The Feulgen reaction at the electron microscopy level.

The Feulgen reaction has been the first specific method for detecting DNA available at light microscopy since 1924. However, a similar specific method was proposed for electron microscopy only 50 years later. Here, we discuss the problems encountered in finding the electrondense reagent capable of taking advantage of the extremely high resolution offered by electron microscopy as well as some applications of the method.

Electron videography of a lipid-protein tango.

Biological phenomena, from enzymatic catalysis to synaptic transmission, originate in the structural transformations of biomolecules and biomolecular assemblies in liquid water. However, directly imaging these nanoscopic dynamics without probes or labels has been a fundamental methodological challenge. Here, we developed an approach for "electron videography"-combining liquid phase electron microscopy with molecular modeling-with which we filmed the nanoscale structural fluctuations of individual, suspended, and unlabeled membrane protein nanodiscs in liquid. Systematic comparisons with biochemical data and simulation indicate the graphene encapsulation involved can afford sufficiently reduced effects of the illuminating electron beam for these observations to yield quantitative fingerprints of nanoscale lipid-protein interactions. Our results suggest that lipid-protein interactions delineate dynamically modified membrane domains across unexpectedly long ranges. Moreover, they contribute to the molecular mechanics of the nanodisc as a whole in a manner specific to the protein within. Overall, this work illustrates an experimental approach to film, quantify, and understand biomolecular dynamics at the nanometer scale.

Safari with an Electron Gun: Visualization of Protein and Membrane Interactions in Mitochondria in Natural Environment.

This paper presents new structural data about mitochondria using correlative light and electron microscopy (CLEM) and cryo-electron tomography. These state-of-the-art structural biology methods allow studying biological objects at nanometer scales under natural conditions. Non-invasiveness of these methods makes them comparable to observing animals in their natural environment on a safari. The paper highlights two areas of research that can only be accomplished using these methods. The study visualized location of the Aß42 amyloid aggregates in relation to mitochondria to test a hypothesis of development of mitochondrial dysfunction in Alzheimer's disease. The results showed that the Aß42 aggregates do not interact with mitochondria, although some of them are closely located. Therefore, the study demonstrated that mitochondrial dysfunction is not directly associated with the effects of aggregates on mitochondrial structure. Other processes should be considered as sources of mitochondrial dysfunction. Second unique area presented in this work is high-resolution visualization of the mitochondrial membranes and proteins in them. Analysis of the cryo-ET data reveals toroidal holes in the lamellar structures of cardiac mitochondrial cristae, where ATP synthases are located. The study proposes a new mechanism for sorting and clustering protein complexes in the membrane based on topology. According to this suggestion, position of the OXPHOS system proteins in the membrane is determined by its curvature. High-resolution tomography expands and complements existing ideas about the structural and functional organization of mitochondria. This makes it possible to study the previously inaccessible structural interactions of proteins with each other and with membranes in vivo.

In situ captured antibacterial action of membrane-incising peptide lamellae.

Developing unique mechanisms of action are essential to combat the growing issue of antimicrobial resistance. Supramolecular assemblies combining the improved biostability of non-natural compounds with the complex membrane-attacking mechanisms of natural peptides are promising alternatives to conventional antibiotics. However, for such compounds the direct visual insight on antibacterial action is still lacking. Here we employ a design strategy focusing on an inducible assembly mechanism and utilized electron microscopy (EM) to follow the formation of supramolecular structures of lysine-rich heterochiral ß3-peptides, termed lamellin-2K and lamellin-3K, triggered by bacterial cell surface lipopolysaccharides. Combined molecular dynamics simulations, EM and bacterial assays confirmed that the phosphate-induced conformational change on these lamellins led to the formation of striped lamellae capable of incising the cell envelope of Gram-negative bacteria thereby exerting antibacterial activity. Our findings also provide a mechanistic link for membrane-targeting agents depicting the antibiotic mechanism derived from the in-situ formation of active supramolecules.

Subcellular pathways through VGluT3-expressing mouse amacrine cells provide locally tuned object-motion-selective signals in the retina.

VGluT3-expressing mouse retinal amacrine cells (VG3s) respond to small-object motion and connect to multiple types of bipolar cells (inputs) and retinal ganglion cells (RGCs, outputs). Because these input and output connections are intermixed on the same dendrites, making sense of VG3 circuitry requires comparing the distribution of synapses across their arbors to the subcellular flow of signals. Here, we combine subcellular calcium imaging and electron microscopic connectomic reconstruction to analyze how VG3s integrate and transmit visual information. VG3s receive inputs from all nearby bipolar cell types but exhibit a strong preference for the fast type 3a bipolar cells. By comparing input distributions to VG3 dendrite responses, we show that VG3 dendrites have a short functional length constant that likely depends on inhibitory shunting. This model predicts that RGCs that extend dendrites into the middle layers of the inner plexiform encounter VG3 dendrites whose responses vary according to the local bipolar cell response type.

