Light Microscopy Images Research Articles

Clearing-enabled light sheet microscopy as a novel method for three-dimensional mapping of the sensory innervation of the mouse knee.

A major barrier that hampers our understanding of the precise anatomic distribution of pain sensing nerves in and around the joint is the limited view obtained from traditional two dimensional (D) histological approaches. Therefore, our objective was to develop a workflow that allows examination of the innervation of the intact mouse knee joint in 3D by employing clearing-enabled light sheet microscopy. We first surveyed existing clearing protocols (SUMIC, PEGASOS, and DISCO) to determine their ability to clear the whole mouse knee joint, and discovered that a DISCO protocol provided the optimal transparency for light sheet microscopy imaging. We then modified the DISCO protocol to enhance binding and penetration of antibodies used for labeling nerves. Using the pan-neuronal PGP9.5 antibody, our protocol allowed 3D visualization of innervation in and around the mouse knee joint. We then implemented the workflow in mice intra-articularly injected with nerve growth factor (NGF) to determine whether changes in the nerve density can be observed. Both 3D and 2D analytical approaches of the light sheet microscopy images demonstrated quantifiable changes in midjoint nerve density following 4 weeks of NGF injection in the medial but not in the lateral joint compartment. We provide, for the first time, a comprehensive workflow that allows detailed and quantifiable examination of mouse knee joint innervation in 3D.

Microstructure evolution of AlSi10Mg alloy in RAP process

Abstract With its computer-aided layer-by-layer production approach, additive manufacturing (AM) and powder bed laser fusion (PBLF) paved the way to produce metallic parts more precisely than any other manufacturing technique. However, the combinability and the interaction of this relatively new manufacturing technique with the other near-net shape production techniques is still a mystery. In this study, the recrystallization and partial melting (RAP) behavior, which is a feedstock production approach for semisolid forming methods, investigated on AlSi10Mg parts produced by PBLF and conventional casting were compared in terms of microstructural and hardness evaluations. After the reheating process, the globalization of Si particles and the breakdown of the Si network around the melt pools were displayed with light microscope and scanning electron microscope (SEM) images. The hardness values of the PBLF as-fabricated specimens were found to be significantly higher than the as-cast specimens; however, the values were almost equaled after the RAP treatment and even got lower on the bottom and top regions of the PBLF samples after 20 min of reheating because of the enlarged shrinkage porosities and the coarsened morphology.

An ultrastructure analysis of the developing human anterior cruciate ligament tibial enthesis.

This study aimed to investigate the ultrastructural anatomy of the developing ACL tibial enthesis. We hypothesized that enthesis architecture would progressively mature and remodel, eventually resembling that of the adult by the early postnatal stage. Five fresh-frozen human pediatric cadaveric knees aged 1-36 months underwent anatomical dissection to harvest the ACL insertion and underlying tibial chondroepiphysis. The samples were prepared for scanning electron microscopy (SEM) to examine the ultrastructural anatomy of the enthesis and underwent histological staining for circular polarized light (CPL) and light microscopy imaging. SEM analysis of the 1- and 8-month-old samples revealed a shallow interdigitation between the dense fibrous (ligamentous) tissue and unmineralized chondrogenic tissues, with a minimal transition zone. By 11-month, a more complex transition zone was present. By age 19- and 36-month-old, a progressively more complex and defined fibrocartilage zone was observed. CPL analysis revealed distinct collagen fiber continuity, alignment, and organization changes over time. By 19 and 36 months, the samples exhibited complex fiber arrangements and a progression toward uniform fiber orientation. Similarly, histological analysis demonstrated progressive remodeling of the enthesis with increasing age. Our results suggest that the ACL enthesis of the developing knee begins to mimic that of an adult as early as 19 months of age, as a more complex transition between ligamentous and chondro-epiphyseal tissue can be appreciated. We hypothesize that the observed changes are likely due to mechanical loading of the enthesis with the onset of weightbearing. Future investigations of ACL reconstruction and repair will benefit from improved understanding of the chondro-epiphyseal/ACL regions.

Halamphorahampyeongensis sp. nov. (Amphipleuraceae, Bacillariophyceae), a new marine benthic diatom from a tidal mudflat in Hampyeong Bay, South Korea.

