5,370 results on '"super‐resolution microscopy"'
Search Results
2. Super-resolution microscopy for protein imaging: Unraveling cellular architecture and function
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Rabiee, Navid
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- 2025
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3. Metamaterial-Assisted Illumination Nanoscopy with Exceptional Axial Resolution.
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Lee, Yeon, Li, Shilong, Zhao, Junxiang, Posner, Clara, Zhang, Jin, and Liu, Zhaowei
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bioimaging ,metamaterials ,nanometer‐scale axial localization ,structured illumination microscopy ,super‐resolution microscopy - Abstract
Recent advancements in optical metamaterials have opened new possibilities in the exciting field of super-resolution microscopies. The far-field metamaterial-assisted illumination nanoscopies (MAINs) have, very recently, enhanced the lateral resolution to one-fifteenth of the optical wavelength. However, the axial localization accuracy of fluorophores in the MAINs remains rarely explored. Here, a MAIN with a nanometer-scale axial localization accuracy is demonstrated by monitoring the distance-dependent photobleaching dynamics of the fluorophores on top of an organic hyperbolic metamaterial (OHM) substrate under a wide-field single-objective microscope. With such a regular experimental configuration, 3D imaging of various biological samples with the resolution of ≈40 nm in the lateral dimensions and ≈5 nm in the axial dimension is realized. The demonstrated imaging modality enables the resolution of the 3D morphology of nanoscopic cellular structures with a significantly simplified experimental setup.
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- 2024
4. Localized Plasmonic Structured Illumination Microscopy Using Hybrid Inverse Design
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Wu, Qianyi, Xu, Yihao, Zhao, Junxiang, Liu, Yongmin, and Liu, Zhaowei
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Information and Computing Sciences ,Communications Engineering ,Engineering ,Bioengineering ,Networking and Information Technology R&D (NITRD) ,Machine Learning and Artificial Intelligence ,Deep learning ,Genetic algorithms ,Photonicsinverse design ,Super-resolution microscopy ,Plasmonics ,Structured illumination microscopy ,Photonics inverse design ,Nanoscience & Nanotechnology - Abstract
Super-resolution fluorescence imaging has offered unprecedented insights and revolutionized our understanding of biology. In particular, localized plasmonic structured illumination microscopy (LPSIM) achieves video-rate super-resolution imaging with ∼50 nm spatial resolution by leveraging subdiffraction-limited nearfield patterns generated by plasmonic nanoantenna arrays. However, the conventional trial-and-error design process for LPSIM arrays is time-consuming and computationally intensive, limiting the exploration of optimal designs. Here, we propose a hybrid inverse design framework combining deep learning and genetic algorithms to refine LPSIM arrays. A population of designs is evaluated using a trained convolutional neural network, and a multiobjective optimization method optimizes them through iteration and evolution. Simulations demonstrate that the optimized LPSIM substrate surpasses traditional substrates, exhibiting higher reconstruction accuracy, robustness against noise, and increased tolerance for fewer measurements. This framework not only proves the efficacy of inverse design for tailoring LPSIM substrates but also opens avenues for exploring new plasmonic nanostructures in imaging applications.
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- 2024
5. STORM Super-Resolution Visualization of Self-Assembled γPFD Chaperone Ultrastructures in Methanocaldococcus jannaschii
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Cha, Hee-Jeong, He, Changdong, Glover, Dominic J, Xu, Ke, and Clark, Douglas S
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Biochemistry and Cell Biology ,Physical Sciences ,Biological Sciences ,Nanotechnology ,Bioengineering ,Generic health relevance ,Methanocaldococcus ,Molecular Chaperones ,Archaeal Proteins ,Microscopy ,Fluorescence ,Imaging ,Three-Dimensional ,archaeal chaperone ,prefoldin ,super-resolutionmicroscopy ,self-assembled nanostructures ,super-resolution microscopy ,Nanoscience & Nanotechnology - Abstract
Gamma-prefoldin (γPFD), a unique chaperone found in the extremely thermophilic methanogen Methanocaldococcus jannaschii, self-assembles into filaments in vitro, which so far have been observed using transmission electron microscopy and cryo-electron microscopy. Utilizing three-dimensional stochastic optical reconstruction microscopy (3D-STORM), here we achieve ∼20 nm resolution by precisely locating individual fluorescent molecules, hence resolving γPFD ultrastructure both in vitro and in vivo. Through CF647 NHS ester labeling, we first demonstrate the accurate visualization of filaments and bundles with purified γPFD. Next, by implementing immunofluorescence labeling after creating a 3xFLAG-tagged γPFD strain, we successfully visualize γPFD in M. jannaschii cells. Through 3D-STORM and two-color STORM imaging with DNA, we show the widespread distribution of filamentous γPFD structures within the cell. These findings provide valuable insights into the structure and localization of γPFD, opening up possibilities for studying intriguing nanoscale components not only in archaea but also in other microorganisms.
