Search

Copyright of Journal of Beijing University of Technology is the property of Journal of Beijing University of Technology, Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Hepatic ischemia-reperfusion injury (HIRI) is characterized by mitochondrial dysfunction and oxidative stress. Monitoring mitochondrial hydrogen peroxide (mtH2O2) levels in real-time through super-resolution imaging is crucial for elucidating its distribution in live cells and its mechanism of action during HIRI. However, low-background fluorogenic probes have been overlooked in the context of super-resolution imaging. In this study, we developed a low-background fluorogenic probe (Mito-WG) with the potential for super-resolution morphology-correlated mitochondrial identification to track the fluctuates of mtH2O2 in HIRI. Activation of the desirable fluorescence properties of the probe by mtH2O2 was confirmed using structural illumination microscopy (SIM), enabling high-quality mitochondrial imaging with exceptional specificity and sensitivity. Fluctuations in mtH2O2 levels were successfully observed in both cellular and rat models of HIRI. Furthermore, we associated the decline in mitochondrial redox homeostasis with accelerated mtH2O2 production during HIRI, which triggered mitophagy deficiency and led to cell death. In conclusion, Mito-WG possesses excellent photophysical and low-background properties for SIM imaging, making it a promising tool for mtH2O2 tracking in HIRI research and clinical diagnosis. [ABSTRACT FROM AUTHOR]

The rapid development of super-resolution microscopy has made it possible to observe subcellular structures and dynamic behaviors in living cells with nanoscale spatial resolution, greatly advancing progress in life sciences. As hardware technology continues to evolve, the availability of new fluorescent probes with superior performance is becoming increasingly important. In recent years, fluorescent nanoprobes (FNPs) have emerged as highly promising fluorescent probes for bioimaging due to their high brightness and excellent photostability. This paper focuses on the development and applications of FNPs as probes for live-cell super-resolution imaging. It provides an overview of different super-resolution methods, discusses the performance requirements for FNPs in these methods, and reviews the latest applications of FNPs in the super-resolution imaging of living cells. Finally, it addresses the challenges and future outlook in this field. [ABSTRACT FROM AUTHOR]

Efficient labeling methods for protein visualization with minimal tag size and appropriate photophysical properties are required for single‐molecule localization microscopy (SMLM), providing insights into the organization and interactions of biomolecules in cells at the molecular level. Among the fluorescent light‐up aptamers (FLAPs) originally developed for RNA imaging, RhoBAST stands out due to its remarkable brightness, photostability, fluorogenicity, and rapid exchange kinetics, enabling super‐resolved imaging with high localization precision. Here, we expand the applicability of RhoBAST to protein imaging by fusing it to protein‐binding aptamers. The versatility of such bifunctional aptamers is demonstrated by employing a variety of protein‐binding aptamers and different FLAPs. Moreover, fusing RhoBAST with the GFP‐binding aptamer AP3 facilitates high‐ and super‐resolution imaging of GFP‐tagged proteins, which is particularly valuable in view of the widespread availability of plasmids and stable cell lines expressing proteins fused to GFP. The bifunctional aptamers compare favorably with standard antibody‐based immunofluorescence protocols, as they are 7‐fold smaller than antibody conjugates and exhibit higher bleaching‐resistance. We demonstrate the effectiveness of our approach in super‐resolution microscopy in secondary mammalian cell lines and primary neurons by RhoBAST‐PAINT, an SMLM protein imaging technique that leverages the transient binding of the fluorogenic rhodamine dye SpyRho to RhoBAST. [ABSTRACT FROM AUTHOR]

Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study, we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three‐dimensional Structured Illumination Microscopy (3D‐SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half‐maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. Comparing the techniques readily available to us (above), 3D‐SIM microscopy, in combination with the shortest‐wavelength dyes, was found overall to give the best lateral resolution. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D‐STED to be the most successful method for this. We then combined this 3D‐STED approach with a custom 3D cell segmentation and analysis pipeline using the open‐source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyse their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super‐resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed. [ABSTRACT FROM AUTHOR]

Synaptogenesis involves the transformation of dendritic filopodial contacts into stable connections with the exact apposition of synaptic components. Signalling triggered by Wnt/β-catenin and calcium has been postulated to aid this process. However, it is unclear how such a signalling process orchestrates synapse formation to organise the spatial arrangement of synapses along dendrites. We show that WNT7A is loaded on dynamic dendritic filopodia during spine formation in human cortical neurons. WNT7A is present at the tips of the filopodia and the contact sites with dendrites of neighbouring neurons, triggering spatially restricted localisation of the Wnt co-receptor LRP6. Here, we demonstrate that WNT7A at filopodia tips leads to the induction of calcium transients, the clustering of preand postsynaptic proteins, and the subsequent transformation into mature spines. Although soluble WNT7A protein can also support synaptogenesis, it fails to provide this degree of spatial information for spine formation and calcium transients, and synaptic markers are induced ectopically along the dendrites. Our data suggest that dendritic filopodia are WNT7A-bearing cytonemes required for focal calcium signalling and initiation of synapse formation, and provide an elegant mechanism for orchestrating the positioning of synapses along dendrites. [ABSTRACT FROM AUTHOR]

