Light Microscopy Images Research Articles

Composite inclusion complexes containing sodium alginate composite nanogels for pH-responsive valnemulin hydrochloride release

To increase the sustained and controlled release of valnemulin hydrochloride to the infected site of Staphylococcus aureus small colony variants (SCVs), pH-responsive valnemulin hydrochloride-loaded sodium alginate (SA) composite nanogels were firstly explored. The valnemulin hydrochloride was incorporated into the β-Cyclodextrins (CD) to form valnemulin hydrochloride-β-CD-inclusion complexes (IC) and IC might be taken up into SA hydrogels to form valnemulin hydrochloride-loaded SA composite nanogels. The formulation screening, characteristics, in vitro release, antibacterial activity, therapeutic effect, and biosafety of the valnemulin hydrochloride composite nanogels were performed. The optimized formula was made up of 10 mg/mL (SA) and 0.5 mg/mL (Ca2+). The mean particle diameter, polydispersity index, zeta potential, encapsulation efficiency, and loading capacity of the optimized formulation were 229.4±1.5 nm, 0.34±0.03, -33.2±2.2 mv, 84.3%±4.1%, and 8.5%±1.2%, respectively. Light microscope and scanning electron microscope images revealed that a cross-linked polymeric network was successfully prepared. The fourier transform infrared and powder X-ray diffraction showed that valnemulin hydrochloride composite nanogels were successfully formulated by complexation and ionic crosslinking. The valnemulin hydrochloride composite nanogels exhibited a biphasic pattern with sustained release of 61.4%±1.8% after 48 h at pH 5.5. The valnemulin hydrochloride composite nanogels might be used as a biocompatible preparation because of their non-toxic effect on the renal and liver. This study provides promising valnemulin hydrochloride composite nanogels to improve the treatment of the SCVs infection.

Rapid, automated nerve histomorphometry through open-source artificial intelligence

We aimed to develop and validate a deep learning model for automated segmentation and histomorphometry of myelinated peripheral nerve fibers from light microscopic images. A convolutional neural network integrated in the AxonDeepSeg framework was trained for automated axon/myelin segmentation using a dataset of light-microscopic cross-sectional images of osmium tetroxide-stained rat nerves including various axonal regeneration stages. In a second dataset, accuracy of automated segmentation was determined against manual axon/myelin labels. Automated morphometry results, including axon diameter, myelin sheath thickness and g-ratio were compared against manual straight-line measurements and morphometrics extracted from manual labels with AxonDeepSeg as a reference standard. The neural network achieved high pixel-wise accuracy for nerve fiber segmentations with a mean (± standard deviation) ground truth overlap of 0.93 (± 0.03) for axons and 0.99 (± 0.01) for myelin sheaths, respectively. Nerve fibers were identified with a sensitivity of 0.99 and a precision of 0.97. For each nerve fiber, the myelin thickness, axon diameter, g-ratio, solidity, eccentricity, orientation, and individual x -and y-coordinates were determined automatically. Compared to manual morphometry, automated histomorphometry showed superior agreement with the reference standard while reducing the analysis time to below 2.5% of the time needed for manual morphometry. This open-source convolutional neural network provides rapid and accurate morphometry of entire peripheral nerve cross-sections. Given its easy applicability, it could contribute to significant time savings in biomedical research while extracting unprecedented amounts of objective morphologic information from large image datasets.

Advances in studying whole mouse brain vasculature using high-resolution 3D light microscopy imaging.

.SignificanceThe cerebrovasculature has become increasingly recognized as a major player in overall brain health and many brain disorders. Although there have been several landmark studies to understand details of these crucially important structures in an anatomically defined area, brain-wide examination of the whole cerebrovasculature, including microvessels, has been challenging. However, emerging techniques, including tissue processing and three-dimensional (3D) microscopy imaging, enable neuroscientists to examine the total vasculature in the entire mouse brain.AimHere, we aim to highlight advances in these high-resolution 3D mapping methods including block-face imaging and light sheet fluorescent microscopy.ApproachWe summarize latest mapping tools to understand detailed anatomical arrangement of the cerebrovascular network and the organizing principles of the neurovascular unit (NVU) as a whole. ResultsWe discuss biological insights gained from studies using these imaging methods and how these tools can be used to advance our understanding of the cerebrovascular network and related cell types in the entire brain.ConclusionsThis review article will help to understand recent advance in high-resolution NVU mapping in mice and provide perspective on future studies.

