High-resolution Microscopy Research Articles

Effects of Sup35 overexpression on the formation, morphology, and physiological functions of intracellular Sup35 assemblies.

The role of condensates in living cells is often studied by overexpression. For understanding their physiological role, this can be problematic. Overexpression can shift cellular functions, thereby changing the system under study, and overexpression can also affect the phase behavior of condensates by shifting the position of the system in the underlying phase diagram. Our detailed study of overexpression of Sup35 in S. cerevisiae shows the interplay between these factors and highlights basic features of intracellular condensation such as the balance between condensation and aggregation as well as how cellular localization and responsiveness depend on protein levels. We also apply super-resolution microscopy to highlight details within the cells.

Neuronal α-synuclein toxicity is the key driver of neurodegeneration in multiple system atrophy.

Multiple system atrophy (MSA) is a rare, rapidly progressing neurodegenerative disorder often misdiagnosed as Parkinson's disease (PD). While both conditions share some clinical features, MSA is distinct in its pathological hallmark: oligodendroglial cytoplasmic α-synuclein (α-Syn) inclusions, known as glial cytoplasmic inclusions (GCIs). These GCIs are pathognomonic for MSA, but they do not lead to significant oligodendroglial cell loss. Instead, MSA is characterised by a substantially greater loss of non-dopaminergic neurons in the nigrostriatal and olivopontocerebellar systems compared to PD. This widespread neuronal degeneration, which is not seen to the same extent in PD, plays a critical role in MSA's clinical presentation and is important to consider if PD is to be redefined as a neuronal α-Syn disease. It also raises the question of differences in the potential toxicity of lesions in MSA and the underlying cause of neuronal death in MSA. By combining an N-terminus α-Syn antibody that reveals more α-Syn pathology and super-resolution microscopy, we identified α-Syn fibrils in MSA neurons penetrating the nucleus from the cytoplasm, leading to nuclear destruction and neuronal death. Our data indicate an early invasion of neuronal nuclei by α-Syn pathology in MSA, precipitating rapid nuclear envelope destruction, as observed through significant structural damage, including the loss of Lamin integrity. Although the progression of α-Syn pathology from the cytoplasm to the nucleus may be similar in oligodendroglia and neurons, the aggregation state of the α-Syn proteoforms involved differs as proteolytic resistance of α-Syn inclusions is significantly higher in neurons and the nucleus is destroyed. We describe the progressive impact of α-Syn nuclear pathology on MSA neurons and show that this is a more detrimental and rapid pathology driving neurodegeneration. Our data suggest that oligodendroglial inclusions contain more soluble, less toxic α-Syn proteoforms, consistent with two distinct α-Syn filaments in MSA. We propose renaming MSA as a neuronal nuclear and oligodendroglial α-synucleinopathy to better reflect these two distinct pathologies.

Super-resolution microscopy based on the inherent fluctuations of dye molecules

Fluorescence microscopy is a critical tool across various disciplines, from materials science to biomedical research, yet it is limited by the diffraction limit of resolution. Advanced super-resolution techniques such as localization microscopy and stimulated-emission-depletion microscopy often demand considerable resources. These methods depend heavily on elaborate sample-staining, complex optical systems, or prolonged acquisition periods, and their application in 3D and multicolor imaging presents significant experimental challenges. In the current work, we provide a complete demonstration of a widely accessible super-resolution imaging approach capable of 3D and multicolor imaging based on super-resolution optical fluctuation imaging (SOFI). We replace the confocal pinhole with an array of single-photon avalanche diodes and use the microsecond-scale fluctuations of dye molecules as a contrast mechanism. This contrast is transformed into a super-resolved image using a robust and deterministic algorithm. Our technique utilizes natural fluctuations inherent to organic dyes, thereby it does not require engineering of the blinking statistics. Our robust, versatile super-resolution method opens the way to next-generation multimodal imaging and facilitates on-demand super-resolution within a confocal architecture.

