Cryogenic Super-Resolution Fluorescence and Electron Microscopy Correlated at the Nanoscale.

We review the emerging method of super-resolved cryogenic correlative light and electron microscopy (srCryoCLEM). Super-resolution (SR) fluorescence microscopy and cryogenic electron tomography (CET) are both powerful techniques for observing subcellular organization, but each approach has unique limitations. The combination of the two brings the single-molecule sensitivity and specificity of SR to the detailed cellular context and molecular scale resolution of CET. The resulting correlative data is more informative than the sum of its parts. The correlative images can be used to pinpoint the positions of fluorescently labeled proteins in the high-resolution context of CET with nanometer-scale precision and/or to identify proteins in electron-dense structures. The execution of srCryoCLEM is challenging and the approach is best described as a method that is still in its infancy with numerous technical challenges. In this review, we describe state-of-the-art srCryoCLEM experiments, discuss the most pressing challenges, and give a brief outlook on future applications.

Brachiaria brizantha Grass as a Feedstock for Ethanol Production

HIGHLIGHTS Brachiaria brizantha proved to be a promising biomass for ethanol production. Fermentation was not impaired by the inhibitors furfural and hydroxymethylfurfural.

Abstract Different lignocellulosic biomasses are found worldwide and each country has its own important industrial crop that can be converted into high-value products, such as ethanol. Therefore, evaluation of new biomasses to be used in biorefineries is important to decrease the dependence on non-renewable resources and to guarantee sustainable development. This work evaluated Brachiaria brizantha, a grass commonly used as animal forage, and the standard biomass for 2G-ethanol, sugarcane bagasse. The chemical compositions of both biomasses were determined and different times and temperature of acid pretreatment were tested. Morphological analysis via scanning electron microscopy showed more deconstructed fibers after harsher biomass pretreatments. Simultaneous saccharification and fermentation of pretreated Brachiaria brizantha presented higher efficiency than when using sugarcane bagasse as the carbon source. A biomass conversion of 46 % was achieved when Brachiaria brizantha grass was pretreated with 2% sulfuric acid for 60 minutes. Moreover, fermentation was not impaired by the inhibitors furfural and hydroxymethylfurfural. It was concluded that Brachiaria brizantha is a promising biomass for ethanol production.

Diagnostic Electron Microscopy of Viruses With Low-voltage Electron Microscopes.

Diagnostic electron microscopy is a useful technique for the identification of viruses associated with human, animal, or plant diseases. The size of virus structures requires a high optical resolution (i.e., about 1 nm), which, for a long time, was only provided by transmission electron microscopes operated at 60 kV and above. During the last decade, low-voltage electron microscopy has been improved and potentially provides an alternative to the use of high-voltage electron microscopy for diagnostic electron microscopy of viruses. Therefore, we have compared the imaging capabilities of three low-voltage electron microscopes, a scanning electron microscope equipped with a scanning transmission detector and two low-voltage transmission electron microscopes, operated at 25 kV, with the imaging capabilities of a high-voltage transmission electron microscope using different viruses in samples prepared by negative staining and ultrathin sectioning. All of the microscopes provided sufficient optical resolution for a recognition of the viruses tested. In ultrathin sections, ultrastructural details of virus genesis could be revealed. Speed of imaging was fast enough to allow rapid screening of diagnostic samples at a reasonable throughput. In summary, the results suggest that low-voltage microscopes are a suitable alternative to high-voltage transmission electron microscopes for diagnostic electron microscopy of viruses.

Robust ultraclean atomically thin membranes for atomic-resolution electron microscopy.

The fast development of high-resolution electron microscopy (EM) demands a background-noise-free substrate to support the specimens, where atomically thin graphene membranes can serve as an ideal candidate. Yet the preparation of robust and ultraclean graphene EM grids remains challenging. Here we present a polymer- and transfer-free direct-etching method for batch fabrication of robust ultraclean graphene grids through membrane tension modulation. Loading samples on such graphene grids enables the detection of single metal atoms and atomic-resolution imaging of the iron core of ferritin molecules at both room- and cryo-temperature. The same kind of hydrophilic graphene grid allows the formation of ultrathin vitrified ice layer embedded most protein particles at the graphene-water interface, which facilitates cryo-EM 3D reconstruction of archaea 20S proteasomes at a record high resolution of ~2.36 Å. Our results demonstrate the significant improvements in image quality using the graphene grids and expand the scope of EM imaging.

