Complementary imaging of nanoclusters interacting with mitochondria via stimulated emission depletion and scanning transmission electron microscopy.

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.

Advanced Microscopy Techniques for Molecular Biophysics.

Though microscopy is most often intended as a technique for providing qualitative assessment of cellular and subcellular properties, when coupled with other instruments such as wavelength selectors, lasers, photoelectric devices and computers, it can perform a wide variety of quantitative measurements, which are demanding in establishing relationships between the properties and structures of biological material in all their spatial and temporal complexities. These combinations of instruments are a powerful approach to improve non-destructive investigations of cellular and subcellular properties (both physical and chemical) at a macromolecular scale resolution. Since many subcellular compartments in living cells are characterized by structurally organized molecules, this review deals with three advanced microscopy techniques well-suited for these kind of investigations, i.e., microspectrophotometry (MSP), super-resolution localization microscopy (SRLM) and holotomographic microscopy (HTM). These techniques can achieve an insight view into the role intracellular molecular organizations such as photoreceptive and photosynthetic structures and lipid bodies play in many cellular processes as well as their biophysical properties. Microspectrophotometry uses a set-up based on the combination of a wide-field microscope and a polychromator, which allows the measurement of spectroscopic features such as absorption spectra. Super resolution localization microscopy combines dedicated optics and sophisticated software algorithms to overcome the diffraction limit of light and allow the visualization of subcellular structures and dynamics in greater detail with respect to conventional optical microscopy. Holotomographic microscopy combines holography and tomography techniques into a single microscopy set-up, and allows 3D reconstruction by means of the phase separation of biomolecule condensates. This review is organized in sections, which for each technique describe some general aspects, a peculiar theoretical aspect, a specific experimental configuration and examples of applications (fish and algae photoreceptors, single labeled proteins and endocellular aggregates of lipids).

Super-Resolution Imaging of Peroxisomal Proteins Using STED Nanoscopy.

Peroxisomes are crucial organelles that occur in almost all eukaryotes. Well known are their roles in various metabolic processes, such as hydrogen peroxide detoxification and lipid metabolism. Recent studies indicated that peroxisomes also have several non-metabolic functions, for instance, in stress response, signaling, and cellular ageing. In mammalian cells, the small size of peroxisomes (~200 nm, near the diffraction limit) hinders unveiling peroxisomal structures by conventional light microscopy. However, in the yeast Hansenula polymorpha, they can reach up to 1.5 µm in diameter, depending on the carbon source. To study the localization of peroxisomal proteins in cells in more detail, super-resolution imaging techniques such as stimulated emission depletion (STED) microscopy can be used. STED enables fast (live-cell) imaging well beyond the diffraction limit of light (30-40 nm in cells), without further data processing. Here, we present optimized protocols for the fluorescent labeling of specific peroxisomal proteins in fixed and living cells. Moreover, detailed measurement protocols for successful STED imaging of human and yeast peroxisomes (using antibodies or genetic tags labeled with dyes) are described, extended with suggestions for individual optimizations.

Fluorescence-based super-resolution-microscopy strategies for chromatin studies.

Super-resolution microscopy (SRM) is a prime tool to study chromatin organisation at near biomolecular resolution in the native cellular environment. With fluorescent labels DNA, chromatin-associated proteins and specific epigenetic states can be identified with high molecular specificity. The aim of this review is to introduce the field of diffraction-unlimited SRM to enable an informed selection of the most suitable SRM method for a specific chromatin-related research question. We will explain both diffraction-unlimited approaches (coordinate-targeted and stochastic-localisation-based) and list their characteristic spatio-temporal resolutions, live-cell compatibility, image-processing, and ability for multi-colour imaging. As the increase in resolution, compared to, e.g. confocal microscopy, leads to a central role of the sample quality, important considerations for sample preparation and concrete examples of labelling strategies applicable to chromatin research are discussed. To illustrate how SRM-based methods can significantly improve our understanding of chromatin functioning, and to serve as an inspiring starting point for future work, we conclude with examples of recent applications of SRM in chromatin research.

