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Numerous imaging techniques are available for observing and interrogating biological samples, and several of them can be used consecutively to enable correlative analysis of different image modalities with varying resolutions and the inclusion of structural or molecular information. Achieving accurate registration of multimodal images is essential for the correlative analysis process, but it remains a challenging computer vision task with no widely accepted solution. Moreover, supervised registration methods require annotated data produced by experts, which is limited. To address this challenge, we propose a general unsupervised pipeline for multimodal image registration using deep learning. We provide a comprehensive evaluation of the proposed pipeline versus the current state-of-the-art image registration and style transfer methods on four types of biological problems utilizing different microscopy modalities. We found that style transfer of modality domains paired with fully unsupervised training leads to comparable image registration accuracy to supervised methods and, most importantly, does not require human intervention.
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Aprendizado Profundo , Humanos , MicroscopiaRESUMO
Most excitatory synapses in the mammalian brain are contacted or ensheathed by astrocyte processes, forming tripartite synapses. Astrocytes are thought to be critical regulators of the structural and functional dynamics of synapses. While the degree of synaptic coverage by astrocytes is known to vary across brain regions and animal species, the reason for and implications of this variability remains unknown. Further, how astrocyte coverage of synapses relates to in vivo functional properties of individual synapses has not been investigated. Here, we characterized astrocyte coverage of synapses of pyramidal neurons in the ferret visual cortex and, using correlative light and electron microscopy, examined their relationship to synaptic strength and sensory-evoked Ca2+ activity. Nearly, all synapses were contacted by astrocytes, and most were contacted along the axon-spine interface. Structurally, we found that the degree of synaptic astrocyte coverage directly scaled with synapse size and postsynaptic density complexity. Functionally, we found that the amount of astrocyte coverage scaled with how selectively a synapse responds to a particular visual stimulus and, at least for the largest synapses, scaled with the reliability of visual stimuli to evoke postsynaptic Ca2+ events. Our study shows astrocyte coverage is highly correlated with structural metrics of synaptic strength of excitatory synapses in the visual cortex and demonstrates a previously unknown relationship between astrocyte coverage and reliable sensory activation.
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Astrócitos , Furões , Córtex Visual Primário , Sinapses , Animais , Astrócitos/fisiologia , Astrócitos/ultraestrutura , Sinapses/fisiologia , Sinapses/ultraestrutura , Córtex Visual Primário/fisiologia , Células Piramidais/fisiologia , Células Piramidais/ultraestrutura , Masculino , Feminino , Potenciais Pós-Sinápticos Excitadores/fisiologia , Cálcio/metabolismo , Córtex Visual/fisiologia , Córtex Visual/citologia , Estimulação Luminosa/métodosRESUMO
Growth cones of oligodendrocyte progenitor cells (OPCs) are challenging to investigate with conventional light microscopy due to their small size. Especially substructures such as filopodia, lamellipodia and their underlying cytoskeleton are difficult to resolve with diffraction limited microscopy. Light microscopy techniques, which surpass the diffraction limit such as stimulated emission depletion microscopy, often require expensive setups and specially trained personnel rendering them inaccessible to smaller research groups. Lately, the invention of expansion microscopy (ExM) has enabled super-resolution imaging with any light microscope without the need for additional equipment. Apart from the necessary resolution, investigating OPC growth cones comes with another challenge: Imaging the topography of membranes, especially label- and contact-free, is only possible with very few microscopy techniques one of them being scanning ion conductance microscopy (SICM). We here present a new imaging workflow combining SICM and ExM, which enables the visualization of OPC growth cone nanostructures. We correlated SICM recordings and ExM images of OPC growth cones captured with a conventional widefield microscope. This enabled the visualization of the growth cones' membrane topography as well as their underlying actin and tubulin cytoskeleton.
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Microscopia , Células Precursoras de Oligodendrócitos , Microscopia/métodos , Cones de Crescimento , Citoesqueleto , MicrotúbulosRESUMO
Super-resolution fluorescence microscopy techniques are powerful tools to investigate polymer systems. In this review, we address how these techniques have been applied to hydrogel nano- and microparticles, so-called nano- or microgels. We outline which research questions on microgels could be addressed and what new insights could be achieved. Studies of the morphology, shape, and deformation of microgels; their internal compartmentalization; the cross-linker distribution and polarity inside them; and their dynamics and diffusion are summarized. In particular, the abilities to super-resolve structures in three dimensions have boosted the research field and have also allowed researchers to obtain impressive 3D images of deformed microgels. Accessing information beyond 3D localization, such as spectral and lifetime properties and correlative imaging or the combination of data with other methods, shines new light onto polymer systems and helps us understand their complexity in detail. Such future trends and developments are also addressed.
