Stimulated emission depletion (STED) microscopy is routinely used to resolve the ultrastructure of cells with a ∼10-fold higher resolution compared to diffraction limited imaging. While STED ...microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides subdiffraction resolution by physically enlarging the sample before microscopy. The expansion of the fixed cells by cross-linking and swelling of hydrogels easily enlarges the sample ∼4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complementary approaches, we combined ExM with STED (ExSTED) and demonstrated an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ∼50 nm isotropic). While the increase in resolution is straightforward, we found that high-fidelity labeling via multi-epitopes is required to obtain emitter densities that allow ultrastructural details with ExSTED to be resolved. Our work provides a robust template for super-resolution microscopy of entire cells in the ten nanometer range.
In recent years, extracellular vesicles (EVs) have become a subject of intense study. These membrane-enclosed spherical structures are secreted by almost every cell type and are engaged in the ...transport of cellular content (cargo) from parental to target cells. The impact of EVs transfer has been observed in many vital cellular processes including cell-to-cell communication and immune response modulation; thus, a fast and precise characterization of EVs may be relevant for both scientific and diagnostic purposes. In this review, the most popular analytical techniques used in EVs studies are presented with the emphasis on exosomes and microvesicles characterization.
Three mitochondrial metabolic pathways are required for efficient energy production in eukaryotic cells: the electron transfer chain (ETC), fatty acid β-oxidation (FAO), and the tricarboxylic acid ...cycle. The ETC is organized into inner mitochondrial membrane supercomplexes that promote substrate channeling and catalytic efficiency. Although previous studies have suggested functional interaction between FAO and the ETC, their physical interaction has never been demonstrated. In this study, using blue native gel and two-dimensional electrophoreses, nano-LC-MS/MS, immunogold EM, and stimulated emission depletion microscopy, we show that FAO enzymes physically interact with ETC supercomplexes at two points. We found that the FAO trifunctional protein (TFP) interacts with the NADH-binding domain of complex I of the ETC, whereas the electron transfer enzyme flavoprotein dehydrogenase interacts with ETC complex III. Moreover, the FAO enzyme very-long-chain acyl-CoA dehydrogenase physically interacted with TFP, thereby creating a multifunctional energy protein complex. These findings provide a first view of an integrated molecular architecture for the major energy-generating pathways in mitochondria that ensures the safe transfer of unstable reducing equivalents from FAO to the ETC. They also offer insight into clinical ramifications for individuals with genetic defects in these pathways.
Exopolysaccharides produced by lactic acid bacteria are widely used to improve the sensory properties of yogurt. The relation between the physical properties of the microbial exopolysaccharides and ...the structural and rheological properties of the yogurt are incompletely understood to date. To address this knowledge gap, we studied how two distinct exopolysaccharides influence the microstructure, rheological properties, and syneresis of yogurt. The effect of a negatively charged, capsular exopolysaccharide produced by Streptococcus thermophilus and a neutral, non-capsular exopolysaccharide produced by Lactococcus lactis were investigated. Using quantitative microstructural analysis, we examined yogurt samples prepared with either the capsular or the non-capsular exopolysaccharide, and with mixtures of the two. Confocal laser scanning microscopy and stimulated emission depletion microscopy were employed to visualize the microstructures, revealing differences in pore size distribution, protein domain size, and casein interconnectivity that were not apparent through visual inspection alone. Additionally, variations in rheological properties were observed among the different yogurt types. In the yogurt fermented with both bacterial strains, we observed a combined impact of the two exopolysaccharide types on relevant microstructural and rheological properties. The negatively charged capsular exopolysaccharide enhanced casein interconnectivity and gel stiffness, while the neutral non-capsular exopolysaccharide led to thicker protein domains, an abundance of small pores, and a lower loss tangent. These factors collectively hindered syneresis, resulting in improved structural integrity. Our study not only provides valuable insights into the influence of different exopolysaccharides on yogurt properties, but also presents the first demonstration and quantification of the effect of multiple types of exopolysaccharides on casein interconnectivity. These findings offer guidance for the production of yogurts with customized microstructure, rheological properties, and resistance to syneresis.
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•Image analysis detects subtle differences in protein domains and pore distribution.•αs1-Casein link density highest in yogurt with mixture of exopolysaccharide types.•Yogurt with capsular exopolysaccharides display higher storage and loss modulus.
Super-resolution fluorescence microscopy has become an invaluable, powerful approach to study biomolecular dynamics and interactions via selective labeling and observation of specific molecules in ...living cells, tissues and even entire organisms. In this perspective, we present a brief overview of the main techniques and their application to cellular biophysics. We place special emphasis on super-resolution imaging via single-molecule localization microscopy and stimulated emission depletion/reversible saturable optical fluorescence transitions microscopy, and we also briefly address fluorescence fluctuation approaches, notably raster image correlation spectroscopy, as tools to record fast diffusion and transport.
