Modeling the Temperature-Dependent Size Change of Polydisperse Nano-objects using a Deep Generative Model.

Modern microscopy techniques can be used to investigate soft nano-objects at the nanometer scale. However, time-consuming microscopy measurements combined with low numbers of observable polydisperse objects often limit the statistics. We propose a method for identifying the most representative objects from their respective point clouds. These point cloud data are obtained, for example, through the localization of single emitters in super-resolution fluorescence microscopy. External stimuli, such as temperature, can cause changes in the shape and properties of adaptive objects. Due to the demanding and time-consuming nature of super-resolution microscopy experiments, only a limited number of temperature steps can be performed. Therefore, we propose a deep generative model that learns the underlying point distribution of temperature-dependent microgels, enabling the reliable generation of unlimited samples with an arbitrary number of localizations. Our method greatly cuts down the data collection effort across diverse experimental conditions, proving invaluable for soft condensed matter studies.

A nanoscale visual exploration of the pathogenic effects of bacterial extracellular vesicles on host cells.

BACKGROUND: Bacterial extracellular vesicles (EVs) are pivotal mediators of intercellular communication and influence host cell biology, thereby contributing to the pathogenesis of infections. Despite their significance, the precise effects of bacterial EVs on the host cells remain poorly understood. This study aimed to elucidate ultrastructural changes in host cells upon infection with EVs derived from a pathogenic bacterium, Staphylococcus aureus (S. aureus). RESULTS: Using super-resolution fluorescence microscopy and high-voltage electron microscopy, we investigated the nanoscale alterations in mitochondria, endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and microtubules of skin cells infected with bacterial EVs. Our results revealed significant mitochondrial fission, loss of cristae, transformation of the ER from tubular to sheet-like structures, and fragmentation of the Golgi apparatus in cells infected with S. aureus EVs, in contrast to the negligible effects observed following S. epidermidis EV infection, probably due to the pathogenic factors in S. aureus EV, including protein A and enterotoxin. These findings indicate that bacterial EVs, particularly those from pathogenic strains, induce profound ultrastructural changes of host cells that can disrupt cellular homeostasis and contribute to infection pathogenesis. CONCLUSIONS: This study advances the understanding of bacterial EV-host cell interactions and contributes to the development of new diagnostic and therapeutic strategies for bacterial infections.

Local Water Content in Polymer Gels Measured with Super-Resolved Fluorescence Lifetime Imaging.

Water molecules play an important role in the structure, function, and dynamics of (bio-) materials. A direct access to the number of water molecules in nanoscopic volumes can thus give new molecular insights into materials and allow for fine-tuning their properties in sophisticated applications. The determination of the local water content has become possible by the finding that H2 O quenches the fluorescence of red-emitting dyes. Since deuterated water, D2 O, does not induce significant fluorescence quenching, fluorescence lifetime measurements performed in different H2 O/D2 O-ratios yield the local water concentration. We combined this effect with the recently developed fluorescence lifetime single molecule localization microscopy imaging (FL-SMLM) in order to nanoscopically determine the local water content in microgels, i.e. soft hydrogel particles consisting of a cross-linked polymer swollen in water. The change in water content of thermo-responsive microgels when changing from their swollen state at room temperature to a collapsed state at elevated temperature could be analyzed. A clear decrease in water content was found that was, to our surprise, rather uniform throughout the entire microgel volume. Only a slightly higher water content around the dye was found in the periphery with respect to the center of the swollen microgels.

Wide-Field Super-Resolution Optical Fluctuation Imaging through Dynamic Near-Field Speckle Illumination.

Stochastic optical fluctuation imaging (SOFI) generates super-resolution fluorescence images by emphasizing the positions of fluorescent emitters via statistical analysis of their on-and-off blinking dynamics. In SOFI with speckle illumination (S-SOFI), the diffraction-limited grain size of the far-field speckles prevents independent blinking of closely located emitters, becoming a hurdle to realize the full super-resolution granted by SOFI processing. Here, we present a surface-sensitive super-resolution technique exploiting dynamic near-field speckle illumination to bring forth the full super-resolving power of SOFI without blinking fluorophores. With our near-field S-SOFI technique, up to 2.8- and 2.3-fold enhancements in lateral spatial resolution are demonstrated with computational and experimental fluorescent test targets labeled with conventional fluorophores, respectively. Fluorescent beads separated by 175 nm are also super-resolved by near-field speckles of 150 nm grain size, promising sub-100 nm resolution with speckle patterns of much smaller grain size.