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Most of the time, the deep analysis of a biological sample requires the acquisition of images at different time points, using different modalities and/or different stainings. This information gives morphological, functional, and physiological insights, but the acquired images must be aligned to be able to proceed with the co-localisation analysis. Practically speaking, according to Aristotle's principle, "The whole is greater than the sum of its parts", multi-modal image registration is a challenging task that involves fusing complementary signals. In the past few years, several methods for image registration have been described in the literature, but unfortunately, there is not one method that works for all applications. In addition, there is currently no user-friendly solution for aligning images that does not require any computer skills. In this work, DS4H Image Alignment (DS4H-IA), an open-source ImageJ/Fiji plugin for aligning multimodality, immunohistochemistry (IHC), and/or immunofluorescence (IF) 2D microscopy images, designed with the goal of being extremely easy to use, is described. All of the available solutions for aligning 2D microscopy images have also been revised. The DS4H-IA source code; standalone applications for MAC, Linux, and Windows; video tutorials; manual documentation; and sample datasets are publicly available.
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Ciência de Dados , Documentação , Imuno-Histoquímica , Microscopia de Fluorescência , ImunofluorescênciaRESUMO
Understanding the solid electrolyte interphase (SEI) formation and (de)lithiation phenomena at silicon (Si) electrodes is key to improving the performance and lifetime of Si-based lithium-ion batteries. However, these processes remain somewhat elusive, and, in particular, the role of Si surface termination merits further consideration. Here, scanning electrochemical cell microscopy (SECCM) is used in a glovebox, followed by secondary ion mass spectrometry (SIMS) at identical locations to study the local electrochemical behavior and associated SEI formation, comparing Si (100) with a native oxide layer (SiOx /Si) and etched with hydrofluoric acid (HF-Si). HF-Si shows greater spatial electrochemical heterogeneity and inferior lithiation reversibility than SiOx /Si. This is attributed to a weakly passivating SEI and irreversible lithium trapping at the Si surface. Combinatorial screening of charge/discharge cycling by SECCM with co-located SIMS reveals SEI chemistry as a function of depth. While the SEI thickness is relatively independent of the cycle number, the chemistry - particularly in the intermediate layers - depends on the number of cycles, revealing the SEI to be dynamic during cycling. This work serves as a foundation for the use of correlative SECCM/SIMS as a powerful approach to gain fundamental insights on complex battery processes at the nano- and microscales.
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Developments in cryo-electron microscopy (cryo-EM) have been interwoven with the study of viruses ever since its first applications to biological systems. Following the success of single particle cryo-EM in the last decade, cryo-electron tomography (cryo-ET) is now rapidly maturing as a technology and catalysing great advancement in structural virology as its application broadens. In this review, we provide an overview of the use of cryo-ET to study viral infection biology, discussing the key workflows and strategies used in the field. We highlight the vast body of studies performed on purified viruses and virus-like particles (VLPs), as well as discussing how cryo-ET can characterise host-virus interactions and membrane fusion events. We further discuss the importance of in situ cellular imaging in revealing previously unattainable details of infection and highlight the need for validation of high-resolution findings from purified ex situ systems. We give perspectives for future developments to achieve the full potential of cryo-ET to characterise the molecular processes of viral infection.
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Viroses , Vírus , Humanos , Tomografia com Microscopia Eletrônica/métodos , Microscopia Crioeletrônica/métodosRESUMO
Correlative super-resolution microscopy has the potential to accurately visualize and validate new biological structures past the diffraction limit. However, combining different super-resolution modalities, such as deterministic stimulated emission depletion (STED) and stochastic single-molecule localization microscopy (SMLM), is a challenging endeavour. For correlative STED and SMLM, the following poses a significant challenge: (1) the photobleaching of the fluorophores in STED; (2) the subsequent reactivation of the fluorophores for SMLM and (3) finding the right fluorochrome and imaging buffer for both imaging modalities. Here, we highlight how the deep ultraviolet (DBUE) wavelengths of the Mercury (Hg) arc lamp can help recover STED bleaching and allow for the reactivation of single molecules for SMLM imaging. We also show that Alexa Fluor 594 and the commercially available Prolong Diamond to be excellent fluorophores and imaging media for correlative STED and SMLM.