Differentiable pixel-super-resolution lensless imaging

Conventional lensless imaging systems require complex phase diversity measurements and sequential processing steps, limiting their practical application despite their compact design. We present a differentiable end-to-end pixel-super-resolution (dPSR) technique that unifies PSR hologram synthesis, autofocusing, and complex-field reconstruction within a single optimization framework. By jointly optimizing these traditionally separate processes, our method eliminates both phase diversity requirements and error accumulation from sequential processing. Our method achieves superior position estimation accuracy (mean error 0.0282 pixels versus 0.1172 pixels with conventional methods), delivering precise autofocusing with accuracy better than 0.3 µm, and enabling a twofold resolution enhancement beyond the sensor’s native pixel size. This robust performance is validated through both simulated and experimental results, including challenging phase objects and label-free cell imaging, establishing dPSR as a practical solution for high-resolution microscopy applications.

Microscopy methods for the in vivo study of nanoscale nuclear organization.

Eukaryotic genomes are highly compacted within the nucleus and organized into complex 3D structures across various genomic and physical scales. Organization within the nucleus plays a key role in gene regulation, both facilitating regulatory interactions to promote transcription while also enabling the silencing of other genes. Despite the functional importance of genome organization in determining cell identity and function, investigating nuclear organization across this wide range of physical scales has been challenging. Microscopy provides the opportunity for direct visualization of nuclear structures and has pioneered key discoveries in this field. Nonetheless, visualization of nanoscale structures within the nucleus, such as nucleosomes and chromatin loops, requires super-resolution imaging to go beyond the ~220 nm diffraction limit. Here, we review recent advances in imaging technology and their promise to uncover new insights into the organization of the nucleus at the nanoscale. We discuss different imaging modalities and how they have been applied to the nucleus, with a focus on super-resolution light microscopy and its application to in vivo systems. Finally, we conclude with our perspective on how continued technical innovations in super-resolution imaging in the nucleus will advance our understanding of genome structure and function.

Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.

Live imaging of excitable axonal microdomains in ankyrin-G-GFP mice.

The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine in vivo live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native Ank3 gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings in vitro, ex vivo, and in vivo, we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible in vivo labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in vivo in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.

3D Single-Molecule Super-Resolution Imaging of Microfabricated Multiscale Fractal Substrates for Calibration and Cell Imaging.

Microstructures arrayed over a substrate have shown increasing interest due to their ability to provide advanced 3D cellular models, which open up new possibilities for cell culture, proliferation, and differentiation. Still, the mechanisms by which physical cues impact the cell phenotype are not fully understood, hence the necessity to interrogate cell behavior at the highest resolution. However, cell 3D high-resolution optical imaging on such microstructured substrates remains challenging due to their complexity as well as axial calibration issues. In this work, we address this issue by leveraging the geometrical characteristics of fractal-like structures, which serve as axial calibration tools and modulate cell growth. To this end, we use multiscale 3D SiO2 substrates consisting of spatially arrayed octahedral features of a few micrometers to hundreds of nanometers. Through optimizations of both the structures and optical imaging conditions, we demonstrate the potential of these 3D multiscale structures as an alternative to electron microscopy for material imaging but also as calibration tools for 3D super-resolution microscopy. We used their multiscale and known geometry to perform lateral and axial calibrations in 3D single-molecule localization microscopy (SMLM) and assess imaging resolutions. We then utilized these substrates as a platform for high-resolution bioimaging. As a proof of concept, we cultivate human mesenchymal stem cells on these substrates, revealing very different growth patterns compared to flat glass. Specifically, the spatial distribution of cytoskeleton proteins is vastly modified, as we demonstrate with a 3D SMLM assessment.

Abstract TP344: Ketamine Modulates Astrocyte-Secreted Proteins to Promote Recovery in a Mouse Model of Stroke