[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.

Studying SARS-CoV-2 with Fluorescence Microscopy.

The COVID-19 pandemic caused by SARS-CoV-2 coronavirus deeply affected the world community. It gave a strong impetus to the development of not only approaches to diagnostics and therapy, but also fundamental research of the molecular biology of this virus. Fluorescence microscopy is a powerful technology enabling detailed investigation of virus-cell interactions in fixed and live samples with high specificity. While spatial resolution of conventional fluorescence microscopy is not sufficient to resolve all virus-related structures, super-resolution fluorescence microscopy can solve this problem. In this paper, we review the use of fluorescence microscopy to study SARS-CoV-2 and related viruses. The prospects for the application of the recently developed advanced methods of fluorescence labeling and microscopy-which in our opinion can provide important information about the molecular biology of SARS-CoV-2-are discussed.

Quantification of the HIV-1 virus-like particle production process by super-resolution imaging: From VLP budding to nanoparticle analysis.

Virus-like particles (VLPs) offer great promise in the field of nanomedicine. Enveloped VLPs are a class of these nanoparticles and their production process occurs by a budding process, which is known to be the most critical step at intracellular level. In this study, we developed a novel imaging method based on super-resolution fluorescence microscopy (SRFM) to assess the generation of VLPs in living cells. This methodology was applied to study the production of Gag VLPs in three animal cell platforms of reference: HEK 293-transient gene expression (TGE), High Five-baculovirus expression vector system (BEVS) and Sf9-BEVS. Quantification of the number of VLP assembly sites per cell ranged from 500 to 3,000 in the different systems evaluated. Although the BEVS was superior in terms of Gag polyprotein expression, the HEK 293-TGE platform was more efficient regarding the assembly of Gag as VLPs. This was translated into higher levels of non-assembled Gag monomer in BEVS harvested supernatants. Furthermore, the presence of contaminating nanoparticles was evidenced in all three systems, specifically in High Five cells. The SRFM-based method here developed was also successfully applied to measure the concentration of VLPs in crude supernatants. The lipid membrane of VLPs and the presence of nucleic acids alongside these nanoparticles could also be detected using common staining procedures. Overall, a complete picture of the VLP production process was achieved in these three production platforms. The robustness and sensitivity of this new approach broaden the applicability of SRFM toward the development of new detection, diagnosis and quantification methods based on confocal microscopy in living systems.

Simultaneous multicolour imaging using quantum dot structured illumination microscopy.

Multicolour structured illumination microscopy (SIM) is a powerful tool used for the investigation of the dynamic interaction between subcellular structures. Nevertheless, most of the multicolour SIM schemes are currently limited by conventional fluorescent dyes and wavelength-dependent optical systems, and can only sequentially record images of different colour channels instead of obtaining multicolour datasets simultaneously. To address these issues, we present a novel multicolour SIM scheme referred to as quantum dot structured illumination microscopy (QD-SIM). QD-SIM enables simultaneously excitation and collection of multicolour fluorescent signals. We also propose a theoretical analysis of the image formation in two-dimensional multicolour SIM to help combine the optically sectioned and super-resolution attributes of SIM. Based on this theory, QD-SIM enables optically sectioned, super-resolution, multicolour simultaneous imaging at a single plane.

High spatiotemporal resolution and low photo-toxicity fluorescence imaging in live cells and in vivo.

Taking advantage of high contrast and molecular specificity, fluorescence microscopy has played a critical role in the visualization of subcellular structures and function, enabling unprecedented exploration from cell biology to neuroscience in living animals. To record and quantitatively analyse complex and dynamic biological processes in real time, fluorescence microscopes must be capable of rapid, targeted access deep within samples at high spatial resolutions, using techniques including super-resolution fluorescence microscopy, light sheet fluorescence microscopy, and multiple photon microscopy. In recent years, tremendous breakthroughs have improved the performance of these fluorescence microscopies in spatial resolution, imaging speed, and penetration. Here, we will review recent advancements of these microscopies in terms of the trade-off among spatial resolution, sampling speed and penetration depth and provide a view of their possible applications.