The abundance and variety of benthic diatoms inhabiting tidal flats is widely acknowledged, although it has received relatively less attention than other research areas. In this investigation, we provide a formal description of a benthic diatom found in the tidal mudflat of South Korea, based on morphological and molecular characteristics and the similarities and differences between Halamphorahampyeongensis sp. nov., with morphologically similar Halamphora species are also discussed. Morphological characteristics are described from light and electron microscopy images. H.hampyeongensis is distinguished by its wide ventral sides of the valve, small and rounded areolae present across the whole valve face, and dense dorsal striae biseriate (34-38 in 10 μm). Phylogenetic analysis based on 18S rDNA and rbcL sequence data revealed that H.hampyeongensis is related to H.montana, H.mosensis, and H.specensa. The results (morphometric and molecular) provide sufficient elements to support and propose this taxon as a new species.

Correlative microscopy - illuminating the endomembrane system of plant seeds.

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

Concordance of Whole-Slide Imaging and Conventional Light Microscopy for Assessment of Pathologic Response Following Neoadjuvant Therapy for Lung Cancer

Pathologic response is an end point in many ongoing clinical trials for neoadjuvant regimens, including immune checkpoint blockade and chemotherapy. Whole-slide scanning of glass slides generates high-resolution digital images and allows for remote review and potential measurement with image analysis tools, but concordance of pathologic response assessment on digital scans compared with that on glass slides has yet to be evaluated. Such a validation goes beyond previous concordance studies, which focused on establishing surgical pathology diagnoses, as it requires quantitative assessment of tumor, necrosis, and regression. Further, as pathologic response assessment is being used as an end point, such concordance studies have regulatory implications. The purpose of this study was 2-fold, which was as follows: first, to determine the concordance between pathologic response assessed on glass slides and that assessed on digital scans, and second, to determine if pathologists benefited from using measurement tools when determining pathologic response. To that end, hematoxylin and eosin–stained glass slides from 64 non–small cell lung carcinoma specimens were visually assessed for percent residual viable tumor (%RVT). The sensitivity and specificity for digital vs glass reads of pathologic complete response (0% RVT) and major pathologic response (≤10% RVT) were all >95%. When %RVT was considered as a continuous variable, the intraclass correlation coefficient of digital vs glass reads was 0.94. The visual assessments of pathologic response were supported by pathologist annotations of residual tumor and tumor bed areas. In a separate subset of hematoxylin and eosin–stained glass slides, several measurement approaches to quantifying %RVT were performed. Pathologist estimates strongly reflected measured %RVT. This study demonstrates the high level of concordance between glass slides evaluated using light microscopy and digital whole-slide images for pathologic response assessments. Pathologists did not require measurement tools to generate robust %RVT values from slide annotations. These findings have broad implications for improving clinical workflows and multisite clinical trials.

Studies on type material from Kützing’s diatom collection VIII. Species assigned to the genera Epithemia Brébisson ex Kützing and Rhopalodia O. Müller

We detail the diatom taxa described by F.T. Kützing across three of his publications that have been referred to the diatom genera Epithemia and Rhopalodia. Taxa considered include Frustulia adnata, F. picta, E. alpestris, E. musculus, E. porcellus, E. proboscidea, E. sorex, E. ventricosa, E. vertagus, E. ventricosa β. gregaria and E. reticulata. For these 12 taxa, this represents the first time Kützing’s specimens have been illustrated with light microscopy. Most of the material consulted here is from the Natural History Museum, London, though we also reviewed material from the Academy of Natural Sciences for E. adnata. We present light microscope images of the Kützing taxa and complement these observations with images of the type slides, material packets and both herbarium (unpublished) and published drawings. In some cases, modern concepts of Kützing species are supported, but in others it is likely that species concepts will be changed by the specimens in the type material. Lectotypes are designated for Epithemia adnata and Epithemia reticulata.

Reevaluation of pollen differentiation in Altingiaceae: Challenges in distinguishing deciduous (Liquidambar) and evergreen (Altingia) types using multivariate statistics and machine learning