Nanoscale myelinogenesis image in developing brain via super-resolution nanoscopy by near-infrared emissive curcumin-BODIPY derivatives.

Understanding the intricate nanoscale architecture of neuronal myelin during central nervous system development is of utmost importance. However, current visualization methods heavily rely on electron microscopy or indirect fluorescent method, lacking direct and real-time imaging capabilities. Here, we introduce a breakthrough near-infrared emissive curcumin-BODIPY derivative (MyL-1) that enables direct visualization of myelin structure in brain tissues. The remarkable compatibility of MyL-1 with stimulated emission depletion nanoscopy allows for unprecedented super-resolution imaging of myelin ultrastructure. Through this innovative approach, we comprehensively characterize the nanoscale myelinogenesis in three dimensions over the course of brain development, spanning from infancy to adulthood in mouse models. Moreover, we investigate the correlation between myelin substances and Myelin Basic Protein (MBP), shedding light on the essential role of MBP in facilitating myelinogenesis during vertebral development. This novel material, MyL-1, opens up new avenues for studying and understanding the intricate process of myelinogenesis in a direct and non-invasive manner, paving the way for further advancements in the field of nanoscale neuroimaging.

Evolution of Virology: Science History through Milestones and Technological Advancements.

The history of virology, which is marked by transformative breakthroughs, spans microbiology, biochemistry, genetics, and molecular biology. From the development of Jenner's smallpox vaccine in 1796 to 20th-century innovations such as ultrafiltration and electron microscopy, the field of virology has undergone significant development. In 1898, Beijerinck laid the conceptual foundation for virology, marking a pivotal moment in the evolution of the discipline. Advancements in influenza A virus research in 1933 by Richard Shope furthered our understanding of respiratory pathogens. Additionally, in 1935, Stanley's determination of viruses as solid particles provided substantial progress in the field of virology. Key milestones include elucidation of reverse transcriptase by Baltimore and Temin in 1970, late 20th-century revelations linking viruses and cancer, and the discovery of HIV by Sinoussi, Montagnier, and Gallo in 1983, which has since shaped AIDS research. In the 21st century, breakthroughs such as gene technology, mRNA vaccines, and phage display tools were achieved in virology, demonstrating its potential for integration with molecular biology. The achievements of COVID-19 vaccines highlight the adaptability of virology to global health.

A semi-automatic method for extracting mitochondrial cristae characteristics from 3D focused ion beam scanning electron microscopy data.

Mitochondria are the main suppliers of energy for cells and their bioenergetic function is regulated by mitochondrial dynamics: the constant changes in mitochondria size, shape, and cristae morphology to secure cell homeostasis. Although changes in mitochondrial function are implicated in a wide range of diseases, our understanding is challenged by a lack of reliable ways to extract spatial features from the cristae, the detailed visualization of which requires electron microscopy (EM). Here, we present a semi-automatic method for the segmentation, 3D reconstruction, and shape analysis of mitochondria, cristae, and intracristal spaces based on 2D EM images of the murine hippocampus. We show that our method provides a more accurate characterization of mitochondrial ultrastructure in 3D than common 2D approaches and propose an operational index of mitochondria's internal organization. With an improved consistency of 3D shape analysis and a decrease in the workload needed for large-scale analysis, we speculate that this tool will help increase our understanding of mitochondrial dynamics in health and disease.

The HEAT repeat protein HPO-27 is a lysosome fission factor.

Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.

Blueprinting extendable nanomaterials with standardized protein blocks.

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures1. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.

GAUSS-EM, guided accumulation of ultrathin serial sections with a static magnetic field for volume electron microscopy.

Serial sectioning electron microscopy (EM) of millimeter-scale three-dimensional (3D) anatomical volumes requires the collection of thousands of ultrathin sections. Here, we report a high-throughput automated approach, GAUSS-EM (guided accumulation of ultrathin serial sections-EM), utilizing a static magnetic field to collect and densely pack thousands of sections onto individual silicon wafers. The method is capable of sectioning hundreds of microns of tissue per day at section thicknesses down to 35 nm. Relative to other automated volume EM approaches, GAUSS-EM democratizes the ability to collect large 3D EM volumes because it is simple and inexpensive to implement. We present two exemplar EM volumes of a zebrafish eye and mouse olfactory bulb collected with the method.

Electron Microscopy Methods for Phage-Based Study.