[General Troubleshooting and Preventive Maintenance of the Digestive Electron Microscope System].

OBJECTIVE: Improve the integrity of the digestive electron microscope equipment and reduce the cost of equipment failure maintenance. METHODS: By studying the composition and function of the digestive electron microscope system and analyzing the causes of common faults, a targeted preventive maintenance plan is developed, equipment users are graded, and a training system is established. RESULTS: The user of the device can skillfully analyze the cause of the malfunction and timely deal with the sudden failure of the diagnosis and treatment, thereby reduce the risk of diagnosis and treatment and the investment in hospital maintenance. CONCLUSIONS: Through the analysis and processing of the digestive electron microscope system, point detection leakage, grading training, preventive maintenance can significantly improve the equipment integrity rate, reduce the risk of clinical diagnosis and treatment, effectively reduce the number of equipment failures, and reduce maintenance costs.

Microbialite-like structures in Cladophora sp. (Ulvophyceae) mats from a subtropical Andean basin: ecological implications / Estructuras de tipo microbialito en Cladophora sp. (Ulvophyceae) de una cuenca andina subtropical: implicaciones ecológicas

Abstract The solubility equilibrium of calcite is influenced by physicochemical, climatic and biological factors. Annual cycles of exceptionally prolonged drought, in conjunction with naturally occurring diffuse organic pollution, generate the unique conditions for the precipitation of lithified carbonate structures (microbialites). The aim of this article is to analyze the possible implications of calcite precipitation produced in mats of Cladophora sp. in an Andean subtropical basin, considering it is the first time this phenomenon is described for the region. We collected samples from selected sites at the Lules River Basin, in four sampling dates between the years 2003 and 2004, within a monitoring work of 15 years. Samples were analyzed using an electron microscope and X-ray diffraction analysis. We found that Gomphonema sp. attached to Cladophora sp. contributes to precipitation of calcite and formation of microbialite like structures, in the studied area. This work presents an initial discussion of the discovery of microbialites-like structures attached to Cladophora sp. mats in a subtropical Andean stream and the environmental conditions that lead to their production, as well as the possible ecological implications of these microbialites.(AU)

Resumen La fase sólida de las sales de los cuerpos de agua juega un papel importante en la concentración de los elementos mayoritarios. El equilibrio de solubilidad de la calcita está influenciado por factores fisicoquímicos, climáticos y biológicos. Los ciclos anuales de sequía excepcionalmente prolongada, junto con la contaminación orgánica difusa de origen natural, generan condiciones únicas para la precipitación de estructuras de carbonato litificado (microbialitos). El objetivo de este artículo es analizar las posibles implicaciones de la precipitación de calcita producida en Cladophora sp. en una cuenca subtropical andina, considerando que es la primera vez que se describe este fenómeno para la región. Recolectamos muestras en sitios seleccionados en la Cuenca del Río Lules, en cuatro fechas de muestreo entre 2003 y 2004, en el marco de un trabajo de monitoreo de quince años. Usamos red Surber para realizar los muestreos. Las muestras de algas, una vez libres de macroinvertebrados, se secaron y pesaron. Analizamos utilizando microscopía electrónica y difracción de rayos X. Encontramos que Gomphonema sp. unido a Cladophora contribuye a la precipitación de calcita y la formación de estructuras similares a microbialitos, en el área estudiada. Este trabajo presenta una discusión inicial sobre el descubrimiento de estructuras similares a microbialitos unidas a filamentos de Cladophora sp. en una cuenca andina subtropical y las condiciones ambientales que conducen a su producción, así como las posibles implicaciones ecológicas de lo mencionado anteriormente.(AU)

Correlative light and electron microscopy is a powerful tool to study interactions of extracellular vesicles with recipient cells.