Altingiaceae, a family of woody plants, comprising evergreen Altingia and deciduous Liquidambar groups, exhibits distinct leaf morphology, yet both groups overlap in geographical range and climatic conditions. While some tropical Altingia species are confined to Myanmar, Thailand, Cambodia and India without Liquidambar, and some temperate Liquidambar species to northern China without Altingia. Their fossil pollen have significant implications in reconstructing palaeoclimate and historical biogeography, based on classification of Altingia-type and Liquidambar-type. However, the results of previous studies to differentiate pollen types of evergreen Altingia and deciduous Liquidambar were based on limited pollen specimens. Therefore pollen morphology of Altingiaceae and differentiation of above mentioned types needs reevaluation using more specimens from wider geographical range.In this study, we present new findings on Altingiaceae pollen morphology from extensive collection of specimens and reassess the diagnostic features to distinguish evergreen and deciduous types. To improve the credibility of palaeoecological and palaeoclimatic interpretations, we applied multivariate statistical analyses to pollen size, number of pores, pollen wall thickness, and size and density of ornamental elements from light microscopy (LM) and Scanning Electronic Microscope (SEM) images. Additionally, random forest classification models were applied to test the accuracy of differentiating Altingiaceae pollen types. Our results reveal significant morphological overlap between the pollen of evergreen Altingia and deciduous Liquidambar, with classification models showing limited accuracy and explainability. Thus, fossil pollen of Altingiaceae cannot be confidently classified into evergreen or deciduous types, highlighting challenges in using their pollen morphology for taxonomic classification in palaeoecological research.

Foliar epidermal micro-morphology of selected brinjal varieties: A study on leaf surface characteristics

Studies on the leaf epidermal morphological characteristics were conducted on the three varieties of brinjal (Solanum melongena L.) belonging to the family Solanaceae. The varieties used in the study were Bhavani Gold, Purple Round and Haritha. Fresh mature brinjal leaves were randomly selected from the three experimental genotypes, and the foliar epidermal features were studied by using light microscopy and SEM imaging. Measurements were taken with an ocular and stage micrometer using 10X and 40X magnifications. The photographs were taken from mounted slides. The stomatal count and each measurement represent the average of ten readings. The upper and lower epidermal surfaces of leaf were studied for stomatal features including, stomatal distribution, stomatal type, stomatal index, stomatal pore length, stomatal pore width, guard cell length, guard cell width, subsidiary cell length and width, epidermal cell type, epidermal cell length and width, trichome type, trichome length and width. Amphistomatous, anisocytic, and with more stomata on the lower epidermal surface than the upper epidermal surface were present in all leaf varieties. On the leaves, the abaxial stomatal index ranged from 68.19 mm-2 (Bhavani Gold) to 56.67 mm-2 (Purple Round) to 58.8 mm-2 (Haritha). While on the adaxial surface, it varied from 59.67 mm-2 (Bhavani Gold), 53.46 mm-2 (Purple Round) and 56.67 mm-2 (Haritha). The three varieties of this study exhibited similarities in the pattern of epidermal cell on the adaxial and abaxial surfaces and were irregular in shape. Among the three varieties epidermal cell become irregular in outline and trichomes were present on both leaf surfaces. It becomes stellate, non-glandular, glandular and 8-12 in number. The highest trichome length was obtained in Bhavani Gold (63 μm) followed by Haritha (57.5 μm) and Purple Round (43 μm) on the upper epidermis. On the lower epidermal region maximum length was obtained in Bhavani Gold (65.5 μm) followed by Haritha (53 μm) and Purple Round (44 μm).

Large-volume fully automated cell reconstruction generates a cell atlas of plant tissues.

The geometric shape and arrangement of individual cells play a role in shaping organ functions. However, analyzing multicellular features and exploring their connectomes in centimeter-scale plant organs remain challenging. Here, we established a set of frameworks named Large-Volume Fully Automated Cell Reconstruction (LVACR), enabling the exploration of three-dimensional (3D) cytological features and cellular connectivity in plant tissues. Through benchmark testing, our framework demonstrated superior efficiency in cell segmentation and aggregation, successfully addressing the inherent challenges posed by light sheet fluorescence microscopy (LSFM) imaging. Using LVACR, we successfully established a cell atlas of different plant tissues. Cellular morphology analysis revealed differences of cell clusters and shapes in between different poplar (P. simonii Carr. and P. canadensis Moench.) seeds, whereas topological analysis revealed that they maintained conserved cellular connectivity. Furthermore, LVACR spatiotemporally demonstrated an initial burst of cell proliferation, accompanied by morphological transformations at an early stage in developing the shoot apical meristem. During subsequent development, cell differentiation produced anisotropic features, thereby resulting in various cell shapes. Overall, our findings provided valuable insights into the precise spatial arrangement and cellular behavior of multicellular organisms, thus enhancing our understanding of the complex processes underlying plant growth and differentiation.