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Many researchers have turned their attention to understanding microplastic interaction with marine fauna. Efforts are being made to monitor exposure pathways and concentrations and to assess the impact such interactions may have. To answer these questions, it is important to select appropriate experimental parameters and analytical protocols. This study focuses on medusae of Cassiopea andromeda jellyfish: a unique benthic jellyfish known to favor (sub-)tropical coastal regions which are potentially exposed to plastic waste from land-based sources. Juvenile medusae were exposed to fluorescent poly(ethylene terephthalate) and polypropylene microplastics (<300 µm), resin embedded, and sectioned before analysis with confocal laser scanning microscopy as well as transmission electron microscopy and Raman spectroscopy. Results show that the fluorescent microplastics were stable enough to be detected with the optimized analytical protocol presented and that their observed interaction with medusae occurs in a manner which is likely driven by the microplastic properties (e.g., density and hydrophobicity).
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Microplásticos , Poluentes Químicos da Água , Plásticos/análise , Análise Espectral Raman , Fluxo de Trabalho , Microscopia Eletrônica , Monitoramento Ambiental , Poluentes Químicos da Água/análiseRESUMO
Superresolution fluorescence microscopy and cryogenic electron tomography (CET) are powerful imaging methods for exploring the subcellular organization of biomolecules. Superresolution fluorescence microscopy based on covalent labeling highlights specific proteins and has sufficient sensitivity to observe single fluorescent molecules, but the reconstructions lack detailed cellular context. CET has molecular-scale resolution but lacks specific and nonperturbative intracellular labeling techniques. Here, we describe an imaging scheme that correlates cryogenic single-molecule fluorescence localizations with CET reconstructions. Our approach achieves single-molecule localizations with an average lateral precision of 9 nm, and a relative registration error between the set of localizations and CET reconstruction of â¼30 nm. We illustrate the workflow by annotating the positions of three proteins in the bacterium Caulobacter crescentus: McpA, PopZ, and SpmX. McpA, which forms a part of the chemoreceptor array, acts as a validation structure by being visible under both imaging modalities. In contrast, PopZ and SpmX cannot be directly identified in CET. While not directly discernable, PopZ fills a region at the cell poles that is devoid of electron-dense ribosomes. We annotate the position of PopZ with single-molecule localizations and confirm its position within the ribosome excluded region. We further use the locations of PopZ to provide context for localizations of SpmX, a low-copy integral membrane protein sequestered by PopZ as part of a signaling pathway that leads to an asymmetric cell division. Our correlative approach reveals that SpmX localizes along one side of the cell pole and its extent closely matches that of the PopZ region.
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Proteínas de Bactérias/metabolismo , Caulobacter crescentus/ultraestrutura , Imagem Individual de Molécula/métodos , Proteínas de Bactérias/ultraestrutura , Caulobacter crescentus/metabolismo , Tomografia com Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Transporte ProteicoRESUMO
Physical properties and chemical composition are fundamentally defining and interconnected surface characteristics. However, few techniques are able to capture both in a correlative fashion at the same sample location and orientation. This is especially important for complex materials such as dentin, which is an inner tooth structure and is a heterogeneous, composite inorganic-organic material with open channels (tubules) that extend toward the tooth pulp. Here, a combined microscope system consisting of an atomic force microscope and a confocal Raman spectrometer was used to study the correlative physical and chemical properties of human dentin. The local hardness of dentin was highly correlated with the Raman signal ratio of inorganic to organic material, and this was enhanced in the peritubular regions of dentin. When the samples were etched with citric acid, Young's modulus, hardness, and inorganic-to-organic material ratio decreased significantly, collagen fibrils on the surface were exposed, the peritubular regions were removed, and the tubule diameters increased. Thus, the combined atomic force microscopy (AFM)-Raman approach allows for comprehensive and correlative physical-chemical analysis of material surfaces and will be invaluable for evaluating oral therapeutic strategies.