Electron microscopy (EM) techniques play a vital role in virology research including phage discovery and their identification. The use of different staining protocols based on the concept of negative staining is one of the most important steps in the EM processing. This chapter will summarize the widely used EM protocols in phage research, their advantages, and limitations. Phage-based therapy, especially recently developed nanoparticle-phage conjugates, are expected to find clinical significance in the antimicrobial resistance (AMR) epidemic. EM techniques are important to characterize these conjugates and we will also discuss the methods here.

Cryo-electron Microscopy and Molecular Modeling Methods to Characterize the Dynamics of Tau Bound to Microtubules.

The electron microscopy metainference integrative structural biology method enables the combination of cryo-electron microscopy electron density maps with molecular modeling techniques such as molecular dynamics to unveil the atomistic biomolecular structural ensemble and the error in the map data in an efficient manner. Here we illustrate the electron microscopy metainference protocol and analysis used to elucidate the atomistic structural ensemble of the microtubule-associated protein tau bound to microtubules by using state-of-the-art molecular mechanic force field and the electron density map.

Photo-Excited Dyes: Emerging Technique Against Tau Protein Aggregation.

Tau aggregates are considered a pathological hallmark of Alzheimer's disease. The screening of molecules against Tau aggregation is a novel strategy for Alzheimer's disease. The photo-excited molecules have proven to be effective as a therapeutic agent in several diseases. In recent studies, the photo-excited dyes showed an inhibitory effect on Alzheimer's disease-related Tau protein aggregation and toxicity. The present chapter deals with the effect of rose bengal on the aggregation of Tau. The in vitro studies carried out with the help of electron microscopy, ThS fluorescence, and circular dichroism suggested that RB attenuated the Tau aggregation under in vitro conditions, whereas PE-RB disaggregated the mature Tau fibrils. Photo-excited rose bengal and the classical rose bengal induced a low degree of toxicity in cells. Thus, for the treatment of Alzheimer's disease, the rose bengal could be considered a potential molecule.

Structural Plasticity of GABAergic Pallidothalamic Terminals in MPTP-Treated Parkinsonian Monkeys: A 3D Electron Microscopic Analysis.

The internal globus pallidus (GPi) is a major source of tonic GABAergic inhibition to the motor thalamus. In parkinsonism, the firing rate of GPi neurons is increased, and their pattern switches from a tonic to a burst mode, two pathophysiological changes associated with increased GABAergic pallidothalamic activity. In this study, we used high-resolution 3D electron microscopy to demonstrate that GPi terminals in the parvocellular ventral anterior nucleus (VApc) and the centromedian nucleus (CM), the two main GPi-recipient motor thalamic nuclei in monkeys, undergo significant morphometric changes in parkinsonian monkeys including (1) increased terminal volume in both nuclei; (2) increased surface area of synapses in both nuclei; (3) increased number of synapses/GPi terminals in the CM, but not VApc; and (4) increased total volume, but not number, of mitochondria/terminals in both nuclei. In contrast to GPi terminals, the ultrastructure of putative GABAergic nonpallidal terminals was not affected. Our results also revealed striking morphological differences in terminal volume, number/area of synapses, and volume/number of mitochondria between GPi terminals in VApc and CM of control monkeys. In conclusion, GABAergic pallidothalamic terminals are endowed with a high level of structural plasticity that may contribute to the development and maintenance of the abnormal increase in pallidal GABAergic outflow to the thalamus in the parkinsonian state. Furthermore, the evidence for ultrastructural differences between GPi terminals in VApc and CM suggests that morphologically distinct pallidothalamic terminals from single pallidal neurons may underlie specific physiological properties of pallidal inputs to VApc and CM in normal and diseased states.

NeuronBridge: an intuitive web application for neuronal morphology search across large data sets.

BACKGROUND: Neuroscience research in Drosophila is benefiting from large-scale connectomics efforts using electron microscopy (EM) to reveal all the neurons in a brain and their connections. To exploit this knowledge base, researchers relate a connectome's structure to neuronal function, often by studying individual neuron cell types. Vast libraries of fly driver lines expressing fluorescent reporter genes in sets of neurons have been created and imaged using confocal light microscopy (LM), enabling the targeting of neurons for experimentation. However, creating a fly line for driving gene expression within a single neuron found in an EM connectome remains a challenge, as it typically requires identifying a pair of driver lines where only the neuron of interest is expressed in both. This task and other emerging scientific workflows require finding similar neurons across large data sets imaged using different modalities. RESULTS: Here, we present NeuronBridge, a web application for easily and rapidly finding putative morphological matches between large data sets of neurons imaged using different modalities. We describe the functionality and construction of the NeuronBridge service, including its user-friendly graphical user interface (GUI), extensible data model, serverless cloud architecture, and massively parallel image search engine. CONCLUSIONS: NeuronBridge fills a critical gap in the Drosophila research workflow and is used by hundreds of neuroscience researchers around the world. We offer our software code, open APIs, and processed data sets for integration and reuse, and provide the application as a service at http://neuronbridge.janelia.org .