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- 2024
6. A framework for the simulation of individual glycan coordinates to analyze spatial relationships within the glycocalyx.
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Fritsche, Sarah and Möckl, Leonhard
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DRY eye syndromes ,GLYCOCALYX ,SIALIC acids ,CELL anatomy ,EMBRYO implantation - Abstract
The glycocalyx is a dense and dynamic layer of glycosylated species that covers every cell in the human body. It plays crucial roles in various cellular processes in health and disease, such as cancer immune evasion, cancer immune therapy, blastocyst implantation, and functional attenuation of membrane protein diffusion. In addition, alterations in glycocalyx structure may play an important role in ocular surface diseases, e.g., dry eye disease. Despite the emerging importance of the glycocalyx, various aspects of its functional organization remain elusive to date. A central reason for this elusiveness is the nanoscale dimension of the glycocalyx in conjunction with its high structural complexity, which is not accessible to observation with conventional light microscopy. Recent advances in super-resolution microscopy have enabled resolutions down to the single-digit nanometer range. In order to fully leverage the potential of these novel methods, computational frameworks that allow for contextualization of the resulting experimental data are required. Here, we present a simulation-based approach to analyze spatial relationships of glycan components on the cell membrane based on known geometrical parameters. We focus on sialic acids in this work, but the technique can be adapted to any glycan component of interest. By integrating data from mass spectrometry and quantitative biological studies, these simulations aim to model possible experimental outcomes, which can then be used for further analysis, such as spatial point statistics. Importantly, we include various experimental considerations, such as labeling and detection efficiency. This approach may contribute to establishing a new standard of connection between geometrical and molecular-resolution data in service of advancing our understanding of the functional role of the glycocalyx in biology as well as its clinical potential. [ABSTRACT FROM AUTHOR]
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- 2025
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7. Fast and Long‐Term Super‐Resolution Imaging of Endoplasmic Reticulum Nano‐structural Dynamics in Living Cells Using a Neural Network.
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Rahm, Johanna V., Balakrishnan, Ashwin, Wehrheim, Maren, Kaminer, Alexandra, Glogger, Marius, Kessler, Laurell F., Kaschube, Matthias, Barth, Hans‐Dieter, and Heilemann, Mike
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IMAGE reconstruction , *STIMULATED emission , *IMAGE denoising , *MICROSCOPY , *AUTOPHAGY - Abstract
Stimulated emission depletion (STED) microscopy is a super‐resolution technique that surpasses the diffraction limit and has contributed to the study of dynamic processes in living cells. However, high laser intensities induce fluorophore photobleaching and sample phototoxicity, limiting the number of fluorescence images obtainable from a living cell. Herein, these challenges are addressed by using ultra‐low irradiation intensities and a neural network for image restoration, enabling extensive imaging of single living cells. The endoplasmic reticulum (ER) is chosen as the target structure due to its dynamic nature over short and long timescales. The reduced irradiation intensity combined with denoising permits continuous ER dynamics observation in living cells for up to 7 h with a temporal resolution of seconds. This allows for quantitative analysis of ER structural features over short (seconds) and long (hours) timescales within the same cell, and enabled fast 3D live‐cell STED microscopy. Overall, the combination of ultralow irradiation with image restoration enables comprehensive analysis of organelle dynamics over extended periods in living cells. [ABSTRACT FROM AUTHOR]
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- 2025
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8. MINFLUX fluorescence nanoscopy in biological tissue.
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Moosmayer, Thea, Kiszka, Kamila A., Westphal, Volker, Pape, Jasmin K., Leutenegger, Marcel, Steffens, Heinz, Grant, Seth G. N., Sahl, Steffen J., and Hell, Stefan W.