Introduction: Astrocytes are non-neuronal cells in the CNS that play crucial roles in synaptic formation, maturation, and maintenance through the synthesis and secretion of various proteins. We identified Chordin-like 1 (Chrdl1) as an astrocyte-secreted protein that limits synaptic plasticity by stabilizing GluA2-containing AMPA receptors (GluA2-AMPARs) at synaptic sites in the cortex. In a mouse model of ischemic stroke, we found that Chrdl1 is excessively upregulated in astrocytes in the peri-infarct area during the post-stroke plasticity window (2–30 days post-stroke in the mouse). We found that Chrdl1 KO mice displayed enhanced synaptic plasticity, and in response to stroke, deficits such as dendritic spine degeneration, impaired GluA2-AMPAR distribution, and motor dysfunction were significantly alleviated. Seeking a pharmacological approach to mitigate stroke-induced Chrdl1 upregulation, we explored the use of sub-anesthetic ketamine, known to enhance plasticity and alter astrocytic function, potentially affecting protein secretion. However, its impact on post-stroke astrocyte-regulated plasticity and recovery has not yet been explored. We hypothesize that sub-anesthetic ketamine may restrict the upregulation of astrocytic Chrdl1 during the post-stroke plasticity window and reduce stroke-induced deficits. Methods: To generate ischemia in vivo , we used photothrombosis to induce distal middle cerebral artery occlusion (PT-dMCAO). Male and female adult mice (2 months old) received a daily dose of sub-anesthetic ketamine (10mg/kg) via intranasal injection for one week. We used single molecule fluorescence in situ hybridization (smFISH) and immunohistochemistry to assess Chrdl1 mRNA and protein levels in peri-infarct astrocytes, synaptic staining and high-resolution confocal microscopy to analyze GluA2-AMPARs distribution, Golgi stainings to study dendritic branching and spine density, and several behavioral tests to assess motor performance, sensorimotor function, and learning and memory. Results: We found that sub-anesthetic ketamine treatment reverts stroke-induced Chrdl1 upregulation in peri-infarct astrocytes and mitigates molecular, structural and behavioral deficits. Conclusions: Our study suggests that sub-anesthetic ketamine treatment is a promising approach to limit astrocyte-driven molecular deficits during the critical post-stroke plasticity window, which may otherwise hinder recovery, and holds great promise for clinical translation.

Tetraspanin CD9 alters cellular trafficking and endocytosis of tetraspanin CD63, affecting CD63 packaging into small extracellular vesicles.

Small extracellular vesicles (sEVs) are particles secreted from cells that play vital roles both in normal physiology and in human disease. sEVs are highly enriched in tetraspanin proteins, such as CD9 and CD63, and contain tetraspanin-enriched membrane microdomains involved in loading of sEVs with macromolecule cargoes and in sEV biogenesis. However, the precise roles of individual tetraspanins in sEV biogenesis and cargo loading remain poorly understood. Here, we report that CD9 negatively regulated CD63 trafficking to tetraspanin-enriched microdomains and its subsequent packaging into small EVs, whereas CD63 had no discernable effect on CD9 localization or packaging. Using super resolution microscopy of individual vesicles, we showed that CD9 governs the fraction of sEVs that are loaded with CD63. Interestingly, CD9-dependent suppression of CD63 packaging was rescued by pharmacological blockade of endocytosis. Together, our data support a model where CD9 contributes to the regulation and secretion of CD63 in an endocytosis-dependent manner to reprogram the contents of sEVs and tetraspanin-enriched microdomains.

FluProCAD: a computational screening workflow for fluorescent protein variants

Fluorescent proteins are the backbone of modern high-resolution microscopy, but natural variants often require optimisation to perform effectively. While directed evolution is commonly used for such optimisation, predicting mutation effects requires a deep understanding of photophysics and photochemistry at the atomic scale. Computational chemistry provides a route to obtain such insights from first principles but requires significant expertise. To address this, we developed FluProCAD, a command-line-based workflow that automates system setup and computation of key properties of fluorescent protein mutants using established atomistic models, without the need for prior modeling experience. We applied FluProCAD to two case studies. First, we evaluated the optical and thermodynamic properties of Aequorea victoria Green Fluorescent Protein (avGFP) mutants, successfully reproducing changes in optical responses and folding and dimerisation free energies for five variants. Second, we predicted structural changes in 14 rsGreen0.7 protein variants and validated these models against experimental crystal structures. These results demonstrate the potential of FluProCAD to streamline the optimisation of fluorescent proteins, and expand the computational toolkit for advancing their performance.

Structural glycobiology - from enzymes to organelles.