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Dentina , Análise Espectral Raman , Humanos , Microscopia de Força Atômica , Módulo de Elasticidade , DurezaRESUMO
By collecting simultaneously optical and chemical/morphological data from nanoscale volumes, the Photonic Atom Probe (PAP) can be applied not only to the study of the relationship between optical and structural properties of quantum emitter but also to evaluate the influence of other factors, such as the presence of point defects, on the photoluminescence. Through the analysis of multiple layers of InGaN/GaN quantum dots (QDs), grown so that the density of structural defects is higher with increasing distance from the substrate, we establish that the light emission is higher in the regions exhibiting a higher presence of structural defects. While the presence of intrinsic point defects with non-radiative recombination properties remains elusive, our result is consistent with the fact that QD layers closer to the substrate behave as traps for non-radiative point defects. This result demonstrates the potential of the PAP as a technique for the study of the optical properties of defects in semiconductors.
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Correlative microscopy is a powerful technique that combines the advantages of multiple imaging modalities to achieve a comprehensive understanding of investigated samples. For example, fluorescence microscopy provides unique functional contrast by imaging only specifically labeled components, especially in biological samples. However, the achievable structural information on the sample in its full complexity is limited. Here, the intrinsic label-free carbon contrast of water window soft X-ray microscopy can complement fluorescence images in a correlative approach ultimately combining nanoscale structural resolution with functional contrast. However, soft X-ray microscopes are complex and elaborate, and are usually installed on large-scale synchrotron radiation sources due to the demanding photon flux requirements. Yet, with modern high-power lasers it has become possible to generate sufficient photon flux from laser-produced plasmas, thus enabling laboratory-based setups. Here, we present a compact table-top soft X-ray microscope with an integrated epifluorescence modality for "in situ" correlative imaging. Samples remain in place when switching between modalities, ensuring identical measurement conditions and avoiding sample alteration or destruction. We demonstrate our new method by multimodal images of several exemplary samples ranging from nanoparticles to various multicolor labeled cell types. A structural resolution of down to 50 nm was reached.
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This work analyzes the intracellular fate of protein-based nanocarriers along their endolysosomal pathway by means of correlative light and electron microscopy methods. To unambiguously identify the nanocarriers and their degradation remnants in the cellular environment, they are labeled with fluorescent, inorganic nanoplatelets. This allows tracking the nanocarriers on their intracellular pathway by means of electron microscopy imaging. From the present data, it is possible to identify different cell compartments in which the nanocarriers are processed. Finally, three different terminal routes for the intracellular destiny of the nanocarriers are presented. These findings are important to reveal the degradation process of protein nanocapsules and contribute to the understanding of the therapeutic success of an encapsulated drug.
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Nanocápsulas , Nanopartículas , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos , Endossomos/metabolismo , Lisossomos/metabolismoRESUMO
Packing biomolecules inside virus capsids has opened new avenues for the study of molecular function in confined environments. These systems not only mimic the highly crowded conditions in nature, but also allow their manipulation at the nanoscale for technological applications. Here, green fluorescent proteins are packed in virus-like particles derived from P22 bacteriophage procapsids. The authors explore individual virus cages to monitor their emission signal with total internal reflection fluorescence microscopy while simultaneously changing the microenvironment with the stylus of atomic force microscopy. The mechanical and electronic quenching can be decoupled by ≈10% each using insulator and conductive tips, respectively. While with conductive tips the fluorescence quenches and recovers regardless of the structural integrity of the capsid, with the insulator tips quenching only occurs if the green fluorescent proteins remain organized inside the capsid. The electronic quenching is associated with the coupling of the protein fluorescence emission with the tip surface plasmon resonance. In turn, the mechanical quenching is a consequence of the unfolding of the aggregated proteins during the mechanical disruption of the capsid.
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Imagem Individual de Molécula , Proteínas Virais , Capsídeo/química , Proteínas do Capsídeo/química , Proteínas de Fluorescência Verde , Microscopia de Força Atômica , Proteínas Virais/químicaRESUMO
Mesenchymal stem cell therapy after stroke is a promising option investigated in animal models and clinical trials. The intravenous route is commonly used in clinical settings guaranteeing an adequate safety profile although low yields of engraftment. In this report, rats subjected to ischemic stroke were injected with adipose-derived stem cells (ADSCs) labeled with superparamagnetic iron oxide nanoparticles (SPIONs) applying an external magnetic field in the skull to retain the cells. Although most published studies demonstrate viability of ADSCs, only a few have used ultrastructural techniques. In our study, the application of a local magnetic force resulted in a tendency for higher yields of SPION-ADSCs targeting the brain. However, grafted cells displayed morphological signs of death, one day after administration, and correlative microscopy showed active microglia and astrocytes associated in the process of scavenging. Thus, we conclude that, although successfully targeted within the brain, SPION-ADSCs viability was rapidly compromised.