Extracellular vesicles (EVs) and their interactions with recipient cells constitute a rapidly growing research area. However, due to the limitations in current methodologies, the mechanisms of these interactions are still unclear. Microscopic studies of EVs are challenging, because their typical diameter is near the resolution limit of light microscopy, and electron microscopy has restricted possibilities for protein labelling. The objective of this study was to combine these two techniques to demonstrate in detail the interactions of EVs by recipient cells. Hyaluronan synthase 3 (HAS3) is an integral transmembrane protein that is enriched in EVs. In this work, GFP-HAS3 was utilized to study the interactions of EVs with the recipient cells. Surprisingly, confocal analysis correlation with scanning electron microscopy (SEM) revealed that most of the EVs were indeed lying on the recipient cell's plasma membrane, while the level of EV-derived intracellular signal was low. Immunoelectron microscopy supported this finding. Furthermore, hyaluronan oligosaccharides decreased the numbers of bound EVs, suggesting that CD44 participates in the regulation of their binding. This study indicates that correlative light and electron microscopy is a reliable method to analyze EV interactions with recipient cells. Detailed 3D confocal imaging of EV carrying a GFP-label on their plasma membrane combined with high-resolution electron microscopy provides significantly more information than either of the techniques alone. In the future studies it is crucial to utilize these techniques and their combinations to solve in detail the ambiguous fate of EV in target cells. Furthermore, live cell imaging at high resolution will be required to obtain definite answers on the detailed mechanisms of binding, fusion and endocytosis of EVs.

Correlative Light and Electron Microscopy of Autophagosomes.

Live-cell imaging has been widely used to study autophagosome biogenesis and maturation. When combined with correlative electron microscopy, this approach can be extended to reveal ultrastructural details in three dimensions. The resolution of electron microscopy is needed when membrane contact sites and tubular connections between organelles are studied.

Measuring Autophagy in Pancreatitis.

Acute pancreatitis is one of the first pathological processes where autophagy has been described in a human tissue. Autophagy, autodigestion, and cell death are early cellular events in acute pancreatitis. Recent advances in understanding autophagy highlight its importance in pathological conditions. However, methods for monitoring autophagic activity during complex diseases, involving highly differentiated secretory cells, are complicated, and the results are sometimes misinterpreted. Here, we describe methods used to identify autophagic structures and to measure autophagic flux in cultured cell models and animal models of pancreatitis. We also briefly describe the pancreas specific autophagy mouse model that was useful to understand the actual role of autophagy in pancreatitis and to identify a novel selective autophagy pathway named zymophagy. Lastly, we describe the immunomagnetic isolation of autophagosomes and the detection of autophagy in pancreatic tissue samples derived from humans.

Porous Silicon Nanoneedles Modulate Endocytosis to Deliver Biological Payloads.

Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane-nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state-of-the-art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin-coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae- and clathrin-mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle-mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell-manipulation technologies.

Survey of the analysis of continuous conformational variability of biological macromolecules by electron microscopy.

Single-particle analysis by electron microscopy is a well established technique for analyzing the three-dimensional structures of biological macromolecules. Besides its ability to produce high-resolution structures, it also provides insights into the dynamic behavior of the structures by elucidating their conformational variability. Here, the different image-processing methods currently available to study continuous conformational changes are reviewed.

Visualization of SNARE-Mediated Organelle Membrane Hemifusion by Electron Microscopy.

SNARE-mediated membrane fusion is required for membrane trafficking as well as organelle biogenesis and homeostasis. The membrane fusion reaction involves sequential formation of hemifusion intermediates, whereby lipid monolayers partially mix on route to complete bilayer merger. Studies of the Saccharomyces cerevisiae lysosomal vacuole have revealed many of the fundamental mechanisms that drive the membrane fusion process, as well as features unique to organelle fusion. However, until recently, it has not been amenable to electron microscopy methods that have been invaluable for studying hemifusion in other model systems. Herein, we describe a method to visualize hemifusion intermediates during homotypic vacuole membrane fusion in vitro by transmission electron microscopy (TEM), electron tomography, and cryogenic electron microscopy (cryoEM). This method facilitates acquisition of invaluable ultrastructural data needed to comprehensively understand how fusogenic lipids and proteins contribute to SNARE-mediated membrane fusion-by-hemifusion and the unique features of organelle versus small-vesicle fusion.

Use of 3D imaging for providing insights into high-order structure of mitotic chromosomes.

The high-order structure of metaphase chromosomes remains still under investigation, especially the 30-nm structure that is still controversial. Advanced 3D imaging has provided useful information for our understanding of this detailed structure. It is evident that new technologies together with improved sample preparations and image analyses should be adequately combined. This mini review highlights 3D imaging used for chromosome analysis so far with future imaging directions also highlighted.

How Seeing Became Knowing: The Role of the Electron Microscope in Shaping the Modern Definition of Viruses.

