZOE sealer following the addition of nanohydroxyapatite-tyrosine amino acid. Materials and Methods: Twenty rabbits were used in this investigation, and they were divided into four groups based on the test observation period (3, 7, 21, and 28 days) following surgical implantation. General anesthesia was given for each rabbit with a subcutaneous incision of about 1 cm ± 0.5 cm made along the symphyseal area of the mandible of each rabbit. Each rabbit had four bone cavities generated in the interdental space of the lower jaw between the central and molar teeth, with one longitudinal subcutaneous incision. The ZOE sealers were mixed and directly inserted within the cavities as follows: ZOE alone on the central area of the right side of the jaw (Group A), 10% nHAp and Tyrosine mixed ZOE at the molar area (Group B), empty cavity (−ve control) located at the central area of the left side of the jaw (Group C) and 20%/nHAp and Tyrosine mixed ZOE located at the molar area on the left side of the jaw (Group D). Animals were scarified and bone biopsy samples from the tested parts were collected. Histological analysis was performed using a low-power light microscope and immunohistochemistry using runt-related transcription factor 2 (Runx2) antibody. Data were statistically analyzed utilizing the SPSS software. One-way analysis of variance was used and the post hoc test Duncan’s test was utilized to measure the significance among various groups at P ≤ 0.01. Results: The inflammatory process decreased over time for all experimental groups, associated with an increase in granulation tissue formation which was gradually decreasing over time to permit an increase in new blood vessels and new bone formation for all experimental groups that increased with time. Group D displayed the highest levels of new bone formation. Runx2 antibody expression declined progressively, particularly in Group D on 28 days. Conclusion: Superior new bone formation and biocompatible properties were demonstrated by the 20% nHAp and tyrosine mixed ZOE sealer.

A systematic evaluation of computational methods for cell segmentation.

Cell segmentation is a fundamental task in analyzing biomedical images. Many computational methods have been developed for cell segmentation and instance segmentation, but their performances are not well understood in various scenarios. We systematically evaluated the performance of 18 segmentation methods to perform cell nuclei and whole cell segmentation using light microscopy and fluorescence staining images. We found that general-purpose methods incorporating the attention mechanism exhibit the best overall performance. We identified various factors influencing segmentation performances, including image channels, choice of training data, and cell morphology, and evaluated the generalizability of methods across image modalities. We also provide guidelines for choosing the optimal segmentation methods in various real application scenarios. We developed Seggal, an online resource for downloading segmentation models already pre-trained with various tissue and cell types, substantially reducing the time and effort for training cell segmentation models.

Histological Fixation Process and Fixatives

Tissue monitoring generally includes the stages of fixation, dehydration, clearing, hardening (infiltration), paraffin blocking/paraffin(emmeding), sectioning, water removal, routine staining, and mounting. Fixation is the basic and first step in the microscopic examination of tissues. The histotechnical process, which includes components such as detection, tissue tracking and staining, basically aims to capture and visualize the state of the relationships between tissue parts inside and outside the cell and various cells at a certain time as close as possible to the living state. Maintaining the natural structure of the tissue is important for the follow-up phase. The main feature of a good fixative should protect the sample and make the macromolecules insoluble without changing the chemistry of the sample studied and allowing it to be examined as closely as possible to its living state. In histological tissue analysis including light microscope and electron microscope techniques, an appropriate fixation method is selected for each study. Detection solutions are classified in terms of content. The most commonly used fixative in light microscopic follow-up procedures is 10% formaldehyde. For the electron microscope, the gluteraldehyde-osmium tetraoxide binary is widely used for fixation purposes. Gluteraldehyde acts more slowly and is more expensive than formaldehyde. Formalin is obtained by dissolving formaldehyde in water. In addition, the fixed samples can be stored in the solution for months. With a successful fixation process, the structural properties of the tissue are preserved and thus it is possible to examine the tissue as closely as possible. Thus, better quality sections are obtained from the tissue samples taken. For this reason, it will be more efficient to interpret well-fixed samples by photographing them. In this review, which was created by using various sources, the elements to be considered for an ideal fixation were determined and it was aimed to provide an overview of successful fixation for light microscope and electron microscope.

Floral dimorphism of Elsholtzia angustifolia (Loes.) Kitag. (Lamiaceae)

Gynodioecy is a sexual system in which hermaphrodites coexist with females and is frequently observed in Lamiaceae. The aim of this study was to investigate the floral morphology of Elsholtzia angustifolia (Loes.) Kitag (Lamiaceae) using stereomicroscopy (SM), light microscopy (LM), and scanning electron microscopy (SEM) to identify floral dimorphism in a potential gynodioecious species. Two different floral morphs were found in Elsholtzia angustifolia, hermaphroditic and female individuals, from three natural populations in South Korea (Ihwa Pass, Mt. Joryeong, and Mt. Sokli). However, females rarely occur in natural populations. Statistical analysis revealed significant size differences in the floral organs of the floral morphs. Micromorphological and anatomical characteristics were also examined using LM and SEM to compare the differences between the female and hermaphroditic types, revealing significant differences in the stamens between the two sexual types. Additionally, floral scent analysis was performed to identify floral morph-specific scent components, with elsholtzia ketone and dehydroelsholtzia ketone as the major components.

Mounting agents with low toxicity and with fast curing time for digital pathology in the intraoperative frozen section laboratory

AimsIn intraoperative frozen tissue section laboratories (FS laboratories) conventional practice for mounting coverslips on tissue slides involves the use of xylene-based mounting agents, such as Pertex. However, toxic vapours pose...