Overview of the Caucasian Perla Geoffroy, 1762 (Plecoptera: Perlidae) based on morphological and molecular data with description of two new species

Six species of Caucasian Perla are reviewed, and diagnostic morphological characteristics of all stages of development (where possible) are described, supplemented, and illustrated in detail with comparative light microscope and scanning electron microscopy images. The DNA barcoding of five species is presented. Two new morphologically and genetically distinct species, Perla schapsugica sp. nov. and Perla palatovi sp. nov., are described for both sexes and all life stages in the North Caucasus, Russia, Krasnodar Kray. Reinstatement of Perla persica Zwick, 1975, as a valid species distinct from P. caucasica Guérin-Méneville, 1843, is proposed. A new record of P. persica is reported for the Greater Caucasus, Russia, North-Ossetia-Alania for the first time. Morphologically, these two latter species can be separated in male adults by the shape of the hemitergal hook on terga X, an additional ventral brush on the penis of P. caucasica, wing length, and color.

SynBot is an open-source image analysis software for automated quantification of synapses

The formation of precise numbers of neuronal connections, known as synapses, is crucial for brain function. Therefore, synaptogenesis mechanisms have been one of the main focuses of neuroscience. Immunohistochemistry is a common tool for visualizing synapses. Thus, quantifying the numbers of synapses from light microscopy images enables screening the impacts of experimental manipulations on synapse development. Despite its utility, this approach is paired with low-throughput analysis methods that are challenging to learn, and the results are variable between experimenters, especially when analyzing noisy images of brain tissue. We developed an open-source ImageJ-based software, SynBot, to address these technical bottlenecks by automating the analysis. SynBot incorporates the advanced algorithms ilastik and SynQuant for accurate thresholding for synaptic puncta identification, and the code can easily be modified by users. The use of this software will allow for rapid and reproducible screening of synaptic phenotypes in healthy and diseased nervous systems.

Biological control of ultra-skeleton mineralization in coral

Abstract Understanding the mineralization of coral is significant for the formation of coral reefs and paleoclimatic reconstructions. However, the fundamental mechanisms involved in biomineralization are poorly understood. A combination of Raman spectral and cross-polarized reflected light microscopy imaging was used to examine the three-dimensional spatial distribution of the skeletal ultrastructures and their associated mineral, organic, and water chemistry in coral, which enable insight into the spatial growth features of the ultrastructures and possible formation processes. A possible mechanism is proposed that controls the formation of skeletal ultrastructures, which likely involves compartmentalized calcifying cells and their related cellular activities. This could clarify the association between coral skeletal mineralization and biology, and it may be beneficial to better protection and application of coral reefs.

Shape memory and mechanical properties of ESO modified epoxy/polyurethane semi-interpenetrating polymer networks for smart plaster

While numerous studies have explored the potential of shape memory materials in medical applications, the current research output is not enough for significant productivity in industrial products. In this research, a type of shape memory orthopedic plaster was fabricated by using epoxy/polyurethane interpenetrating networks modified with epoxidized soybean oil (ESO) and then compared with some commercial plasters. Polymer networks that were produced could be tailored to have glass transition temperatures (Tgs) within the range of 70–90 °C by changing the percentage composition of polyurethane and soybean oil. Shape recovery and fixity ratios in all samples were above 95 and 97 %. DMA results showed that IPNs with variable amount of soybean oil had lower glass transition temperature (Tg) and cross-link densities compared to IPNs without soybean oil. Based on the tensile test results, most samples exhibited an elastic modulus in the range of 280–400MPa, a level deemed somewhat acceptable when compared to commercial samples. Light microscope images had shown that the increase of polyurethane led to the phase separation of polymers, which was improved by the addition of soybean oil.

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.

Validation of histopathological chronicity scores in native kidney biopsies using light microscopy and digital morphometry for predicting renal outcome

Quantitative assessment of chronicity changes in native kidney biopsies offer valuable insights in to disease prognosis, despite the strength of qualitative information. Yet, standardization and reproducibility remain challenging. The present study aims to assess and compare the prognostic utility and reproducibility of two chronicity scoring systems based on light microscopy and whole slide imaging with morphometry and also to evaluate the prognostic utility of structural measurements: cortical non-sclerotic glomerular (NSG) density and NSG area/volume. We designed a retrospective longitudinal study involving 101 adult and paediatric patients who underwent native kidney biopsies. Chronicity scoring was performed using two semi-quantitative methods: Method 1 (method proposed in PMID: 28314581) and Method 2 (method proposed in PMID: 32516862), under light microscopy as well as on whole-slide scanned images, and assessed for prognostic utility. Kidney-Failure-Risk-Equation (KFRE) was employed in combination with chronicity-scoring-methods and assessed for predictive capability. Interobserver reproducibility for the two chronicity methods was studied among three renal pathologists. Structural measurements were performed on whole-slide- scanned-images. Both the chronicity scoring methods significantly predicted decline in estimated glomerular filtration rate (eGFR) and persistent need for renal replacement therapy in follow-up. Method 1 combined with KFRE, outperformed Method 2 in predicting renal survival. Method 2 however showed higher interobserver reproducibility. Combined KFRE plus histopathological scoring methods showed better predictive accuracy. The study validates the precision of chronicity scoring using whole slide scanned images. The morphometric structural measurements showed significant correlations with follow-up eGFR, thereby providing supplementary prognostic information.