Measurement of Nucleoid Size Using STED Microscopy.

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 .

[Frontiers and development in live-cell super-resolution fluorescence microscopy].

This paper reviews the research progress on live-cell super-resolution fluorescence microscopy, discusses the current research status and hotspots in this field, and summarizes the technological application of super-resolution fluorescence microscopy for live-cell imaging. To date, this field has gained progress in numerous aspects. Specifically, the structured illumination microscopy, stimulated emission depletion microscopy, and the recently introduced minimal photon fluxes microscopy are the current research hotspots. According to the current progress in this field, future development trend is likely to be largely driven by artificial intelligence as well as advances in fluorescent probes and relevant labelling methods.

Frontiers and development in live-cell super-resolution fluorescence microscopy / 生物医学工程学杂志

This paper reviews the research progress on live-cell super-resolution fluorescence microscopy, discusses the current research status and hotspots in this field, and summarizes the technological application of super-resolution fluorescence microscopy for live-cell imaging. To date, this field has gained progress in numerous aspects. Specifically, the structured illumination microscopy, stimulated emission depletion microscopy, and the recently introduced minimal photon fluxes microscopy are the current research hotspots. According to the current progress in this field, future development trend is likely to be largely driven by artificial intelligence as well as advances in fluorescent probes and relevant labelling methods.

Nanoscopic distribution of VAChT and VGLUT3 in striatal cholinergic varicosities suggests colocalization and segregation of the two transporters in synaptic vesicles.

Striatal cholinergic interneurons (CINs) use acetylcholine (ACh) and glutamate (Glut) to regulate the striatal network since they express vesicular transporters for ACh (VAChT) and Glut (VGLUT3). However, whether ACh and Glut are released simultaneously and/or independently from cholinergic varicosities is an open question. The answer to that question requires the multichannel detection of vesicular transporters at the level of single synaptic vesicle (SV). Here, we used super-resolution STimulated Emission Depletion microscopy (STED) to characterize and quantify the distribution of VAChT and VGLUT3 in CINs SVs. Nearest-neighbor distances analysis between VAChT and VGLUT3-immunofluorescent spots revealed that 34% of CINs SVs contain both VAChT and VGLUT3. In addition, 40% of SVs expressed only VAChT while 26% of SVs contain only VGLUT3. These results suggest that SVs from CINs have the potential to store simultaneously or independently ACh and/or Glut. Overall, these morphological findings support the notion that CINs varicosities can signal with either ACh or Glut or both with an unexpected level of complexity.

One-Dimensional STED Microscopy in Optical Tweezers.

Optical tweezers and fluorescence microscopy are powerful methods for investigating the mechanical and structural properties of biomolecules and for studying the dynamics of the biomolecular processes that these molecules are involved in. Here we provide an outline of the concurrent use of optical tweezers and fluorescence microscopy for analyzing biomolecular processes. In particular, we focus on the use of super-resolution microscopy in optical tweezers, which allows visualization of molecules at the higher molecular densities that are typically encountered in living systems. We provide specific details on the alignment procedures of the optical pathways for confocal fluorescence microscopy and 1D-STED microscopy and elaborate on how to diagnose and correct optical aberrations and STED phase plate misalignments.

Versatile Near-Infrared Super-Resolution Imaging of Amyloid Fibrils with the Fluorogenic Probe CRANAD-2.

CRANAD-2 is a fluorogenic curcumin derivative used for near-infrared detection and imaging in vivo of amyloid aggregates, which are involved in neurodegenerative diseases. We explore the performance of CRANAD-2 in two super-resolution imaging techniques, namely stimulated emission depletion (STED) and single-molecule localization microscopy (SMLM), with markedly different fluorophore requirements. By conveniently adapting the concentration of CRANAD-2, which transiently binds to amyloid fibrils, we show that it performs well in both techniques, achieving a resolution in the range of 45-55 nm. Correlation of SMLM with atomic force microscopy (AFM) validates the resolution of fine features in the reconstructed super-resolved image. The good performance and versatility of CRANAD-2 provides a powerful tool for near-infrared nanoscopic imaging of amyloids in vitro and in vivo.