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GLUTAMATE transporters , *FLUORESCENCE microscopy , *TISSUES , *OPTICAL images , *FLUOROPHORES - Abstract
Optical imaging access to nanometer-level protein distributions in intact tissue is a highly sought-after goal, as it would provide visualization in physiologically relevant contexts. Under the unfavorable signal-to-background conditions of increased absorption and scattering of the excitation and fluorescence light in the complex tissue sample, superresolution fluorescence microscopy methods are severely challenged in attaining precise localization of molecules. We reasoned that the typical use of a confocal detection pinhole in MINFLUX nanoscopy, suppressing background and providing optical sectioning, should facilitate the detection and resolution of single fluorophores even amid scattering and optically challenging tissue environments. Here, we investigated the performance of MINFLUX imaging for different synaptic targets and fluorescent labels in tissue sections of the mouse brain. Single fluorophores were localized with a precision of <5 nm at up to 80 µm sample depth. MINFLUX imaging in two color channels allowed to probe PSD95 localization relative to the spine head morphology, while also visualizing presynaptic vesicular glutamate transporter (VGlut) 1 clustering and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) clustering at the postsynapse. Our two-dimensional (2D) and three-dimensional (3D) two-color MINFLUX results in tissue, with <10 nm 3D fluorophore localization, open up broad avenues to investigate protein distributions on the single-synapse level in fixed and living brain slices. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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9. Multi‐Photon Super‐Linear Image Scanning Microscopy Using Upconversion Nanoparticles.
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Wang, Yao, Liu, Baolei, Ding, Lei, Chen, Chaohao, Shan, Xuchen, Wang, Dajing, Tian, Menghan, Song, Jiaqi, Zheng, Ze, Xu, Xiaoxue, Zhong, Xiaolan, and Wang, Fan
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LIFE sciences , *FLUORESCENCE microscopy , *MULTIPHOTON processes , *MICROSCOPY , *NANOPARTICLES - Abstract
Super‐resolution fluorescence microscopy is of great interest in life science studies for visualizing subcellular structures at the nanometer scale. Among various kinds of super‐resolution approaches, image scanning microscopy (ISM) offers a doubled resolution enhancement in a simple and straightforward manner, based on the commonly used confocal microscopes. ISM is also suitable to be integrated with multi‐photon microscopy techniques, such as two‐photon excitation and second‐harmonic generation imaging, for deep tissue imaging, but it remains the twofold limited resolution enhancement and requires expensive femtosecond lasers. Here, the super‐linear ISM (SL‐ISM) pushes the resolution enhancement beyond the factor of two is presented and experimentally demonstrated, with a single low‐power, continuous‐wave, and near‐infrared laser, by harnessing the emission nonlinearity within the multiphoton excitation process of lanthanide‐doped upconversion nanoparticles (UCNPs). Based on a modified confocal microscope, a resolution of ≈120 nm, 1/8th of the excitation wavelength is achieved. Furthermore, a parallel detection strategy of SL‐ISM with the multifocal structured excitation pattern is demonstrated, to speed up the acquisition frame rate. This method suggests a new perspective for super‐resolution imaging or sensing, multi‐photon imaging, and deep‐tissue imaging with simple, low‐cost, and straightforward implementations. [ABSTRACT FROM AUTHOR]
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- 2024
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10. Global nuclear reorganization during heterochromatin replication in the giant‐genome plant Nigella damascena L.
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Arifulin, Eugene A., Sorokin, Dmitry V., Anoshina, Nadezhda A., Kuznetsova, Maria A., Valyaeva, Anna A., Potashnikova, Daria M., Omelchenko, Denis O., Schubert, Veit, Kolesnikova, Tatyana D., and Sheval, Eugene V.
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NON-coding DNA , *TRANSMISSION electron microscopy , *HETEROCHROMATIN , *GENOME size , *PLANT genomes - Abstract
SUMMARY: Among flowering plants, genome size varies remarkably, by >2200‐fold, and this variation depends on the loss and gain of noncoding DNA sequences that form distinct heterochromatin complexes during interphase. In plants with giant genomes, most chromatin remains condensed during interphase, forming a dense network of heterochromatin threads called interphase chromonemata. Using super‐resolution light and electron microscopy, we studied the ultrastructure of chromonemata during and after replication in root meristem nuclei of Nigella damascena L. During S‐phase, heterochromatin undergoes transient decondensation locally at DNA replication sites. Due to the abundance of heterochromatin, the replication leads to a robust disassembly of the chromonema meshwork and a general reorganization of the nuclear morphology visible even by conventional light microscopy. After replication, heterochromatin recondenses, restoring the chromonema structure. Thus, we show that heterochromatin replication in interphase nuclei of giant‐genome plants induces a global nuclear reorganization. Significant Statement: The replication of giant plant genomes requires a global chromatin reorganization in interphase nuclei. [ABSTRACT FROM AUTHOR]
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- 2024
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11. Super-Resolution Microscopy as a Versatile Tool in Probing Molecular Assembly.