In order to break the diffraction limit and improve the imaging resolution of optical microscope, in this article, we theoretically deduced the influence of phase difference on imaging resolution under coherent illumination. As the phase difference increased, the resolution improved gradually. Inspired by this conclusion, a super-resolution optical imaging system based on phase modulation was proposed and simulated. An optical mask was designed to generate additional phase difference for the adjacent area at the sample's surface, and the influence of its structural parameters was analyzed numerically. The simulation results preliminarily confirm the feasibility of this scheme, laying the foundation for a more optimal and comprehensive super-resolution imaging scheme. Due to its advantages of high resolution, a wide field of view, and being compatible, this non-fluorescence super-resolution imaging scheme is worthy of further research and application. [ABSTRACT FROM AUTHOR]

The transition from optical film to digital image sensors (DIS) in imaging systems has brought great convenience to human life. However, the sampling resolution of DIS is considerably lower than that of optical film due to the limitation that the pixels are significantly larger than the silver halide molecules. How to break DIS's sampling limit and achieve high‐resolution imaging is highly desired for imaging applications. In the research, a novel mechanism that allows for a significant reduction in the smallest sampling unit of DIS to as small as 1/16th of a pixel, or even smaller, through measuring the intra‐pixel quantum efficiency for the first time and recomputing the image — a technique referred to as hyper‐sampling imaging (HSI) is developed. Employing the HSI method, the physical sampling resolution of regular DIS can be enhanced by 4 × 4 times or potentially higher, and detailed object information can be acquired. The HSI method has undergone rigorous testing in real‐world imaging scenarios, demonstrating its robustness and efficiency in overcoming the sampling constraints of conventional DIS. This advancement is particularly beneficial for applications such as remote sensing, long‐range reconnaissance, and astronomical observations, where the ability to capture fine details is paramount. [ABSTRACT FROM AUTHOR]

Self-labeling protein (SLP) tags, such as HaloTag, have gained considerable interest as advanced tools for live cell labeling. However, the chloroalkane-based substrates that can be directly used for protein labeling are limited. Here, we report two bioorthogonal small molecule linkers, chloroalkane-tetrazine (CA-Tz) and chloroalkane-azide (CA-N3), which can penetrate cell membranes and facilitate click chemistry-based labeling in live cells. We compare their labeling capability using two clickable silicon rhodamine dyes (SiR-PEG3-TCO and SiR-PEG4-DBCO). Confocal imaging results demonstrate that using CA-Tz and SiR-PEG3-TCO dye exhibits superior intracellular labeling with low nonspecific signals. We subsequently compared the photostability of SiR dyes with that of green fluorescent proteins (mEmerald). Total internal reflection fluorescence (TIRF) imaging indicates that SiR dyes exhibit superior photostability under identical excitation conditions, making them suitable for long-term cell imaging. Furthermore, SiR dyes labeling also shows high structure retention for the fourth-order super-resolution optical fluctuation imaging (SOFI) compared to fluorescent proteins. This study presents clickable HaloTag linkers as effective tools for live cell labeling and imaging, highlighting the high-quality labeling of chloroalkane linkers and clickable dyes for live cell imaging. [ABSTRACT FROM AUTHOR]

Sensing and control of size are critical for cellular function and survival. A striking example of size sensing occurs during meiosis in the nematode Caenorhabditis elegans. C. elegans chromosomes compare the lengths of the two chromosome "arms" demarcated by the position of their single off-center crossover, and they differentially modify these arms to ensure that sister chromatid cohesion is lost specifically on the shorter arm in the first meiotic division, while the longer arm maintains cohesion until the second division. While many of the downstream steps leading to cohesion loss have been characterized, the length-sensing process itself remains poorly understood. Here, we have used cytological visualization of short and long chromosome arms, combined with quantitative microscopy, live imaging, and simulations, to investigate the principles underlying length-sensitive chromosome partitioning. By quantitatively analyzing short-arm designation patterns on fusion chromosomes carrying multiple crossovers, we develop a model in which a short-arm-determining factor originates at crossover designation sites, diffuses within the synaptonemal complex, and accumulates within crossover-bounded chromosome segments. We demonstrate experimental support for a critical assumption of this model: that crossovers act as boundaries to diffusion within the synaptonemal complex. Further, we develop a discrete simulation based on our results that recapitulates a wide variety of observed partitioning outcomes in both wild-type and previously reported mutants. Our results suggest that the concentration of a diffusible factor is used as a proxy for chromosome length, enabling the correct designation of short and long arms and proper segregation of chromosomes. [Display omitted] • CO-delimited chromosome functional arms are established in mid-meiotic prophase • Both phosphorylated and non-phosphorylated SYP-1 proteins partition to short arms • Multiple COs can act additively on domains to establish "short" or "long" identity • CO sites block diffusion along the SC, enabling potential concentration of factors The holocentric chromosomes of C. elegans lose cohesion at meiosis I in the shorter of the two domains defined by the single crossover. Rodriguez-Reza et al. provide evidence that the sensing of short versus long domains relies on comparing the concentration of crossover-derived factors accumulating within these domains as a proxy for physical length. [ABSTRACT FROM AUTHOR]

Deep learning is capable of greatly promoting the progress of super-resolution imaging technology in terms of imaging and reconstruction speed, imaging resolution, and imaging flux. This paper proposes a deep neural network based on a generative adversarial network (GAN). The generator employs a U-Net-based network, which integrates DenseNet for the downsampling component. The proposed method has excellent properties, for example, the network model is trained with several different datasets of biological structures; the trained model can improve the imaging resolution of different microscopy imaging modalities such as confocal imaging and wide-field imaging; and the model demonstrates a generalized ability to improve the resolution of different biological structures even out of the datasets. In addition, experimental results showed that the method improved the resolution of caveolin-coated pits (CCPs) structures from 264 nm to 138 nm, a 1.91-fold increase, and nearly doubled the resolution of DNA molecules imaged while being transported through microfluidic channels. [ABSTRACT FROM AUTHOR]

In order to break the diffraction limit and improve the imaging resolution of optical microscope, in this article, we theoretically deduced the influence of phase difference on imaging resolution under coherent illumination. As the phase difference increased, the resolution improved gradually. Inspired by this conclusion, a super-resolution optical imaging system based on phase modulation was proposed and simulated. An optical mask was designed to generate additional phase difference for the adjacent area at the sample’s surface, and the influence of its structural parameters was analyzed numerically. The simulation results preliminarily confirm the feasibility of this scheme, laying the foundation for a more optimal and comprehensive super-resolution imaging scheme. Due to its advantages of high resolution, a wide field of view, and being compatible, this non-fluorescence super-resolution imaging scheme is worthy of further research and application.