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Nanopartículas de Magnetita , Acidente Vascular Cerebral , Adipócitos , Animais , Encéfalo , Campos Magnéticos , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita/química , Ratos , Células-Tronco , Acidente Vascular Cerebral/terapiaRESUMO
Zircon crystals from the Jack Hills, Western Australia, are one of the few surviving mineralogical records of Earth's first 500 million years and have been proposed to contain a paleomagnetic record of the Hadean geodynamo. A prerequisite for the preservation of Hadean magnetization is the presence of primary magnetic inclusions within pristine igneous zircon. To date no images of the magnetic recorders within ancient zircon have been presented. Here we use high-resolution transmission electron microscopy to demonstrate that all observed inclusions are secondary features formed via two distinct mechanisms. Magnetite is produced via a pipe-diffusion mechanism whereby iron diffuses into radiation-damaged zircon along the cores of dislocations and is precipitated inside nanopores and also during low-temperature recrystallization of radiation-damaged zircon in the presence of an aqueous fluid. Although these magnetites can be recognized as secondary using transmission electron microscopy, they otherwise occur in regions that are indistinguishable from pristine igneous zircon and carry remanent magnetization that postdates the crystallization age by at least several hundred million years. Without microscopic evidence ruling out secondary magnetite, the paleomagnetic case for a Hadean-Eoarchean geodynamo cannot yet been made.
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Correlative microscopy approaches are attracting considerable interest in several research fields such as materials and battery research. Recent developments regarding X-ray computer tomography have made this technique available in a compact module for scanning electron microscopes (SEMs). Nano-computed tomography (nanoCT) allows morphological analysis of samples in a nondestructive way and to generate 2D and 3D overviews. However, morphological analysis alone is not sufficient for advanced studies, and to draw conclusions beyond morphology, chemical analysis is needed. While conventional SEM-based chemical analysis techniques such as energy-dispersive X-ray spectroscopy (EDS) are adequate in many cases, they are not well suited for the analysis of trace elements and low-Z elements such as hydrogen or lithium. Furthermore, the large information depth in typical SEM-EDS imaging conditions limits the lateral resolution to micrometer length scales. In contrast, secondary ion mass spectrometry (SIMS) can perform elemental mapping with good surface sensitivity, nanoscale lateral resolution, and the possibility to analyze even low-Z elements and isotopes. In this study, we demonstrate the feasibility and compatibility of a novel FIB-SEM-based correlative nanoCT-SIMS imaging approach to correlate morphological and chemical data of the exact same sample volume, using a cathode material of a commercial lithium battery as an example.
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Refractory high-entropy alloys (RHEAs) are promising candidates for next-generation high-temperature materials. RHEAs containing Al, often exhibit a checkered pattern microstructure comprising a combination of disordered BCC and ordered B2 phases. Since the ordered B2 phase is based on the BCC parent matrix, distinguishing these two phases can be rather challenging. Advanced characterization techniques are necessary for a reliable qualitative and quantitative analysis of BCC and B2 phases in RHEAs. Additionally, there is a tendency for transformation of the ordered B2 phase into more complex ordered-omega type phases that are usually deleterious to mechanical properties. The current study focuses on the phase stability of a candidate RHEA, Al0.5Mo0.5NbTa0.5TiZr. Correlative transmission electron microscopy (TEM) and atom probe tomography (APT) have been employed to investigate the phase stability and transformation pathway of this RHEA when isothermally annealed at 800°C. The results show that a metastable two-phase BCC + B2 microstructure formed at the early stages of decomposition, eventually transforming into a three-phase BCC + B2 + hP18 microstructure. The hP18 phase is an ordered omega derivative of the ordered B2 phase. The correlative microscopy techniques (TEM and APT) reveal a very interesting interplay of compositional partitioning between the different phases and their respective stability.
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The renal stone found in the natural mummy of an anonymous nobleman dating to 19th century was investigated using advanced imaging modalities and analytic investigations. By this multidisciplinary approach we were able to identify the chemical components and their distribution throughout the sample. These results allowed to understand the lifestyle habits of the subject, as well as the exact pathogenesis of his disease.