Light and scanning electron microscopic imaging of leaf transverse sections of indigenous fodder grasses of central Punjab, Pakistan.

The significance of grasses as fodder is enormous. However, it has been observed that different fodder grasses are identified locally with the similar vernacular names. Even same species recognized with different names. This happened due to their reduced and overlapped morphological characters. Hence comprehensive taxonomic keys were required to provide authentic identification of this grass flora. Current study established microscopic identification tools on the basis of leaf transverse anatomical features. Results demonstrated a great diversity in ribs and furrows arrangement among all studied grass members. Lolium temulentum, Phalaris minor, Poa annua, Arundo donax, Aristida adscensionis, Desmostachya bipinnata, Cenchrus biflorus and Digitaria ciliaris possessed ribs and furrows only at adaxial surfaces whereas all other species possessed this feature abaxially. Generally all the species contain three types of vascular bundles i.e. small, medium and large. Therefore, this qualitative feature is valuable only at family level, however, it found insignificant in taxa identification. Diversity was observed in bundle sheath type which could be taxonomically significant. Generally bundle sheath was found complete in most of species but in Bromus japonicus Thunb., Dactylis glomerata L., Acrachne racemosa (Heyne ex Roth) Ohwi, Chrysopogon aucheri (Boiss.) Stapf, Dichanthium annulatum (Forssk.) Stapf, Heteropogon contortus (L.) P Beauv. Ex. Roem & Schult. Eragrostis minor Host. and in Agrostis gigantea Roth. had incomplete type of sheath. Results reported great diversity in characters which could be taxonomically viable. RESEARCH HIGHLIGHTS: This research highlighted the significance of microscopic examination of leaf transverse anatomical features in field of taxonomy. Established anatomical tools would be of great importance for the identification of studied species.

On the Peristomes of the Corticolous African Species of Fissidens Hedw. (Fissidentaceae, Bryophyta)

The peristomes of 18 corticolous African Fissidens Hedw. species are described in detail and illustrated with light microscope images. Seventeen belong to F. subgen. Polypodiopsis sect. Antennidens and one to F. subgen. Neoamblyothallia. Most of these peristomes are imperfect. Compared to their presumed ancestral types, they are shorter, narrower, stiffer, undivided, deeply divided or irregularly divided, and in having a reduced hygroscopic capacity. Other sporophytic traits frequently found together with these anomalous peristomes are: narrow, cylindrical capsules with narrow, oblong exothecial cells; short and often papillose setae; and short opercula. Based on literature a strong relation is demonstrated between a corticolous life-style and anomalous peristomes for F. subgen. Polypodiopsis and F. subgen. Neoamblyothallia, whereas in the type subgenus anomalous peristomes are often connected with an aquatic habitat.

Improved Fluorescent Proteins for Dual-Colour Post-Embedding CLEM.

Post-embedding correlative light and electron microscopy (CLEM) has the advantage of high-precision registration and enables light and electron microscopy imaging of the same slice. However, its broad application has been hampered by the limited available fluorescent proteins (FPs) and a low signal-to-background ratio (SBR). Here, we developed a green photoswitchable FP, mEosEM-E with substantially high on/off contrast in EM samples embedded in Epon resin, which maximally preserves cellular structures but quenches the fluorescence of FPs. Taking advantage of the photoswitching property of mEosEM-E, the autofluorescence background from the resin was significantly reduced by a subtraction-based CLEM (sCLEM) method. Meanwhile, we identified a red fluorescent protein (RFP) mScarlet-H that exhibited higher brightness and SBR in resin than previously reported RFPs. With mEosEM-E and mScarlet-H, dual-colour post-Epon-embedding CLEM images with high SBR and no cross-talk signal were successfully performed to reveal the organization of nucleolar proteins. Moreover, a dissection of the influences of different EM sample preparation steps on the fluorescence preservation for several RFPs provides useful guidance for further probe development.