Metastasising Fibroblasts Show an HDAC6-Dependent Increase in Migration Speed and Loss of Directionality Linked to Major Changes in the Vimentin Interactome.

Metastasising cells express the intermediate filament protein vimentin, which is used to diagnose invasive tumours in the clinic. We aimed to clarify how vimentin regulates the motility of metastasising fibroblasts. STED super-resolution microscopy, live-cell imaging and quantitative proteomics revealed that oncogene-expressing and metastasising fibroblasts show a less-elongated cell shape, reduced cell spreading, increased cell migration speed, reduced directionality, and stronger coupling between these migration parameters compared to normal control cells. In total, we identified and compared 555 proteins in the vimentin interactome. In metastasising cells, the levels of keratin 18 and Rab5C were increased, while those of actin and collagen were decreased. Inhibition of HDAC6 reversed the shape, spreading and migration phenotypes of metastasising cells back to normal. Inhibition of HDAC6 also decreased the levels of talin 1, tropomyosin, Rab GDI ß, collagen and emilin 1 in the vimentin interactome, and partially reversed the nanoscale vimentin organisation in oncogene-expressing cells. These findings describe the changes in the vimentin interactome and nanoscale distribution that accompany the defective cell shape, spreading and migration of metastasising cells. These results support the hypothesis that oncogenes can act through HDAC6 to regulate the vimentin binding of the cytoskeletal and cell-extracellular matrix adhesion components that contribute to the defective motility of metastasising cells.

A Super-Resolved View of the Alzheimer's Disease-Related Amyloidogenic Pathway in Hippocampal Neurons.

BACKGROUND: Processing of the amyloid-ß protein precursor (AßPP) is neurophysiologically important due to the resulting fragments that regulate synapse biology, as well as potentially harmful due to generation of the 42 amino acid long amyloid ß-peptide (Aß42), which is a key player in Alzheimer's disease. OBJECTIVE: Our aim was to clarify the subcellular locations of the fragments involved in the amyloidogenic pathway in primary neurons with a focus on Aß42 and its immediate substrate AßPP C-terminal fragment (APP-CTF). To overcome the difficulties of resolving these compartments due to their small size, we used super-resolution microscopy. METHODS: Mouse primary hippocampal neurons were immunolabelled and imaged by stimulated emission depletion (STED) microscopy, including three-dimensional three-channel imaging, and quantitative image analyses. RESULTS: The first (ß-secretase) and second (γ-secretase) cleavages of AßPP were localized to functionally and distally distinct compartments. The ß-secretase cleavage was observed in early endosomes in soma, where we were able to show that the liberated N- and C-terminal fragments were sorted into distinct vesicles budding from the early endosomes. Lack of colocalization of Aß42 and APP-CTF in soma suggested that γ-secretase cleavage occurs in neurites. Indeed, APP-CTF was, in line with Aß42 in our previous study, enriched in the presynapse but absent from the postsynapse. In contrast, full-length AßPP was not detected in either the pre- or the postsynaptic side of the synapse. Furthermore, we observed that endogenously produced and endocytosed Aß42 were localized in different compartments. CONCLUSION: These findings provide critical super-resolved insight into amyloidogenic AßPP processing in primary neurons.

Super-Resolution Fluorescence Microscopy Methods for Assessing Mouse Biology.