In eukaryotic cell nuclei, specific sets of proteins gather in nuclear bodies and facilitate distinct genomic processes. The nucleolus, a nuclear body, functions as a factory for ribosome biogenesis by accumulating constitutive proteins, such as RNA polymerase I and nucleophosmin 1 (NPM1). Although in vitro assays have suggested the importance of liquid--liquid phase separation (LLPS) of constitutive proteins in nucleolar formation, how the nucleolus is structurally maintained with the intranuclear architecture remains unknown. This study revealed that the nucleolus is encapsulated by a single-stranded (ss)DNA-based molecular complex inside the cell nucleus. Super-resolution lattice-structured illumination microscopy (lattice-SIM) showed that there was a high abundance of ssDNA beyond the 'outer shell' of the nucleolus. Nucleolar disruption and the release of NPM1 were caused by in situ digestion of ssDNA, suggesting that ssDNA has a structural role in nucleolar encapsulation. Furthermore, we identified that ssDNA forms a molecular complex with histone H1 for nucleolar encapsulation. Thus, this study illustrates how an ssDNA-based molecular complex upholds the structural integrity of nuclear bodies to coordinate genomic processes such as gene transcription and replication. [ABSTRACT FROM AUTHOR]

Fourier ptychography (FP) is a high resolution wide‐field imaging method based on the extended aperture in the Fourier space, which is synthesized from raw images with varying illumination angles. If FP is extended to coherent nonlinear optical imaging, the resolution could be further improved due to the increase of the cutoff frequency of the synthesized coherent optical transfer function (C‐OTF) with respect to the order of nonlinear optical processes. However, there is a fundamental conflict between wide‐field FP and nonlinear optical imaging, whereby the nonlinear optical imaging typically requires a focused excitation laser beam with high power density. To tackle the problem, in this work, a unique point‐scanning FP (PS‐FP) method is presented for super‐resolution nonlinear optical imaging, in which the nonlinear optical signal is obtained by using focused laser beam, while the conventional FP algorithm can still be used to retrieve the super‐resolution image. PS‐FP coherent anti‐Stokes Raman scattering (PS‐FP‐CARS) imaging on a variety of samples, where a 1.8‐fold expansion of the OTF is achieved experimentally for enhancing vibrational imaging. Further theoretical calculation shows that the C‐OTF of PS‐FP higher‐order CARS (PS‐FP‐HO‐CARS) can be expanded up to ≈4.9‐fold, thereby improving the spatial resolution by ≈3‐fold in comparison with conventional point‐scanning CARS with under tightly focused beams. The generality of PS‐FP method developed in this work can be adapted to other coherent nonlinear optical imaging modalities for super‐resolution imaging in tissue and cells. [ABSTRACT FROM AUTHOR]

Resolving complex three-dimensional (3D) subcellular dynamics noninvasively in live tissues demands imaging tools that balance spatiotemporal resolution, field-of-view and phototoxicity. Image scanning microscopy (ISM), as an advancement of confocal laser scanning microscopy, provides a 2-fold 3D resolution enhancement. Nevertheless, the relatively low imaging speed has been the major obstacle for ISM to be further employed in in vivo imaging of biological tissues. Our proposed solution, multi-confocal image scanning microscopy (MC-ISM), aims to overcome the limitations of existing techniques in terms of spatiotemporal resolution balancing by optimizing pinhole diameter and pitch, eliminating out-of-focus signals, and introducing a frame reduction reconstruction algorithm. The imaging speed is increased by 16 times compared with multifocal structured illumination microscopy. We further propose a single-galvo scan, akin to the Archimedes spiral in spinning disk confocal systems, to ensure a high-speed and high-accuracy scan without the galvanometer's inertial motion. Benefitting from its high photon efficiency, MC-ISM allows continuous imaging of mitochondria dynamics in live cells for 1000 frames without apparent phototoxicity, reaching an imaging depth of 175 μm. Noteworthy, MC-ISM enables the observation of the inner membrane structure of living mitochondria in Arabidopsis hypocotyl for the first time, demonstrating its outstanding performance. [ABSTRACT FROM AUTHOR]

Blood cells are the most integral part of the body, which are made up of erythrocytes, platelets and white blood cells. The examination of subcellular structures and proteins within blood cells at the nanoscale can provide valuable insights into the health status of an individual, accurate diagnosis, and efficient treatment strategies for diseases. Super-resolution microscopy (SRM) has recently emerged as a cutting-edge tool for the study of blood cells, providing numerous advantages over traditional methods for examining subcellular structures and proteins. In this paper, we focus on outlining the fundamental principles of various SRM techniques and their applications in both normal and diseased states of blood cells. Furthermore, future prospects of SRM techniques in the analysis of blood cells are also discussed. [ABSTRACT FROM AUTHOR]