This paper examines the vital role played by electron microscopy toward the modern definition of viruses, as formulated in the late 1950s. Before the 1930s viruses could neither be visualized by available technologies nor grown in artificial media. As such they were usually identified by their ability to cause diseases in their hosts and defined in such negative terms as "ultramicroscopic" or invisible infectious agents that could not be cultivated outside living cells. The invention of the electron microscope, with magnification and resolution powers several orders of magnitude better than that of optical instruments, opened up possibilities for biological applications. The hitherto invisible viruses lent themselves especially well to investigation with this new instrument. We first offer a historical consideration of the development of the instrument and, more significantly, advances in techniques for preparing and observing specimens that turned the electron microscope into a routine biological tool. We then describe the ways in which the electron microscopic images, or micrographs, functioned as forms of new knowledge about viruses and resulted in a paradigm shift in the very definition of these entities. Micrographs were not mere illustrations since they did the work for the electron microscopists. Drawing extensively on primary publications, we adduce the role of the new instrument in understanding the so-called eclipse phase in virus multiplication and the unexpected spinoffs of data from electron microscopy in naming and classifying viruses. Thus, we show that electron microscopy functioned not only to provide evidence, but also arguments in facilitating a reordering of the world that it brought into the visual realm.

Imaging extracellular vesicles: current and emerging methods.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells. They range from 30 nm to several micrometers in diameter, and ferry biological cargos such as proteins, lipids, RNAs and DNAs for local and distant intercellular communications. EVs have since been found to play a role in development, as well as in diseases including cancers. To elucidate the roles of EVs, researchers have established different methods to visualize and study their spatiotemporal properties. However, since EV are nanometer-sized, imaging them demands a full understanding of each labeling strategy to ensure accurate monitoring. This review covers current and emerging strategies for EV imaging for prospective studies.

Correlative light and electron microscopy for complex cellular structures on PDMS substrates with coded micro-patterns.

Fluorescence light microscopy (FLM) is commonly used for localizing specific cellular and subcellular targets. Electron microscopy (EM), on the other hand, can reveal ultrastructural details of cellular architectures beyond the limit of optical resolution. Correlative light and electron microscopy (CLEM) that combines the two techniques has proven valuable in various cell biological applications that require both specificity and resolution. Here, we report an efficient and easy-to-use CLEM system, and its applications in studying neuronal synapses. The system utilizes patterned symbols to encode coordinates on micro-fabricated polydimethylsiloxane (PDMS) substrates, on which dissociated primary hippocampal neurons grow and form synaptic connections. After imaging and localizing specifically labeled synapses with FLM, samples are embedded in resin blocks and sectioned for EM analysis. The patterned symbols on PDMS substrates provide coordinate information, allowing efficient co-registration between FLM and EM images with high precision. A custom-developed software package achieves automated EM image collection, FLM/EM alignment, and EM navigation. With this CLEM system, we have obtained high quality electron tomograms of fluorescently labeled synapses along dendrites of hippocampal neurons and analyzed docking statistics of synaptic vesicles (SVs) in different subtypes of excitatory synapses. This technique provides an efficient approach to combine functional studies with ultrastructural analysis of heterogeneous neuronal synapses, as well as other subcellular structures in general.

Ultrastructural histochemistry in biomedical research: Alive and kicking.

The high-resolution images provided by the electron microscopy has constituted a limitless source of information in any research field of life and materials science since the early Thirties of the last century. Browsing the scientific literature, electron microscopy was especially popular from the 1970's to 80's, whereas during the 90's, with the advent of innovative molecular techniques, electron microscopy seemed to be downgraded to a subordinate role, as a merely descriptive technique. Ultrastructural histochemistry was crucial to promote the Renaissance of electron microscopy, when it became evident that a precise localization of molecules in the biological environment was necessary to fully understand their functional role. Nowadays, electron microscopy is still irreplaceable for ultrastructural morphology in basic and applied biomedical research, while the application of correlative light and electron microscopy and of refined ultrastructural histochemical techniques gives electron microscopy a central role in functional cell and tissue biology, as a really unique tool for high-resolution molecular biology in situ.

Multicolor single-particle reconstruction of protein complexes.

Single-particle reconstruction (SPR) from electron microscopy (EM) images is widely used in structural biology, but it lacks direct information on protein identity. To address this limitation, we developed a computational and analytical framework that reconstructs and coaligns multiple proteins from 2D super-resolution fluorescence images. To demonstrate our method, we generated multicolor 3D reconstructions of several proteins within the human centriole, which revealed their relative locations, dimensions and orientations.