Super-resolution (diffraction unlimited) microscopy was developed 15 years ago; the developers were awarded the Nobel Prize in Chemistry in recognition of their work in 2014. Super-resolution microscopy is increasingly being applied to diverse scientific fields, from single molecules to cell organelles, viruses, bacteria, plants, and animals, especially the mammalian model organism Mus musculus. In this review, we explain how super-resolution microscopy, along with fluorescence microscopy from which it grew, has aided the renaissance of the light microscope. We cover experiment planning and specimen preparation and explain structured illumination microscopy, super-resolution radial fluctuations, stimulated emission depletion microscopy, single-molecule localization microscopy, and super-resolution imaging by pixel reassignment. The final section of this review discusses the strengths and weaknesses of each super-resolution technique and how to choose the best approach for your research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.

The plant nuclear lamina proteins NMCP1 and NMCP2 form a filamentous network with lateral filament associations.

Plant genomes lack genes encoding intermediate filament proteins, including lamins; however, functional lamin analogues are presumed to exist in plants. Plant-specific coiled-coil proteins, that is, nuclear matrix constituent proteins (NMCPs), are the most likely candidates as the structural elements of the nuclear lamina because they exhibit a lamin-like domain arrangement. They are exclusively localized at the nuclear periphery and have functions that are analogous to those of lamins. However, their assembly into filamentous polymers has not yet been confirmed. In this study, we examined the higher-order structure of NMCP1 and NMCP2 in Apium graveolens cells by using stimulated emission depletion microscopy combined with immunofluorescence cell labelling. Our analyses revealed that NMCP1 and NMCP2 form intricate filamentous networks, which include thick segments consisting of filament bundles, forming a dense filamentous layer extending across the nuclear periphery. Furthermore, the outermost chromatin distribution was found to be in the nucleoplasm-facing region of the nuclear lamina. Recombinant Daucus carota NMCP1 with a His-tag produced in Escherichia coli refolded into dimers and self-assembled into filaments and filament bundles. These results suggest that NMCP1 and NMCP2 organize into the nuclear lamina by forming a filamentous network with filament bundles that localize at the nuclear periphery.

Aberration correction in stimulated emission depletion microscopy to increase imaging depth in living brain tissue.

Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberrations and improve the spatial resolution of STED microscopy inside thick biological tissue. Aim: The aim of our work was to develop and validate a method based on adaptive optics to achieve an a priori correction of spherical aberrations as a function of imaging depth. Approach: We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. We then used a spatial light modulator to apply corrective phase shifts and validate this calibration approach by imaging neurons in living brain slices. Results: After quantifying the spatial resolution in depth in phantom samples, we demonstrated that the corrections can substantially increase image quality in living brain slices. Specifically, we could measure structures as small as 80 nm at a depth of 90 µ m inside the biological tissue and obtain a 60% signal increase after correction. Conclusion: We propose a simple and robust approach to calibrate and compensate the distortions of the STED beam profile introduced by spherical aberrations with increasing imaging depth and demonstrated that this method offers significant improvements in microscopy performance for nanoscale cellular imaging in live tissue.

Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes.

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200-250 nm laterally, ~500-700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4',6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

Visualization of the structure of native human pulmonary mucus.

Human respiratory mucus lining the airway epithelium forms a challenging barrier to inhalation therapeutics. Therefore, structural elucidation of hydrated mucus is essential for an efficient drug delivery development. The structure of mucus has been primarily investigated by conventional electron microscopy techniques, which operate under vacuum conditions and require sample preparation steps that might alter the structure of mucus. In this study we investigated the impact of dehydration on mucus and analyzed the structure of mucus in its hydrated state. Cryo-scanning electron microscopy (Cryo-SEM) analysis of mucus showed, that during the process of sublimation, non-porous structure of mucus is transformed into a porous network. Similarly, images acquired by environmental scanning electron microscopy (ESEM), revealed a non-porous structure of hydrated mucus, while further observation at decreasing pressure demonstrated the strong influence of dehydration on mucus structure. We could successfully visualize the structural organization of the major gel forming mucin MUC5B in its hydrated state by employing stimulated emission depletion (STED) microscopy, which allowed resolving the nano-scale patterns of mucin macromolecules within the essentially pore-free mucus structure. The general structural organization of mucus components was addressed by confocal laser scanning microscopy (CLSM), which revealed the heterogeneous and composite structure of mucus. These results provide a novel view on the native structure of mucus and will affect drug delivery development.