Fluorescent Diarylethene Photoswitches-A Universal Tool for Super-Resolution Microscopy in Nanostructured Materials.

Super-resolution fluorescence microscopy allows for unprecedented in situ visualization of biological structures, but its application to materials science has so far been comparatively limited. One of the main reasons is the lack of powerful dyes that allow for labeling and photoswitching in materials science systems. In this study it is shown that appropriate substitution of diarylethenes bearing a fluorescent closed and dark open form paves the way for imaging nanostructured materials with three of the most popular super-resolution fluorescence microscopy methods that are based on different concepts to achieve imaging beyond the diffraction limit of light. The key to obtain optimal resolution lies in a proper control over the photochemistry of the photoswitches and its adaption to the system to be imaged. It is hoped that the present work will provide researchers with a guide to choose the best photoswitch derivative for super-resolution microscopy in materials science, just like the correct choice of a Swiss Army Knife's tool is essential to fulfill a given task.

Coupling between chromosome intermingling and gene regulation during cellular differentiation.

In this article, we summarize current findings for the emergence of biophysical properties such as nuclear stiffness, chromatin compaction, chromosome positioning, and chromosome intermingling during stem cell differentiation, which eventually correlated with the changes of gene expression profiles during cellular differentiation. An overview is first provided to link stem cell differentiation with alterations in nuclear architecture, chromatin compaction, along with nuclear and chromatin dynamics. Further, we highlight the recent biophysical and molecular approaches, imaging methods and computational developments in characterizing transcription-related chromosome organization especially chromosome intermingling and nano-scale chromosomal contacts. Finally, the article ends with an outlook towards the emergence of a functional roadmap in setting up chromosome positioning and intermingling in a cell type specific manner during cellular differentiation.

Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy.

Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction. In addition, these tools allow 3D mapping of specific DNA/RNA sequences and nuclear proteins relative to the 3D chromatin compaction maps and comparisons of multiple cell nuclei. The tools are available in the free and open source R packages nucim and bioimagetools. We discuss the use of masks for the segmentation of nuclei and the use of DNA stains, such as DAPI, as a proxy for local differences in chromatin compaction. We further discuss the limitations of 3D maps of the nuclear landscape as well as problems of the biological interpretation of such data.

Nanoscopic Visualization of Cross-Linking Density in Polymer Networks with Diarylethene Photoswitches.

The in situ nanoscopic imaging of soft matter polymer structures is of importance to gain knowledge of the relationship between structure, properties, and functionality on the nanoscopic scale. Cross-linking of polymer chains effects the viscoelastic properties of gels. The correlation of mechanical properties with the distribution and amount of cross-linkers is relevant for applications and for a detailed understanding of polymers on the molecular scale. We introduce a super-resolution fluorescence-microscopy-based method for visualizing and quantifying cross-linker points in polymer systems. A novel diarylethene-based photoswitch with a highly fluorescent closed and a non-fluorescent open form is used as a photoswitchable cross-linker in a polymer network. As an example for its capability to nanoscopically visualize cross-linking, we investigate pNIPAM microgels as a system known with variations in internal cross-linking density.

The molecular architecture of hemidesmosomes, as revealed with super-resolution microscopy.

Hemidesmosomes have been extensively studied with immunofluorescence microscopy, but owing to its limited resolution, the precise organization of hemidesmosomes remains poorly understood. We studied hemidesmosome organization in cultured keratinocytes with two- and three-color super-resolution microscopy. We observed that, in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments and that ß4 integrin (also known as ITGB4) is distributed along, rather than under, keratin filaments. By applying innovative methods to quantify molecular distances, we demonstrate that the hemidesmosomal plaque protein plectin interacts simultaneously and asymmetrically with ß4 integrin and keratin. Furthermore, we show that BP180 (BPAG2, also known as collagen XVII) and BP230 (BPAG1e, an epithelial splice variant of dystonin) are characteristically arranged within hemidesmosomes with BP180 surrounding a central core of BP230 molecules. In skin cross-sections, hemidesmosomes of variable sizes could be distinguished with BP230 and plectin occupying a position in between ß4 integrin and BP180, and the intermediate filament system. In conclusion, our data provide a detailed view of the molecular architecture of hemidesmosomes in cultured keratinocytes and skin.

Progress in the Correlative Atomic Force Microscopy and Optical Microscopy.