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•Fluorescence microscopy enables imaging of specific biomolecules in live specimens under near-physiological conditions.•Key approaches to achieve super-resolution in fluorescence microscopy are discussed.•Fluorescence fluctuation spectroscopy techniques allow quantification of fast biomolecular dynamics.•How to choose the optimal fluorescence marker for a specific imaging technique.•Where are we going with advanced optical imaging?
Several methodologies have been developed over the past several years for super-resolution fluorescence microscopy including saturated structured-illumination microscopy (SSIM), stimulated emission ...depletion microscopy (STED), photoactivated localization microscopy (PALM), fluorescence photoactivation localization microscopy (FPALM), and stochastic optical reconstruction microscopy (STORM). While they have shown great promise for biological research, these techniques all have individual strengths and weaknesses. This review will describe the basic principles for achieving super resolution, demonstrate some applications in biology, and provide an overview of technical considerations for implementing these methods.
Kidney ischemia–reperfusion injury is a major cause of acute kidney injury (AKI). Following reduced kidney perfusion, the pathological overproduction of reactive oxygen and reactive nitrogen species ...play a substantial role in the development of kidney ischemia–reperfusion injury. Arginase 2 (ARG2) competes with nitric oxide synthase for the same substrate, L-arginine, and is implicated in the regulation of reactive nitrogen species. Therefore, we investigated the role of ARG2 in kidney ischemia–reperfusion injury using human proximal tubule cells (HK-2) and a mouse model of kidney ischemia–reperfusion injury. ARG2 was predominantly expressed in kidney tubules of the cortex, which was increased after ischemia–reperfusion injury. In HK-2 cells, ARG2 was expressed in punctate form in the cytoplasm and upregulated after hypoxia–reoxygenation. ARG2 knockdown reduced the level of reactive oxygen species and 3-nitrotyrosine after hypoxia–reoxygenation injury compared with control siRNA. Consistent with these results, in Arg2 knockout mice, abnormal kidney function and the increased acute tubular necrosis score induced by ischemia–reperfusion injury was significantly reduced without any obvious blood pressure changes. Additionally, an accumulation of 3-nitrotyrosine and apoptosis of renal tubule cells were attenuated in Arg2 knockout mice compared with wild-type mice. Inhibition of arginase by Nω-hydroxy-nor-L-arginine alleviated kidney ischemia–reperfusion injury like the results found in Arg2 knockout mice. Thus, ARG2 plays a pivotal role in ischemia–reperfusion-induced AKI by means of nitrosative stress. Hence, an ARG2-specific inhibitor may effectively treat kidney ischemia–reperfusion injury.
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The emerging stress caused by nanomaterials in the environment is of great concern because they can have toxic effects on organisms. However, thorough study of the interactions between cells and ...diverse nanoparticles (NPs) using a unified approach is challenging. Here, we present a novel approach combining stimulated emission depletion (STED) microscopy and scanning transmission electron microscopy (STEM) for quantitative assessment, real-time tracking, and in situ imaging of the intracellular behavior of gold–silver nanoclusters (AuAgNCs), based on their fluorescence and electron properties. The results revealed an aggregated state of AuAgNCs within the mitochondria and an increase in sulfur content in AuAgNCs, presumably owing to their reaction with thiol-containing molecules inside the mitochondria. Moreover, AuAgNCs (100 μg/mL) induced a 75% decline in mitochondrial membrane potential and a 12-fold increase of mitochondrial reactive oxygen species in comparison to control. This mitochondrial damage may be triggered by the reaction of AuAgNCs with thiol, which provides direct imaging evidence for uncovering the action mechanism of AuAgNCs on the mitochondria. The proposed dual-imaging strategy using STED and STEM is a potential tool to offer valuable insights into cytotoxicity between subcellular structures and diverse NPs, and can serve as a key strategy for nanomaterial biosafety assessment.
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•Dual-imaging of STED and STEM can be used for visualizing nano-cell interactions.•STED imaging tracked the AuAgNCs localized within mitochondria in living cells.•STEM imaging exhibited the aggregating AuAgNCs within mitochondria.•STEM-EDS analysis showed an increase in sulfur contents in AuAgNCs.•The aggregating AuAgNCs within mitochondria led to mitochondrial damage.
Mitochondria are equipped with their own DNA (mtDNA), which is packed into structures termed nucleoids . While nucleoids can be visualized in situ by fluorescence microscopy , the advent of ...super-resolution microscopy , and in particular of stimulated emission depletion (STED), has recently enabled the visualization of nucleoids at sub-diffraction resolution. Super-resolution microscopy has proved an invaluable tool for addressing fundamental questions in mitochondrial biology. In this chapter I describe how to achieve efficient labeling of mtDNA and how to quantify nucleoid diameter using an automated approach in fixed cultured cells by STED microscopy .
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