Fluorescence microscopic imaging is essentially a convolution process distorted by random noise, limiting critical parameters such as imaging speed, duration, and resolution. Though algorithmic compensation has shown great potential to enhance these pivotal aspects, its fidelity remains questioned. Here we develop a physics-rooted computational resolution extension and denoising method with ensured fidelity. Our approach employs a multi-resolution analysis (MRA) framework to extract the two main characteristics of fluorescence images against noise: across-edge contrast, and along-edge continuity. By constraining the two features in a model-solution framework using framelet and curvelet, we develop MRA deconvolution algorithms, which improve the signal-to-noise ratio (SNR) up to 10 dB higher than spatial derivative based penalties, and can provide up to two-fold fidelity-ensured resolution improvement rather than the artifact-prone Richardson-Lucy inference. We demonstrate our methods can improve the performance of various diffraction-limited and super-resolution microscopies with ensured fidelity, enabling accomplishments of more challenging imaging tasks. [ABSTRACT FROM AUTHOR]

Volumetric subcellular imaging has long been essential for studying structures and dynamics in cells and tissues. However, due to limited imaging speed and depth of field, it has been challenging to perform live-cell imaging and single-particle tracking. Here we report a 2.5D fluorescence microscopy combined with highly inclined illumination beams, which significantly reduce not only the image acquisition time but also the out-of-focus background by ∼2-fold compared to epi-illumination. Instead of sequential z-scanning, our method projects a certain depth of volumetric information onto a 2D plane in a single shot using multi-layered glass for incoherent wavefront splitting, enabling high photon detection efficiency. We apply our method to multi-color immunofluorescence imaging and volumetric super-resolution imaging, covering ∼3–4 µm thickness of samples without z-scanning. Additionally, we demonstrate that our approach can substantially extend the observation time of single-particle tracking in living cells. [ABSTRACT FROM AUTHOR]

Hemorrhagic hydrocephalus is a common pathology in neonates with high mortality and morbidity. Current imaging approaches fail to capture the mechanisms behind its pathogenesis. Here, we discuss the processes underlying this pathology, the metabolic dysfunction that occurs as a result, and the ways in which these metabolic changes inform novel methods of clinical imaging. The imaging advances described allow earlier detection of the cellular and metabolic changes, leading to better outcomes for affected neonates. [ABSTRACT FROM AUTHOR]

In vivoimaging of protein complexes is a powerful method for understanding the underlying biological function of these key biomolecules. Though the engineering of small, high affinity nanobodies have become more prevalent, the off-rates of these tags may result in incomplete or partial labeling of proteins in live cells. The SpyCatcher003 and SpyTag split protein system allow for irreversible, covalent binding to a short target peptide unlike nanobody-affinity based probes. However, delivering these tags into a cell without disrupting its normal function is a key challenge. Cell penetrating peptides (CPPs) are short peptide sequences that facilitate the transduction of otherwise membrane-impermeable 'cargo' , such as proteins, into cells. Here we report on our efforts to engineer and characterize CPP-SpyCatcher003 fusions as modular imaging probes. We selected three CPPs, CUPID, Pentratin, and pVEC, to engineer fusion protein probes for superresolution microscopy, with the aim to eliminate prior permeabilization treatments that could introduce imaging artifacts. We find that fusing the CPP sequences to SpyCatcher003 resulted in dimer and multimer formation as determined by size exclusion chromatography, dynamic light scattering, and SDS resistant dimers on SDS-PAGE gels. By isolating and labeling the monomeric forms of the engineered protein, we show these constructs retained their ability to bind SpyTag and all three CPP sequences remain membrane active, as assessed by CD spectroscopy in the presence of SDS detergent. Using fluorescence and super resolution Lattice structured illumination microscopy (Lattice SIM) imaging we show that the CPPs did not enhance uptake of SpyCatcher byE. coli,however withCaulobacter crescentuscells, we show that Penetratin, and to a lesser degree CUPID, does enhance uptake. Our results demonstrate the ability of the CPP-SpyCatcher003 to label targets within living cells, providing the groundwork for using split protein systems for targetedin vivoimaging.

Summary: Background: Neovascularisation of carotid plaques contributes to their vulnerability. Current imaging methods such as contrast-enhanced ultrasound (CEUS) usually lack the required spatial resolution and quantification capability for precise neovessels identification. We aimed at quantifying plaque vascularisation with ultrasound localization microscopy (ULM) and compared the results to histological analysis. Methods: We conducted a prospective, monocentric, study involving patients who were undergoing carotid endarterectomy (CEA) for carotid artery stenosis. The day before CEA ultrasound examination coupled with the injection of microbubbles (MB) as a contrast agent (CEUS) to image the MB circulating within and around the carotid plaque was performed. CEUS images analysis classified patients into 2 groups: absence of neovascularisation (group A) or presence of neovascularisation (group B). ULM was performed by localising and tracking individual MB centres to reconstruct the neovessels structure with a resolution of around 60 μm. Plaques were manually segmented on the images to quantify the number of neovessels and various haemodynamic metrics inside the plaques. Histological analysis of the excised carotid plaque specimens classified patients into 2 groups: absence of neovascularisation (group I) or presence of neovascularisation (group II). Findings: Among the 26 patients included, classification was as follows: group I: n = 8 and group II: n = 18, 18 patients had analysable CEUS images and were classified as follows: group A: n = 10, group B: n = 8. The median (Q1-Q3) number of MB tracked per second inside the plaque was 0.03 (0–0.37) for patients in group I and 0.51 (0–3) for patients in group A versus (vs.) 3.55 (1.26–17.68) for patients in group II and 9.69 (5.83–34.68) for patients in group B (p = 0.00049; p = 0.010 respectively). The length of the MB tracks was 0.02 mm (0–0.16) in group I vs. 0.29 mm (0.22–0.45) in group II (p = 0.0069). The study also showed that flow in the neovessels was greater during systole than during diastole period: 9.38 (1.67–19.17) MB tracked per second vs. 1.35 (0.28–6.56) (p = 0.021). Interpretation: ULM allows the detection of neovessels within the carotid atherosclerotic plaque. Thus, ULM provides a precise picture of plaque neovascularisation in patients and could be used as a non-invasive imaging technique to assess carotid plaque vulnerability. Funding: The study was sponsored and funded by Assistance Publique–Hôpitaux de Paris (CRC 1806 APHP INNOVATION 2018). Co-funding by ART (Technological Research Accelerator) biomedical ultrasound program of INSERM, France.