Atomic force microscopy (AFM) has evolved from the originally morphological imaging technique to a powerful and multifunctional technique for manipulating and detecting the interactions between molecules at nanometer resolution. However, AFM cannot provide the precise information of synchronized molecular groups and has many shortcomings in the aspects of determining the mechanism of the interactions and the elaborate structure due to the limitations of the technology, itself, such as non-specificity and low imaging speed. To overcome the technical limitations, it is necessary to combine AFM with other complementary techniques, such as fluorescence microscopy. The combination of several complementary techniques in one instrument has increasingly become a vital approach to investigate the details of the interactions among molecules and molecular dynamics. In this review, we reported the principles of AFM and optical microscopy, such as confocal microscopy and single-molecule localization microscopy, and focused on the development and use of correlative AFM and optical microscopy.

Do mechanical forces contribute to nanoscale membrane organisation in T cells?

Mechanotransduction describes how a cell senses and interacts with its environment. The concept originated in adhesion biology where adhesion receptors, integrins, facilitate force transmission between the extracellular matrix and the intracellular actin cytoskeleton. Indeed, during any adhesive contacts, cells do exert mechanical force. Hence, the probing of the local environment by cells results in mechanical cues that contribute to cellular functions and cell fate decisions such as migration, proliferation, differentiation and apoptosis. On the molecular level, mechanical forces can rearrange proteins laterally within the membrane, regulate their activity by inducing conformational changes and probe the mechanical properties and bond strength of receptor-ligands. From this point of view, it appears surprising that molecular forces have been largely overlooked in membrane organisation and ligand discrimination processes in lymphocytes. During T cell activation, the T cell receptor recognises and distinguishes antigenic from benign endogenous peptides to initiate the reorganisation of membrane proteins into signalling clusters within the immunological synapse. In this review, we asked whether characteristics of fibroblast force sensing could be applied to immune cell antigen recognition and signalling, and outline state-of-the-art experimental strategies for studying forces in the context of membrane organisation. This article is part of a Special Issue entitled: Nanoscale membrane orgainisation and signalling.

Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution.

Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.

Nanoscale single-vesicle analysis: High-throughput approaches through AI-enhanced super-resolution image analysis.

The analysis of membrane vesicles at the nanoscale level is crucial for advancing the understanding of intercellular communication and its implications for health and disease. Despite their significance, the nanoscale analysis of vesicles at the single particle level faces challenges owing to their small size and the complexity of biological fluids. This new vesicle analysis tool leverages the single-molecule sensitivity of super-resolution microscopy (SRM) and the high-throughput analysis capability of deep-learning algorithms. By comparing classical clustering methods (k-means, DBSCAN, and SR-Tesseler) with deep-learning-based approaches (YOLO, DETR, Deformable DETR, and Faster R-CNN) for the analysis of super-resolution fluorescence images of exosomes, we identified the deep-learning algorithm, Deformable DETR, as the most effective. It showed superior accuracy and a reduced processing time for detecting individual vesicles from SRM images. Our findings demonstrate that image-based deep-learning-enhanced methods from SRM images significantly outperform traditional coordinate-based clustering techniques in identifying individual vesicles and resolving the challenges related to misidentification and computational demands. Moreover, the application of the combined Deformable DETR and ConvNeXt-S algorithms to differently labeled exosomes revealed its capability to differentiate between them, indicating its potential to dissect the heterogeneity of vesicle populations. This breakthrough in vesicle analysis suggests a paradigm shift towards the integration of AI into super-resolution imaging, which is promising for unlocking new frontiers in vesicle biology, disease diagnostics, and the development of vesicle-based therapeutics.

Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles.

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

Super-resolution fluorescence microscopic imaging in pathogenesis and drug treatment of neurological disease.

Since super-resolution fluorescence microscopic technology breaks the diffraction limit that has existed for a long time in optical imaging, it can observe the process of synapses formed between nerve cells and the protein aggregation related to neurological disease. Thus, super-resolution fluorescence microscopic imaging has significantly impacted several industries, including drug development and pathogenesis research, and it is anticipated that it will significantly alter the future of life science research. Here, we focus on several typical super-resolution fluorescence microscopic technologies, introducing their benefits and drawbacks, as well as applications in several common neurological diseases, in the hope that their services will be expanded and improved in the pathogenesis and drug treatment of neurological diseases.