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants.

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Micromorphological characterization and electron microscopic study of parasitic species of the family Loranthaceae Juss.

The Loranthaceae Juss. family includes parasitic species that invade important trees such as fruit trees. In Saudi Arabia, Loranthaceae comprises four genera, which include six species that grow in the western, southwestern, and northern regions: Tapinanthus globifer (A.Rich.) Tiegh, Oncocalyx glabratus (Engl.) M. G. Gilbert, Loranthella deflersii (Tiegh.) S. Blanco & C. E. Wetze, Phragmanthera austroarabica A. G. Mill. & J. Nyberg, Plicosepalus curviflorus (Benth.ex Oliv.) Tiegh. and Plicosepalus acaciae (Zucc.). The species present in the Kingdom of Saudi Arabia have not been the subject of enough studies. This work aims to screen and evaluate the taxonomic importance of the micromorphological traits of leaves and fruits in Loranthaceae species native to Saudi Arabia (SA) using scanning electron microscope (SEM). In this study, cluster dendrogram (CD), principal component analysis (PCA) and analysis of variance (ANOVA) were used to evaluate the ability to discriminate Loranthaceae species using micromorphological characteristics. Most of the micromorphological characteristics of the leaf and fruit surfaces used reflected significant variation between the species of Loranthaceae. The type of stomata, trichome, lenticels, fine relief of the cell wall and wax form were the most taxonomically important characteristics. In addition, the cluster dendrogram of morphological characteristics showed species distribution within branches based on affiliation to subtribes Tapinanthinae and Emelianthinae. To the best of our knowledge, the fruit and leaves of the species under study have never been described using electron microscopy, and this study is considered the first of its kind. It also contributes to solving the classification problems of the family Loranthaceae in general and confirms the importance of the characteristics and methods used as tools for characterizing parasitic species that infect trees and helps to verify their identities. This will help to improve resistance efforts and put effective control plans in place.

RNA as a component of scrapie fibrils.

Recently, electron cryo-microscopy (cryo-EM) maps of fibrils from the brains of mice and hamsters with five infectious scrapie strains have been published and deposited in the electron microscopy data bank (EMDB). As noted by the primary authors, the fibrils contain a second component other than protein. The aim of the present study was to identify the nature of this second component in the published maps using an in silico approach. Extra densities (EDs) containing this component were continuous, straight, axial, at right angles to protein rungs and within hydrogen-bonding distance of protein, consistent with a structural role. EDs co-located with strips of basic residues, notably lysines, and formed a conspicuous cladding over parts of the N-terminal lobe of the protein. A Y-shaped polymer consistent with RNA was found, in places forming a single chain and at one location forming a duplex, comprising two antiparallel chains, and raising the intriguing possibility of replicative behaviour. To reflect the monotonous nature of the protein interface, it is suggested that the RNA may be a short tandem repeat. Fibrils from brains of patients with Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and other neurodegenerations also contain EDs and may be of a similar aetiology.

The assessment of neuronal plasticity following sciatic nerve injuries in rats using electron microscopy and stereological methods.

The transmission of signals to the cell body from injured axons induces significant alterations in primary sensory neurons located in the ganglion tissue, the site of the perikaryon of the affected nerve fibers. Disruption of the continuity between the proximal and distal ends leads to substantial adaptability in ganglion cells and induces macrophage-like activity in the satellite cells. Research findings have demonstrated the plasticity of satellite cells following injury. Satellite cells work together with sensory neurons to extend the interconnected surface area in order to permit effective communication. The dynamic cellular environment within the ganglion undergoes several alterations that ultimately lead to differentiation, transformation, or cell death. In addition to necrotic and apoptotic cell morphology, phenomena such as histomorphometric alterations, including the development of autophagic vacuoles, chromatolysis, cytosolic degeneration, and other changes, are frequently observed in cells following injury. The use of electron microscopic and stereological techniques for assessing ganglia and nerve fibers is considered a gold standard in terms of investigating neuropathic pain models, regenerative therapies, some treatment methods, and quantifying the outcomes of pharmacological and bioengineering interventions. Stereological techniques provide observer-independent and reliable results, which are particularly useful in the quantitative assessment of three-dimensional structures from two-dimensional images. Employing the fractionator and disector techniques within stereological methodologies yields unbiased data when assessing parameters such as number. The fundamental concept underlying these methodologies involves ensuring that each part of the structure under evaluation has an equal opportunity of being sampled. This review describes the stereological and histomorphometric evaluation of dorsal root ganglion neurons and satellite cells following nerve injury models.