Deep learning is capable of greatly promoting the progress of super-resolution imaging technology in terms of imaging and reconstruction speed, imaging resolution, and imaging flux. This paper proposes a deep neural network based on a generative adversarial network (GAN). The generator employs a U-Net-based network, which integrates DenseNet for the downsampling component. The proposed method has excellent properties, for example, the network model is trained with several different datasets of biological structures; the trained model can improve the imaging resolution of different microscopy imaging modalities such as confocal imaging and wide-field imaging; and the model demonstrates a generalized ability to improve the resolution of different biological structures even out of the datasets. In addition, experimental results showed that the method improved the resolution of caveolin-coated pits (CCPs) structures from 264[Formula: see text]nm to 138[Formula: see text]nm, a 1.91-fold increase, and nearly doubled the resolution of DNA molecules imaged while being transported through microfluidic channels.

Kinesin-1 ensembles maneuver vesicular cargoes through the three-dimensional (3D) intracellular microtubule (MT) network. To define how such cargoes navigate MT intersections, we first determined how many kinesins from an ensemble on a lipid-based cargo simultaneously engage a MT, and then determined the directional outcomes (straight, turn, terminate) for liposome cargoes at perpendicular MT intersections. Run lengths of 350-nm diameter liposomes decorated with up to 20, constitutively active, truncated kinesin-1 KIF5B (K543) were longer than single motor transported cargo, suggesting multiple motor engagement. However, detachment forces of lipid-coated beads with ~20 kinesins, measured using an optical trap, showed no more than three simultaneously engaged motors, with a single engaged kinesin predominating, indicating anticooperative MT binding. At two-dimensional (2D) and 3D in vitro MT intersections, liposomes frequently paused (~2 s), suggesting kinesins simultaneously bind both MTs and engage in a tug-of-war. Liposomes showed no directional outcome bias in 2D (1.1 straight:turn ratio) but preferentially went straight (1.8 straight:turn ratio) in 3D intersections. To explain these data, we developed a mathematical model of liposome transport incorporating the known mechanochemistry of kinesins, which diffuse on the liposome surface, and have stiff tails in both compression and extension that impact how motors engage the intersecting MTs. Our model predicts the ~3 engaged motor limit observed in the optical trap and the bias toward going straight in 3D intersections. The striking similarity of these results to our previous study of liposome transport by myosin Va suggests a "universal" mechanism by which cargoes navigate 3D intersections. [ABSTRACT FROM AUTHOR]

In the rapidly evolving landscape of molecular imaging, super‐resolution techniques emerge as indispensable tools, revolutionizing our capacity to decipher the intricacies of protein aggregation, and paving the way for molecular‐level understanding of the progression of neurodegenerative disorders. In this review article we provide an overview of the diverse super‐resolution imaging techniques applied to study amyloids at high resolution. We outline the strengths and limitations of each technique, offering insights into their applicability to different amyloid systems. We next delve into the diverse strategies employed for labeling amyloids in conjunction with super‐resolution imaging. From small organic dyes to fluorescent proteins and advanced chemical probes, the discussion encompasses the strengths and considerations associated with each labeling method. The comparative analysis not only evaluates the impact of labeling on resolution and specificity but also highlights emerging trends and future directions in the field. [ABSTRACT FROM AUTHOR]

The recent outbreak of monkeypox virus (MPXV) was unprecedented in its size and distribution. Those living with uncontrolled HIV and low CD4 T cell counts might develop a fulminant clinical mpox course with increased mortality, secondary infections, and necrotizing lesions. Fatal cases display a high and widespread MPXV tissue burden. The underlying pathomechanisms are not fully understood. We report here the pathological findings of an MPXV‐driven abscess in gastrocnemius muscle requiring surgery in an immunocompromised patient with severe mpox. Presence of virus particles and infectivity were confirmed by electron microscopy, expansion microscopy, and virus culture, respectively. MPXV tissue distribution by immunohistochemistry (IHC) showed a necrotic core with infection of different cell types. In contrast, at the lesion rim fibroblasts were mainly infected. Immune cells were almost absent in the necrotic core, but were abundant at the infection rim and predominantly macrophages. Further, we detected high amounts of alternatively activated GPNMB+‐macrophages at the lesion border. Of note, macrophages only rarely colocalized with virus‐infected cells. Insufficient clearance of infected cells and infection of lesion‐associated fibroblasts sustained by the abundance of profibrotic macrophages might lead to the coalescing of lesions and the severe and persistent clinical mpox course observed in immunocompromised patients. [ABSTRACT FROM AUTHOR]