Biological carbohydrate polymers represent some of the most complex molecules in life, enabling their participation in a huge range of physiological functions. The complexity of biological carbohydrates arises from an extensive enzymatic repertoire involved in their construction, deconstruction and modification. Over the past decades, structural studies of carbohydrate processing enzymes have driven major insights into their mechanisms, supporting associated applications across medicine and biotechnology. Despite these successes, our understanding of how multienzyme networks function to create complex polysaccharides is still limited. Emerging techniques such as super-resolution microscopy and cryo-electron tomography are now enabling the investigation of native biological systems at near molecular resolutions. Here, we review insights from classical in vitro studies of carbohydrate processing, alongside recent in situ studies of glycosylation-related processes. While considerable technical challenges remain, the integration of molecular mechanisms with true biological context promises to transform our understanding of carbohydrate regulation, shining light upon the processes driving functional complexity in these essential biomolecules.

Co-linear Hexa-Mirror-Based Multi-Periodic Structured Illumination Microscopy.

Structured illumination microscopy (SIM) is a robust wide-field optical nanoscopy technique. Several approaches are implemented to improve SIM's resolution capability (∼2-fold). However, achieving a high resolution with a large field of view (FOV) is still challenging. We present tilt-mirror-based multi-periodic SIM for large-FOV super-resolution microscopy. The sample is illuminated by a multi-periodic structured pattern generated by six-beam interference using a custom-designed mirror mount. We achieve 3.16-fold resolution improvement while using a 20×/0.40 numerical-aperture objective that supports a large FOV (0.53 mm × 0.34 mm). This overcomes the high-space-bandwidth product challenge, achieving 9.98-fold improvement. mMP-SIM decouples illumination and collection paths, enabling scalable super-resolution over a large FOV. By using a 28×/0.80 numerical-aperture objective lens, an optical resolution of 170 nm over a 0.40 mm × 0.25 mm imaging area is demonstrated. The proof-of-principle experimental demonstration is performed for both fluorescent beads and a biosample like U2OS (human bone osteosarcoma) cells.

Hyperbolic polariton-coupled emission optical microscopy

Abstract A new type of optical microscopy based on hyperbolic polariton-coupled emission (HPCE) is demonstrated. By employing hyperbolic metamaterials as the substrate, we show a nearly 6-fold increase in fluorescence intensity in the HPCE microscope compared to total internal reflection fluorescence (TIRF) on glass substrates. Moreover, we achieve precise, time-dependent control of the fluorescence intensity by modulating the incidence angle with a galvo scanner. This tunability offers extensive potential for applications in super-resolution fluorescence microscopy and high-sensitivity sensing, enabling real-time fluorescence intensity adjustment.

Carbon Nanodots as a Red Emissive Fluorescent Probe for the Super‐Resolution Microscopy of DNA Dynamics during Paclitaxel Treatment

Paclitaxel is a commonly used frontline chemotherapeutic drug for cancer treatment. It is known to be functional by arresting the microtubule disassembly during mitosis. Recently, a nonmitotic pathway has been evolving and thus contemplating the mitotic mechanism. Herein, using super‐resolution microscopy (SRM), the nuclear dynamics is directly visualized and the mechanism of paclitaxel treatment is unveiled. A new class of nontoxic, biocompatible, and highly fluorescent carbon nanodots (CNDs) are used as a fluorescent probe that are highly capable to directly stain the nuclear DNA and capture the SRM imaging of chromosomes and the chromatin structures. Apart from SRM imaging of chromosomes during all stages of normal mitotic cell division, CNDs successfully visualize the formation of lagging, mis‐segregated, and bridging chromosomes, leading to the multi‐micronucleus formation upon paclitaxel treatment. A detailed chromatin remodeling analysis suggests that heterochromatin plays an important role in the formation of condensed multi‐micronucleus, ultimately leading to cell death.

High-resolution microscopy of biofilm and bacteria on titanium implants

Abstract Implant-associated spine infection (IASI) is related to bacteria and the formation of biofilm. Numerous analyses on the formation mechanism of biofilm have been reported based on in vitro studies. However, the available literature is lacking detailed high-resolution characterization of biofilms on surgically removed implants. The present work deals with the development of a simplified fixation protocol applied on ex-situ Ti-6Al-4V implants. High-resolution scanning electron microscopy was applied for detailed characterization of various types of bacteria as well as biofilm structures. Erythrocytes and Staphylococci as well as fibrin networks were observed on implants removed from different patients. Moreover, the application of the new fixation protocol together with high-resolution microscopy revealed fully developed biofilms totally covering the bacteria as well as very fine structures attaching the bacteria to the implant surfaces, corresponding to early stages of biofilm formation.