Combining cross-coupling reaction and Knoevenagel condensation in the synthesis of glyco-BODIPY probes for DC-SIGN super-resolution bioimaging.

Lectins are involved in a wide range of carbohydrate mediated recognition processes. Therefore, the availability of highly performant fluorescent tools tailored for lectin targeting and able to efficiently track events related to such key targets is in high demand. We report here on the synthesis of the glyco-BODIPYs 1 and 2, based on the efficient combination of a Heck-like cross coupling and a Knoevenagel condensation, which revealed efficient in addressing lectins. In particular, glyco-BODIPY 1 has two glycosidase stable C-mannose residues, which act as DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) targeting modules. By using live-cell fluorescence microscopy, we proved that BODIPY-mannose 1 was efficiently taken up by immune cells expressing DC-SIGN receptors. Super-resolution stimulated emission depletion (STED) microscopy further revealed that the internalized 1 localized in membranes of endosomes, proving that 1 is a reliable tool also in STED applications. Of note, glyco-BODIPY 1 contains an aryl-azido group, which allows further functionalization of the glycoprobe with bioactive molecules, thus paving the way for the use of 1 for tracking lectin-mediated cell internalization in diverse biological settings.

Expansion STED microscopy (ExSTED).

The recently developed expansion microscopy method (ExM) allows for the resolution of structures below the diffraction limit of light not by sophisticated instrumentation, but rather by physically expanding the molecular structure of cells. This happens by crosslinking the protein in the sample to a hydrogel that is polymerized in situ and subsequently expanded, tearing the proteins apart in a nearly isotropic manner. In the resulting, larger facsimile of the original sample, the fluorescence-labeled molecules of interest can be optically separated by conventional fluorescence microscopy since the intermolecular distances are enlarged by a factor ranging from ~4 to 20 depending on the chemistry used for the hydrogel. The achieved improvement in resolution thus corresponds to the expansion factor. Further increase in resolution beyond this value may be achieved by combining ExM with established super-resolution microscopy methods. Indeed, this is possible using structured illumination microscopy (SIM) (Halpern et al., 2017; Wang et al., 2018), single molecule localization microscopy (SMLM) (Zwettler et al., 2020) and stimulated emission depletion (STED), as we and others have shown recently (Gambarotto et al., 2019; Gao et al., 2018; Kim, Kim, Lee, & Shim, 2019; Unnersjö-Jess et al., 2016). Here, we provide a protocol, for our method, called ExSTED, which enabled us to achieve an increase in resolution of up to 30-fold compared to conventional microscopy, well beyond what is possible with conventional STED microscopy. Our protocol includes a strategy to achieve very high intensity fluorescence labeling, which is essential for optimal signal retention during the expansion process for ExSTED.

Recent Advances on Organic Fluorescent Probes for Stimulated Emission Depletion (STED) Microscopy.

Stimulated emission depletion (STED) microscopy has become a useful tool for visualization and dynamic monitoring at an ultra-high resolution in biological research and material science. For STED technology, fluorescent probes are irreplaceable in the imaging process. Among these probes, organic fluorescent probes have superior photo-stability, high brightness, large Stokes' shifts and excellent biocompatibility, thus are widely applied in STED microscopy. Based on this consideration, this review presents the recent advances on organic fluorescent probes for STED microscopy, including typical organic fluorescent probes, aggregation-induced emission luminogens (AIEgens), polymer dots, and other nanoparticles. The applications of organic fluorescent probes in biological imaging, such as in live-cell, live-tissue, and in vivo imaging, as well as in material monitoring at the nanoscale using STED microscopy, are also included. This review provides the guidelines for the design of new materials that can be used to enhance the imaging performance of STED microscopy, thus leading to real-world applications.