Stimulated emission depletion (STED) microscopy, as a popular super-resolution imaging technique, has been widely used in bio-structure analysis and resolving the dynamics of biological processes beyond the diffraction limit. The performance of STED critically depends on the optical properties of the fluorescent probes. Ideally, the probe should process high brightness and good photostability, and exhibit a sensitive response to the depletion beam. Organic dyes and fluorescent proteins, as the most widely used STED probes, suffer from low brightness and exhibit rapid photobleaching under a high excitation power. Recently, luminescent nanoparticles (NPs) have emerged as promising fluorescent probes in biological imaging due to their high brightness and good photostability. STED imaging using various kinds of NPs, including quantum dots, polymer dots, carbon dots, aggregation-induced emission dots, etc., has been demonstrated. This review will comprehensively review recent advances in fluorescent NP-based STED probes, discuss their advantages and pitfalls, and outline the directions for future development. [ABSTRACT FROM AUTHOR]

Introducing super‐resolution techniques to imaging through scattering media potentially revolutionizes the technical analysis for many exotic applications, such as cell structures behind biological tissues. The main challenge is scattering media's inhomogeneous structures, which scramble the light path and create noise‐like speckle patterns, hindering object's visualization even at a low‐resolution level. Here, a computational method is proposed relying on the object's spatial and temporal fluctuation to visualize nanoscale objects through scattering media non‐invasively. Taking advantage of the optical memory effect and multiple frames, the point spreading function (PSF) of scattering media is estimated. Multiple images of fluctuating objects are obtained by deconvolution; then, the super‐resolution image is achieved by computing the higher‐order cumulants. Non‐linearity of high order cumulant significantly suppresses artifacts in the resulting images and enhances resolution by a factor of N$\sqrt N $, where N is the cumulant order. The proof‐of‐concept demonstrates a resolution of 266 nm at the 6th‐order cumulant with numerical aperture (NA) of 0.42, breaking the diffraction limit by a factor of 2.45. An adaptive approach is also demonstrated for imaging through dynamic scattering media. The non‐invasive super‐resolution speckle fluctuation imaging (NISFFI) presents a nanoscopy technique with straightforward imaging hardware configuration to visualize samples behind scattering media. [ABSTRACT FROM AUTHOR]

The present study aimed to explore the clinical applicability of ultrasound super-resolution imaging (US SRI) for assessing renal microcirculation in patients with acute kidney injury (AKI). A total of 62 patients with sepsis were enrolled in the present study—38 with AKI and 24 control patients—from whom renal ultrasounds and clinical data were obtained. SonoVue contrast (1.5 mL) was administered through the elbow vein and contrast-enhanced ultrasound (CEUS) images were obtained on a Mindray Resona A20 ultrasound unit for 2 min. The renal perfusion time-intensity curve (TIC) was analyzed and, after 15 min, additional images were obtained to create a microscopic blood flow map. Microvascular density (MVD) was calculated and its correlation with serum creatinine (Scr) levels was analyzed. There were significant differences in heart rate, Scr, blood urea nitrogen, urine volume at 24 h, and glomerular filtration rate between the two groups (p < 0.01), whereas other characteristics, such as renal morphology, did not differ significantly between the AKI group and control group (p > 0.05). The time to peak and mean transit times of the renal cortex in the AKI group were prolonged compared to those in the control group (p < 0.01), while the peak intensity and area under the TIC were lower than those in the control group (p < 0.05). The MVD of the renal cortex in the AKI group was lower than that in the control group (18.46 ± 5.90% vs. 44.93 ± 11.65%; p < 0.01) and the MVD in the AKI group showed a negative correlation with Scr (R = −0.84; p < 0.01). Based on the aforementioned results, US SRI can effectively assess renal microcirculation in patients with AKI and is a noninvasive technique for the diagnosis of AKI and quantitative evaluation of renal microcirculation. [ABSTRACT FROM AUTHOR]

Understanding the operando defect-tuning performance of catalysts is critical to establish an accurate structure-activity relationship of a catalyst. Here, with the tool of single-molecule super-resolution fluorescence microscopy, by imaging intermediate CO formation/oxidation during the methanol oxidation reaction process on individual defective Pt nanotubes, we reveal that the fresh Pt ends with more defects are more active and anti-CO poisoning than fresh center areas with less defects, while such difference could be reversed after catalysis-induced step-by-step creation of more defects on the Pt surface. Further experimental results reveal an operando volcano relationship between the catalytic performance (activity and anti-CO ability) and the fine-tuned defect density. Systematic DFT calculations indicate that such an operando volcano relationship could be attributed to the defect-dependent transition state free energy and the accelerated surface reconstructing of defects or Pt-atom moving driven by the adsorption of the CO intermediate. These insights deepen our understanding to the operando defect-driven catalysis at single-molecule and subparticle level, which is able to help the design of highly efficient defect-based catalysts. [ABSTRACT FROM AUTHOR]