TiO2-ZnPc nanoparticles functionalized with folic acid as a target photosensitizer for photodynamic therapy against glioblastoma cells

The use of TiO2 as a photosensitizer in photodynamic therapy is limited due to TiO2 generates reactive oxygen species only under UV irradiation. The TiO2 surface has been modified with different functional groups to achieve activation at longer wavelengths (visible light). This work reports the synthesis, characterization, and biological toxicity assay of TiO2 nanoparticles functionalized with folic acid and combined with a zinc phthalocyanine to obtain a nano-photosensitizer for its application in photodynamic therapy for glioblastoma cancer treatment. The nano-photosensitizer was prepared using the sol-gel method. Folic acid and zinc phthalocyanine were added during the hydrolysis and condensation of titanium butoxide, which was the TiO2 precursor. The samples obtained were characterized by several microscopy and spectroscopy techniques. An in vitro toxicity test was performed using the MTT assay and the C6 cellular line. The results of the characterization showed that the structure of the nanoparticles corresponds mainly to the anatase phase. Successful functionalization with folic acid and an excellent combination with phthalocyanine was also achieved. Both folic acid-functionalized TiO2 and phthalocyanine-functionalized TiO2 had no cytotoxic effect on C6 cells (even at high concentrations) in comparison to Cis-Pt, which was very toxic to C6 cells. The materials behaved similarly to the control (untreated cells). The cell viability and light microscopy images suggest that both materials could be considered biocompatible and mildly phototoxic in these cells when activated by light.Graphical

Deep Learning‐Based Classification of Histone–DNA Interactions Using Drying Droplet Patterns

Developing scalable and accurate predictive analytical methods for the classification of protein‐DNA binding is critical for advancing our understanding of molecular biology, disease mechanisms, and a wide spectrum of biotechnological and medical applications. It is discovered that histone–DNA interactions can be stratified based on stain patterns created by the deposition of various nucleoprotein solutions onto a substrate. In this study, a deep‐learning neural network is applied to categorize polarized light microscopy images of drying droplet deposits originating from different histone–DNA mixtures. These DNA stain patterns featured high reproducibility across different species and thus enabled comprehensive DNA categorization (100% accuracy) and accurate prediction of their respective binding affinities to histones. Eukaryotic DNA, which has a higher binding affinity to mammalian histones than prokaryotic DNA, is associated with a higher overall prediction accuracy. For a given species, the average prediction accuracy increased with DNA size. To demonstrate generalizability, a pre‐trained CNN is challenged with unknown images that originated from DNA samples of species not included in the training set. The CNN classified these unknown histone‐DNA samples as either strong or medium binders with 84.4% and 96.25% accuracy, respectively.

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.

Scale Characteristics of Six Fish Species of the Genus Cyprinion (Teleostei: Cypriniformes): A Microscopic Analysis.

This study investigated the morphological characteristics of scales in six Cyprinion species, using light and scanning electron microscopy focusing on key features such as scale type, key scales, lateral line scales, radius/radii, rostral margin, focus, circuli, lepidonts, tubercles, and scale indices. The research analyzed the scales using ultramicroscopy and light microscopy imaging, categorizing them based on size classes and body regions. The morphological variations in scale characteristics were examined across different species, regions, and size classes. Notable findings included the tetra-sectioned form of scales, representing a unique characteristic of the Cyprinion genus. Morphological changes in scale features were observed with fish growth, particularly in the overall shape, focus shape, and size. Quantitative analysis revealed variations in average relative scale length and width among different species, regions, and size classes. The study utilized canonical discriminant analysis for multivariate assessment, classifying the species into distinct groups based on morphometric indices. The findings contribute to the understanding of scale morphology in Cyprinion species and exploring morphological variation between the examined species.