Automatic Recognition of Ragged Red Fibers in Muscle Biopsy from Patients with Mitochondrial Disorders

Mitochondrial dysfunction is considered to be a major cause of primary mitochondrial myopathy in children and adults, as reduced mitochondrial respiration and morphological changes such as ragged red fibers (RRFs) are observed in muscle biopsies. However, it is also possible to hypothesize the role of mitochondrial dysfunction in aging muscle or in secondary mitochondrial dysfunctions. The recognition of true histological patterns of mitochondrial myopathy can avoid unnecessary genetic investigations. The aim of our study was to develop and validate machine-learning methods for RRF detection in light microscopy images of skeletal muscle tissue. We used image sets of 489 color images captured from representative areas of Gomori’s trichrome-stained tissue retrieved from light microscopy images at a 20× magnification. We compared the performance of random forest, gradient boosting machine, and support vector machine classifiers. Our results suggested that the advent of scanning technologies, combined with the development of machine-learning models for image classification, make neuromuscular disorders’ automated diagnostic systems a concrete possibility.

DICOMization of Proprietary Files Obtained from Confocal, Whole-Slide, and FIB-SEM Microscope Scanners.

The evolution of biomedical imaging technology is allowing the digitization of hundreds of glass slides at once. There are multiple microscope scanners available in the market including low-cost solutions that can serve small centers. Moreover, new technology is being researched to acquire images and new modalities are appearing in the market such as electron microscopy. This reality offers new diagnostics tools to clinical practice but emphasizes also the lack of multivendor system’s interoperability. Without the adoption of standard data formats and communications methods, it will be impossible to build this industry through the installation of vendor-neutral archives and the establishment of telepathology services in the cloud. The DICOM protocol is a feasible solution to the aforementioned problem because it already provides an interface for visible light and whole slide microscope imaging modalities. While some scanners currently have DICOM interfaces, the vast majority of manufacturers continue to use proprietary solutions. This article proposes an automated DICOMization pipeline that can efficiently transform distinct proprietary microscope images from CLSM, FIB-SEM, and WSI scanners into standard DICOM with their biological information maintained within their metadata. The system feasibility and performance were evaluated with fifteen distinct proprietary modalities, including stacked WSI samples. The results demonstrated that the proposed methodology is accurate and can be used in production. The normalized objects were stored through the standard communications in the Dicoogle open-source archive.

A Deep Learning-Based Workflow for Dendritic Spine Segmentation.

The morphological analysis of dendritic spines is an important challenge for the neuroscientific community. Most state-of-the-art techniques rely on user-supervised algorithms to segment the spine surface, especially those designed for light microscopy images. Therefore, processing large dendritic branches is costly and time-consuming. Although deep learning (DL) models have become one of the most commonly used tools in image segmentation, they have not yet been successfully applied to this problem. In this article, we study the feasibility of using DL models to automatize spine segmentation from confocal microscopy images. Supervised learning is the most frequently used method for training DL models. This approach requires large data sets of high-quality segmented images (ground truth). As mentioned above, the segmentation of microscopy images is time-consuming and, therefore, in most cases, neuroanatomists only reconstruct relevant branches of the stack. Additionally, some parts of the dendritic shaft and spines are not segmented due to dyeing problems. In the context of this research, we tested the most successful architectures in the DL biomedical segmentation field. To build the ground truth, we used a large and high-quality data set, according to standards in the field. Nevertheless, this data set is not sufficient to train convolutional neural networks for accurate reconstructions. Therefore, we implemented an automatic preprocessing step and several training strategies to deal with the problems mentioned above. As shown by our results, our system produces a high-quality segmentation in most cases. Finally, we integrated several postprocessing user-supervised algorithms in a graphical user interface application to correct any possible artifacts.

Light Microscopy of Medium-Density Rigid Polyurethane Foams Filled with Nanoclay.

Practical applications and mathematical modelling of the physical and mechanical properties of medium-density rigid polyurethane foams require knowledge of their structure. It is necessary to determine structural characteristics without destroying the foams and measuring each element. A methodology is described for the use of light microscopy on environmentally sustainable, medium-density rigid polyurethane foams (in the density region of ≈210–230 kg/m3), by the analysis of two types of light microscopy images: (1) Cutting surface images; and (2) Through-cutting surface images. The dimensions of structural elements of polyurethane foams, filled with the nanoclay Cloisite-30B at concentrations of 0.0%, 0.25%, 0.50%, 1.0%, 2.0%, 3.0%, and 5.0% from the mass of the filled reacting mixture, are estimated. Probability density functions of projections of bubbles’ diameters and struts’ length are determined using images in three mutually perpendicular planes. A mathematical model is developed for the restoration of the actual dimensions of bubbles’ diameters using data of cutting circles’ diameters. Intercalation and exfoliation of the filler’s Cloisite-30B mono-layers is evaluated via the basal spacing by X-ray diffraction at a 5 wt.% concentration of nanoclay.