Fluorescence labeling of chemically fixed specimens, especially immunolabeling, plays a vital role in super-resolution imaging as it offers a convenient way to visualize cellular structures like mitochondria or the distribution of biomolecules with high detail. Despite the development of various distinct probes that enable super-resolved stimulated emission depletion (STED) imaging of mitochondria in live cells, most of these membrane-potential- dependent fluorophores cannot be retained well in mitochondria after chemical fixation. This lack of suitable mitochondrial probes has limited STED imaging of mitochondria to live cell samples. In this study, we introduce a mitochondria-specific probe, PK Mito Orange FX (PKMO FX), which features a fixation-driven cross-linking motif and accumulates in the mitochondrial inner membrane. It exhibits high fluorescence retention after chemical fixation and efficient depletion at 775 nm, enabling nanoscopic imaging both before and after aldehyde fixation. We demonstrate the compatibility of this probe with conventional immunolabeling and other strategies commonly used for fluorescence labeling of fixed samples. Moreover, we show that PKMO FX facilitates correlative super-resolution light and electron microscopy, enabling the correlation of multicolor fluorescence images and transmission EM images via the characteristic mitochondrial pattern. Our probe further expands the mitochondrial toolkit for multimodal microscopy at nanometer resolutions. [ABSTRACT FROM AUTHOR]

The interaction between lamin A and the cytoplasmic skeleton plays a key role in maintaining nuclear mechanical properties. However, the effect of destruction of the cytoplasmic skeleton on the 3D submicroscopic structure of lamin A has not been elucidated. In this study, we developed an image quantization algorithm to quantify changes in the submicroscopic structure of the intact lamin A 3D network within the nucleus. We used blebbistatin or nocodazole to disrupt the fibrillar structure of F-actin or tubulin, respectively, and then quantified changes in the lamin A super-resolution network structure, the morphological and mechanical properties of the nucleus and the spatial distribution of chromosomes. Ultimately, we found for the first time that disruption of the cytoplasmic skeleton changes the lamin A submicroscopic network and nuclear structural characteristics. In summary, this study contributes to understanding the trans-nuclear membrane interaction characteristics of lamin A and the cytoplasmic skeleton.

Background: The purpose of this study was to examine the feasibility and practical application of ultrasound (US) super-resolution imaging (SRI) in evaluating microvasculature and measuring renal allograft function. Methods: Sixteen consecutive patients who received kidney transplants were prospectively enrolled. The patients were assigned as: normal allograft function (n = 6), and allograft malfunction (n = 10). Localizing each potential contrast signal resulted in super-resolution images (SRI). SRI was utilized to assess micro-vessel density (MVD) and microvascular flow rate, whereas contrast-enhanced (CE) US images were statistically processed to get the time to peak (TTP) and peak intensity. Logistic regression was utilized to evaluate their relationship. Results: US SRI may be utilized effectively on allografts to show microvasculature with significantly higher resolution than typical color Doppler flow and CEUS pictures. In the multivariate analysis, MVD and TTP were significant US markers of renal allograft failure (p = 0.031 and p = 0.045). The combination of MVD and TTP produced an AUC of 0.783 (p

Abstract Fluorescence polarization microscopy is widely used in biology for molecular orientation properties. However, due to the limited temporal resolution of single-molecule orientation localization microscopy and the limited orientation dimension of polarization modulation techniques, achieving simultaneous high temporal-spatial resolution mapping of the three-dimensional (3D) orientation of fluorescent dipoles remains an outstanding problem. Here, we present a super-resolution 3D orientation mapping (3DOM) microscope that resolves 3D orientation by extracting phase information of the six polarization modulation components in reciprocal space. 3DOM achieves an azimuthal precision of 2° and a polar precision of 3° with spatial resolution of up to 128 nm in the experiments. We validate that 3DOM not only reveals the heterogeneity of the milk fat globule membrane, but also elucidates the 3D structure of biological filaments, including the 3D spatial conformation of λ-DNA and the structural disorder of actin filaments. Furthermore, 3DOM images the dipole dynamics of microtubules labeled with green fluorescent protein in live U2OS cells, reporting dynamic 3D orientation variations. Given its easy integration into existing wide-field microscopes, we expect the 3DOM microscope to provide a multi-view versatile strategy for investigating molecular structure and dynamics in biological macromolecules across multiple spatial and temporal scales.

Nanostructural contact sites formed by interorganelle membrane contacts, including mitochondria and lysosome contacts (MLC), facilitate the exchange of substances during various life processes. However, existing bioanalytical technologies have yet to provide accurate information on the exchange of substances, as these techniques exhibit limited spatial and temporal resolution. To address this limitation, a strategy is proposed that combines fluorescence resonance energy transfer (FRET) probes with high spatial resolution detection and super‐resolution microscopes (SRM) that feature time‐resolved imaging. Specifically, a proof‐of‐concept approach is presented for monitoring H+ fluctuations during MLC with a spatial H+ biosensor targeting lysosomes, BDP‐RhB. The biosensor comprises H+‐sensitive rhodamine B as a FRET acceptor connected by a flexible chain to a BODIPY derivative as a donor. The acidity of MLC sites may vary, influencing the spatial distance of the flexible chain and causing a fluorescence transition in BDP‐RhB. Consequently, the spatial distribution of H+ can be identified using SRM. Furthermore, an algorithm has been developed to screen and identify potential compounds that control substance exchange in the MLC. Collectively, this work presents the dynamic of H+ in lysosomes within living cells, which provides a drug screening tool for studying substance exchange through interorganelle membrane contacts. [ABSTRACT FROM AUTHOR]