Super-resolution imaging of proteins inside live mammalian cells with mLIVE-PAINT.

Super-resolution microscopy has revolutionized biological imaging, enabling the visualization of structures at the nanometer length scale. Its application in live cells, however, has remained challenging. To address this, we adapted LIVE-PAINT, an approach we established in yeast, for application in live mammalian cells. Using the 101A/101B coiled-coil peptide pair as a peptide-based targeting system, we successfully demonstrate the super-resolution imaging of two distinct proteins in mammalian cells, one localized in the nucleus, and the second in the cytoplasm. This study highlights the versatility of LIVE-PAINT, suggesting its potential for live-cell super-resolution imaging across a range of protein targets in mammalian cells. We name the mammalian cell version of our original method mLIVE-PAINT.

Context-specific inhibition of mitochondrial ribosomes by phenicol and oxazolidinone antibiotics.

The antibiotics chloramphenicol (CHL) and oxazolidinones, including linezolid (LZD), are known to inhibit mitochondrial translation. This can result in serious, potentially deadly, side effects when used therapeutically. Although the mechanism by which CHL and LZD inhibit bacterial ribosomes has been elucidated in detail, their mechanism of action against mitochondrial ribosomes has yet to be explored. CHL and oxazolidinones bind to the ribosomal peptidyl transfer center (PTC) of the bacterial ribosome and prevent incorporation of incoming amino acids under specific sequence contexts, causing ribosomes to stall only at certain sequences. Through mitoribosome profiling, we show that inhibition of mitochondrial ribosomes is similarly context-specific-CHL and LZD lead to mitoribosome stalling primarily when there is an alanine, serine, or threonine in the penultimate position of the nascent peptide chain. We further validate context-specific stalling through in vitro translation assays. A high-resolution cryo-electron microscopy structure of LZD bound to the PTC of the human mitoribosome shows extensive similarity to the mode of bacterial inhibition and also suggests potential avenues for altering selectivity. Our findings could help inform the rational development of future, less mitotoxic, antibiotics, which are critically needed in the current era of increasing antimicrobial resistance.

Caliber of zebrafish touch-sensory axons is dynamic in vivo.

Cell shape is crucial to cell function, particularly in neurons. The cross-sectional diameter, also known as caliber, of axons and dendrites is an important parameter of neuron shape, best appreciated for its influence on the speed of action potential propagation. Many studies of axon caliber focus on cell-wide regulation and assume that caliber is static. Here, we have characterized local variation and dynamics of axon caliber in vivo using the peripheral axons of zebrafish touch-sensing neurons at embryonic stages, prior to sex determination. To obtain absolute measurements of caliber in vivo, we paired sparse membrane labeling with super-resolution microscopy of neurons in live fish. We observed that axon segments had varicose or "pearled" morphologies, and thus vary in caliber along their length, consistent with reports from mammalian systems. Sister axon segments originating from the most proximal branch point in the axon arbor had average calibers that were uncorrelated with each other. Axon caliber also tapered across the branch point. Varicosities and caliber, overall, were dynamic on the timescale of minutes, and dynamicity changed over the course of development. By measuring the caliber of axons adjacent to dividing epithelial cells, we found that skin cell division is one aspect of the cellular microenvironment that may drive local differences and dynamics in axon caliber. Our findings support the possibility that spatial and temporal variation in axon caliber could significantly influence neuronal physiology.

ResShift-4E: Improved Diffusion Model for Super-Resolution with Microscopy Images

Blind super-resolution algorithms based on diffusion models still face significant challenges at the current stage, including high computational cost, long inference time, and limited cross domain generalization ability. This paper aims to apply super-resolution algorithms to the field of optical microscopy imaging to reveal more microscopic structures and details. Firstly, we proposed a lightweight super-resolution model called ResShift-4E, which is an optimized model from two important aspects: reducing the diffusion steps in ResShift and strengthening the influence of the original residuals on model learning. Secondly, we constructed a dataset of Multimodal High-resolution Microscopy Images (MHMI) including a total of 1220 images, which is available on line. Moreover, we extended our model to application-oriented research on blind image super-resolution of optical microscopy imaging. The experimental results demonstrate that our ResShift-4E model outperforms other models on various microscopy images.