3D characterization of the complex vascular bundle system of Hakea fruits based on X-ray microtomography (µCT) for a better understanding of the opening mechanism

Fruits (follicles) of Hakea salicifolia and Hakea sericea (Proteaceae) are characterised by pronounced lignification and open via a ventral suture and the dorsal side. The opening along both sides is unique within the Proteaceae. Both serotinous species are obligate seeders, whose spreading benefits from bush fire events. The different tissues and the course of the vascular bundles must allow the opening mechanism. While their 2D-arrangements are known to some extent from light-microscopy images of cross-sections, this work presents their three-dimensional structures and discusses their contribution to the opening of Hakea fruits. For this purpose, 3D greyscale images, reconstructed from µCT-projection data of both fruits are segmented, assisted by a deep learning algorithm (AI algorithm). 3D renderings from these segmentations show strongly interconnected vascular bundles that build a double-dome shaped network in each valve of H. salicifolia and a dome shaped honeycomb-structure in each valve of H. sericea. However, the vascular bundles of both species show no interconnection between the two lateral valves of the fruit but leave gaps for predetermined fracture tissues on the ventral and dorsal side. The opening of the fruits after a fire or after separation from the mother plant can be explained by the anisotropic shrinkage in the two valves of the fruit.

Atorvastatin Inhibits Viability and Migration of MCF7 Breast Cancer Cells.

Objective:Atorvastatin is commonly used as a lipid lowering drug. The emerging interest in statins as anticancer agents is based on their pleiotropic effects on cancer cells. Among the statins, atorvastatin, and in cancers, breast malignancies have received less attention in preclinical investigations. In order to enhance the efficacy of cancer treatment, adjuvant, less expensive therapeutic strategies have been recently noticed. In this case, we investigated the in-vitro effect of atorvastatin on viability and migration of MCF7 breast cancer cell line. Methods:We tested the cytotoxicity of atorvastatin on breast cancer cells survival by MTT assay. Annexin-V / PI staining and then flow cytometry of cancer cells in addition to quantitative real-time PCR tests quantified the apoptosis and necrosis of cancer cells. We figured out the impact of atorvastatin on cancer cell migration capability through scratch-wound healing assay and transwell migration examination. Inverted light microscope and fluorescent imaging displayed the morphological changes following treatment of MCF7 cells with atorvastatin.Result:We resulted that atorvastatin can trigger MCF7 cancer cells to undergo necrosis and caspase-dependent apoptosis based on the viable/dead cell number, mitotic cell cycle, gene expression, and morphological assays. The results were dose- and time-dependent and the half- maximal inhibitory concentration of atorvastatin for cancer cells’ viability inhibition was 9.1 μM/L(nM/mL). Moreover, the migration of MCF7 cells were inhibited in the treated group as we figured out in two- and three-dimensional migration methods. Conclusion:In-vitro inspection of drug-cancer cell interactions paves the way for future in-vivo research studies. These in-vitro results revealed that atorvastatin has anti-viability and anti-migration effects on breast cancer cells.

Revisiting Potter Cove, King George Island, Antarctica, 12 years later: new observations of marine benthic diatoms

Abstract Continuous observations of Antarctic benthic diatoms are necessary to detect changes in species composition and biodiversity that may result from environmental changes. The present work provides a systematic list of benthic diatoms from Potter Cove (62.03°S 58.35°W) collected during summer 2015. The new findings are compared with observations made 12 years ago (summer 2003). In total, 80 taxa were found, similar to that encountered earlier, but 17 species (21% of the total species number) were not observed in 2003, and 26 species (31%) of those reported in 2003 did not appear in 2015. The dominant species in 2003 and 2015 was the large epipelic and cosmopolitan Gyrosigma fasciola. Most of the species either newly observed in the present study, or absent in the present study but observed previously, occurred either rarely or very rarely. Gyrosigma arcuatum and Pleurosigma diversestriatum were newly observed in 2015 and not previously reported from polar regions. This study provides systematic and ecological information on all taxa encountered and is illustrated with light microscopy and scanning electron microscopy (SEM) images. We emphasize that ice-inhabiting and other benthic diatoms should be included when considering the biodiversity of polar diatoms. Thus, we hope that the present study will add a piece to the puzzle of climate change effects on the benthic diatoms in this vulnerable region and will complement earlier published species records in the area.