【Objective】This work is to solve the bottleneck of accurately identifying fine structures, and dynamic changes cannot be concurrently met by imaging speed and imaging resolution in the study of plant cell endoplasmic reticulum.【Method】This study employed structured illumination microscopy techniques to achieve super-resolution real-time imaging of the ER in live Arabidopsis materials. Additionally, a self-supervised denoising framework（Blind2Unblind）was optimized to further enhance the signal-to-noise ratio of rapid microscopic imaging. 【Result】Based on the images with high quality, a method for quantitative analysis of ER structures using time-lapse images was established. Moreover, detections of changes in ER structures under environmental stress were conducted to verify the effectiveness of the method. Moreover, correlation analyses of various parameters indicated a significantly positive correlation between the area,length of tubular ER and the number of growth tips and three-way junctions, while the area of ER cisternae and bulk flow had a significantly negative correlation with the area and length of tubules.【Conclusion】The optimized self-supervised denoising framework in this study improves the signal-to-noise ratio of images with structure illumination microscopy in living plant cells, enabling the quantification of complex structures and dynamics, such as tubular ER, cisternae in ER, bulk flow, growth tips, and nodes, with complex correlations among the structures. [ABSTRACT FROM AUTHOR]

The far‐field resolution of traditional optical imaging systems is restricted by the Abbe diffraction limit, a direct result of the wave nature of light. To break this limit, one applicable approach is to reduce the effective size of the point‐spread‐function (PSF). In the past decades, great endeavors have been made to produce an effective super‐resolution PSF by exploiting different mechanisms, including optical nonlinearities and structured light illumination. However, it is difficult to apply these methods to objects at far distance miles away. Here, the study proposes a new approach to achieving super‐resolution imaging for far‐distance objects by utilizing angular magnification. To this end, a new class of metalenses with angular magnification is first theoretically and experimentally validated and demonstrated. Both theoretical and experimental results have demonstrated a more than twofold enhancement beyond the traditional angular‐resolution limit. The proposed approach is of promising potential in the applications of super‐resolution telescopes and remote sensing. [ABSTRACT FROM AUTHOR]

Fluorogenic dyes with high brightness, large turn‐on ratios, excellent photostability, favorable specificity, low cytotoxicity, and high membrane permeability are essential for high‐resolution fluorescence imaging in live cells. In this study, we endowed these desirable properties to a rhodamine derivative by simply replacing the N, N‐diethyl group with a pyrrole substituent. The resulting dye, Rh‐NH, exhibited doubled Stokes shifts (54 nm) and a red‐shift of more than 50 nm in fluorescence spectra compared to Rhodamine B. Rh‐NH preferentially exists in a non‐emissive but highly permeable spirolactone form. Upon binding to lysosomes, the collective effects of low pH, low polarity, and high viscosity endow Rh‐NH with significant fluorescence turn‐on, making it a suitable candidate for wash‐free, high‐contrast lysosome tracking. Consequently, Rh‐NH enabled us to successfully explore stimulated emission depletion (STED) super‐resolution imaging of lysosome dynamics, as well as fluorescence lifetime imaging of lysosomes in live cells. [ABSTRACT FROM AUTHOR]

Glutamate receptors at the postsynaptic side translate neurotransmitter release from presynapses into postsynaptic excitation. They play a role in many forms of synaptic plasticity, e.g., homeostatic scaling of the receptor field, activity-dependent synaptic plasticity and the induction of presynaptic homeostatic potentiation (PHP). The latter process has been extensively studied at Drosophila melanogaster neuromuscular junctions (NMJs). The genetic removal of the glutamate receptor subunit IIA (GluRIIA) leads to an induction of PHP at the synapse. So far, mostly imprecise knockouts of the GluRIIA gene have been utilized. Furthermore, mutated and tagged versions of GluRIIA have been examined in the past, but most of these constructs were not expressed under endogenous regulatory control or involved the mentioned imprecise GluRIIA knockouts. We performed CRISPR/Cas9-assisted gene editing at the endogenous locus of GluRIIA. This enabled the investigation of the endogenous expression pattern of GluRIIA using tagged constructs with an EGFP and an ALFA tag for super-resolution immunofluorescence imaging, including structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). All GluRIIA constructs exhibited full functionality and PHP could be induced by philanthotoxin at control levels. By applying hierarchical clustering algorithms to analyze the dSTORM data, we detected postsynaptic receptor cluster areas of ~0.15 µm2. Consequently, our constructs are suitable for ultrastructural analyses of GluRIIA. [ABSTRACT FROM AUTHOR]

The high azimuth resolution and low computational complexity are significant for scanning radar super-resolution imaging. In this paper, we propose a fast sparse Bayesian learning method with truncated singular value (FSBL-T) to quickly improve the azimuth resolution. Firstly, we truncate the small singular value of the convolution matrix to rewrite the echo signal. Secondly, we introduce the Gaussian-Gamma distribution to model the echo noise and sparse target. Then, the expectation maximization method is utilized to estimate the imaging parameters. To reduce the computational load, we introduce Kailath Variant conversion to reduce the size of the inverse matrix. Finally, the simulations illustrate that the FSBL-T method can effectively enhance the azimuth resolution without reducing the imaging accuracy.

Lipid droplets (LDs) are dynamic organelles interacting with a variety of intracellular organelles. Tracking intracellular LD dynamics employing synthetic small molecules is crucial for biological studies. Fluorescence imaging in the red and near infrared (NIR) region is more suitable for biological imaging due to its low phototoxicity and high signal-to-noise ratio. However, available LD-dyes in the red region with remarkable environmental sensitivity, selectivity for LDs staining are limited. Here, we constructed a red-emission D-π-A-π type LD-dye LD 688P with higher environmental sensitivity and suitable “calculated log P” (Clog P) for LDs dynamic imaging. LD 688P was proved to be highly selective and photostable for tracing LD fusion including multiple consecutive fusions and fusions in a centrosymmetric manner by super-resolution microscopy. We believe that the D-π-A-π skeleton would be an efficient strategy to construct red and even NIR-emission dyes.