Computer-aided characterization of ferroelectric phase transitions and domain structures using polarizing light microscopy

Optical microscopy is an essential characterization technique that is used as an initial step in the study of domain structures and phase transitions of ferroelectric materials. Image analysis aided by advanced computer algorithms is urgently required to enable an improved accuracy and higher efficiency of data processing. In this study, an automatic full-angle light intensity detection algorithm is developed to study polarizing light microscopy images of domain structures and the phase evolution process is decoded using the scale invariant feature transform method. This data processing algorithm is then used to characterize the phase transition process in relaxor ferroelectric PMN-0.29PT single crystals with complex domain structures. An emerging in-plane tetragonal phase is tracked during the rhombohedral (R) to tetragonal (T) phase transition, which is associated with peaks present in the temperature-dependent dielectric curve. Compared to the traditional polarizing light microscopy characterization method, this computer-aided algorithm enables a quicker and more informative analysis of domain evolutions in ferroelectric single crystals.

Acucapito hainanensis sp. nov., the first record of the family Acucapitidae Wiles, 1996 (Acari, Hydrachnidia) from China

We describe a new species Acucapito hainanensis Li and Guo sp. nov. from Hainan Province, P. R. China. In addition to being the first record of the family Acucapitidae Wiles, 1996 for Chinese water mite fauna, it is only the third species of this rare family in the world. Subtle structures are illustrated in detail with line drawings, light microscope images and scanning electron micrographs.

Surface-Engineered Gold Nanoclusters for Stimulated Emission Depletion and Correlated Light and Electron Microscopy Imaging.

Stimulated emission depletion (STED) nanoscopy is an emerging super-resolution imaging platform for the study of the cellular structure. Developing suitable fluorescent probes of small size, good photostability, and easy functionalization is still in demand. Herein, we introduce a new type of surface-engineered gold nanoclusters (Au NCs) that are ultrasmall (1.7 nm) and ultrabright (QY = 60%) for STED bioimaging. A rigid shell formed by l-arginine (l-Arg) and 6-aza-2-thiothymine (ATT) on the Au NC surface enables not only its strong fluorescence in aqueous solution but also its easy chemical modification for specific biomolecule labeling. Au NCs show remarkable performance as STED nanoprobes, including high depletion efficiency, good photobleaching resistance, and low saturation intensity. Super-resolution imaging has been achieved with these Au NCs, and targeted nanoscopic imaging of cellular tubulin has been demonstrated. Moreover, the circular structure of lysosomes in live cells has been revealed. As a Au NC is also an ideal probe for electron microscopy, dual imaging of Aβ42 aggregates with the single labeling probe of Au NCs has been realized in correlative light and electron microscopy (CLEM). This work reports, for the first time, the application of Au NCs as a novel probe in STED and CLEM imaging. With their excellent properties, Au NCs show promising potential for nanoscale bioimaging.

Character analysis and descriptions of Eocene sphodrine fossils (Coleoptera, Carabidae) using light microscopy, micro-CT scanning, and 3D imaging

Of the 12 specimens of Calathus-like sphodrine beetles presently known from Baltic and Rovno amber deposits, 11 specimens were investigated using light microscopy, micro-CT scanning, and 3D imaging techniques. For the first time, many significant diagnostic characters of the external morphology and male and female genitalia of Eocene Sphodrini were studied in detail. Based on these data, three fossil species are diagnosed and placed in a natural group characterized by a derived pattern in elytral chaetotaxy and microsculpture and therefore the genus Quasicalathus Schmidt & Will, gen. nov. is described to comprise these species. Due to the presence of a styloid right paramere, Quasicalathus gen. nov. is considered a member of the sphodrine “P clade” of Ruiz et al. (2009). However, given the absence of synapomorphies of any species group of the P clade, the systematic position of Quasicalathus gen. nov. within this clade remains unresolved. The Baltic amber species Calathus elpis Ortuño & Arillo, 2009 is redescribed based on additional, fossil, non-holotype material and transferred to Quasicalathus gen. nov. Identification of the additional C. elpis fossil material remains slightly uncertain due to the non-availability of the holotype for direct comparison coupled with doubts regarding the accuracy of certain character states presented in its original description. Two species are newly described: Quasicalathus agonicollis Schmidt & Will, sp. nov., from Baltic amber, and Q. conservans Schmidt & Will, sp. nov., from Rovno amber.

Euro-BioImaging – Interdisciplinary research infrastructure bringing together communities and imaging facilities to support excellent research

Euro-BioImaging – Interdisciplinary research infrastructure bringing together communities and imaging facilities to support excellent research

Ultrastructural imaging reveals vascular remodeling in migraine patients.

Migraine is a neurological disorder and one of the most common pain conditions worldwide. Despite its prevalence, the basic biology and underlying mechanisms contributing to the development of migraine are still poorly understood. It is still unclear, for instance, whether the vasculature, both extra and intracranial, plays a significant role in the generation of migraine pain. Neuroimaging data, indeed, have reported conflicting results on blood vessels abnormalities like vasodilation, while functional studies suggest that vessels dysfunction may extend beyond vasodilation. Here we combined light and electron microscopy imaging to investigate the fine structure of superficial temporal (STA) and occipital arteries (OA) from patients that underwent minimally invasive surgery for migraine. Using optical microscopy, we observed that both STA and OA vessels showed marked endothelial thickening and internal elastic lamina fragmentation. In the muscular layer, we found profound shape changes of vascular smooth muscle cells (VSMCs), abundant extracellular matrix, and the presence of clear extracellular vacuoles. The electron microscopy analysis confirmed putative VSMCs infiltrated within the intima layer and revealed a consistent shifting of VSMCs from contractile to a synthetically active phenotype. We also report the presence of (i) abundant extracellular vacuoles filled with fine granular material and membranes, (ii) multilamellar structures, (iii) endosome-like organelles, and (iv) bona fide extracellular vesicles in the matrix space surrounding synthetically active cells. As both the endothelial layer and VSMCs coordinate a variety of vascular functions, these results suggest that a significant vascular remodeling is occurring in STA and OA of migraine patients. Thus, this phenomenon may represent an important target for future investigation designed toward the development of new therapeutic approaches.

The Role of Phase Migration of Carbon Nanotubes in Melt-Mixed PVDF/PE Polymer Blends for High Conductivity and EMI Shielding Applications.

In this work, the effects of blend ratio and mixing time on the migration of multi-walled carbon nanotubes (MWCNTs) within poly(vinylidene fluoride) (PVDF)/polyethylene (PE) blends are studied. A novel two-step mixing approach was used to pre-localize MWCNTs within the PE phase, and subsequently allow them to migrate into the thermodynamically favored PVDF phase. Light microscopy images confirm that MWCNTs migrate from PE to PVDF, and transmission electron microscopy (TEM) images show individual MWCNTs migrating fully into PVDF, while agglomerates remained trapped at the PVDF/PE interface. PVDF:PE 50:50 and 20:80 polymer blend nanocomposites with 2 vol% MWCNTs exhibit exceptional electromagnetic interference shielding effectiveness (EMI SE) at 10 min of mixing (13 and 16 dB, respectively-at a thickness of 0.45 mm), when compared to 30 s of mixing (11 and 12 dB, respectively), suggesting the formation of more interconnected MWCNT networks over time. TEM images show that these improved microstructures are concentrated on the PE side of the PVDF/PE interface. A modified version of the “Slim-Fast-Mechanism” is proposed to explain the migration behavior of MWCNTs within the PVDF/PE blend. In this theory, MWCNTs approaching perpendicular to the interface penetrate the PVDF/PE interface, while those approaching in parallel or as MWCNT agglomerates remain trapped. Trapped MWCNTs act as barriers to additional MWCNTs, regardless of geometry. This mechanism is verified via TEM and scanning electron microscopy and suggests the feasibility of localizing MWCNTs at the interface of PVDF/PE blends.