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Sun, Nan, Bai, Shiwei, Dai, Luru, and Jia, Yi
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MOLECULAR probes , *MOLECULAR structure , *MOLECULAR dynamics , *MOLECULAR interactions , *NANOTECHNOLOGY - Abstract
Molecular assembly is promising in the construction of advanced materials, obtaining structures with specific functions. In-depth investigation of the relationships between the formation, dynamics, structure, and functionality of the specific molecular assemblies is one of the greatest challenges in nanotechnology and chemistry, which is essential in the rational design and development of functional materials for a variety of applications. Super-resolution microscopy (SRM) has been used as a versatile tool for investigating and elucidating the structures of individual molecular assemblies with its nanometric resolution, multicolor ability, and minimal invasiveness, which are also complementary to conventional optical or electronic techniques that provide the direct observation. In this review, we will provide an overview of the representative studies that utilize SRM to probe molecular assemblies, mainly focusing on the imaging of biomolecular assemblies (lipid-based, peptide-based, protein-based, and DNA-based), organic–inorganic hybrid assemblies, and polymer assemblies. This review will provide guidelines for the evaluation of the dynamics of molecular assemblies, assembly and disassembly processes with distinct dynamic behaviors, and multicomponent assembly through the application of these advanced imaging techniques. We believe that this review will inspire new ideas and propel the development of structural analyses of molecular assemblies to promote the exploitation of new-generation functional materials. [ABSTRACT FROM AUTHOR]
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- 2024
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12. Modulated illumination microscopy: Application perspectives in nuclear nanostructure analysis.
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Cremer, Christoph, Schock, Florian, Failla, Antonio Virgilio, and Birk, Udo
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FLUORESCENCE yield , *FLUORESCENCE microscopy , *CELL nuclei , *MICROSCOPY , *NUCLEAR structure - Abstract
The structure of the cell nucleus of higher organisms has become a major topic of advanced light microscopy. So far, a variety of methods have been applied, including confocal laser scanning fluorescence microscopy, 4Pi, STED and localisation microscopy approaches, as well as different types of patterned illumination microscopy, modulated either laterally (in the object plane) or axially (along the optical axis). Based on our experience, we discuss here some application perspectives of Modulated Illumination Microscopy (MIM) and its combination with single‐molecule localisation microscopy (SMLM). For example, spatially modulated illumination microscopy/SMI (illumination modulation along the optical axis) has been used to determine the axial extension (size) of small, optically isolated fluorescent objects between ≤ 200 nm and ≥ 40 nm diameter with a precision down to the few nm range; it also allows the axial positioning of such structures down to the 1 nm scale; combined with laterally structured illumination/SIM, a 3D localisation precision of ≤1 nm is expected using fluorescence yields typical for SMLM applications. Together with the nanosizing capability of SMI, this can be used to analyse macromolecular nuclear complexes with a resolution approaching that of cryoelectron microscopy. [ABSTRACT FROM AUTHOR]
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- 2024
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13. Correlative super‐resolution microscopy with deep UV reactivation.
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Prakash, Kirti
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STIMULATED emission , *SINGLE molecules , *MORPHOLOGY , *FLUOROPHORES , *MICROSCOPY - Abstract
Correlative super‐resolution microscopy has the potential to accurately visualize and validate new biological structures past the diffraction limit. However, combining different super‐resolution modalities, such as deterministic stimulated emission depletion (STED) and stochastic single‐molecule localization microscopy (SMLM), is a challenging endeavour. For correlative STED and SMLM, the following poses a significant challenge: (1) the photobleaching of the fluorophores in STED; (2) the subsequent reactivation of the fluorophores for SMLM and (3) finding the right fluorochrome and imaging buffer for both imaging modalities. Here, we highlight how the deep ultraviolet (DBUE) wavelengths of the Mercury (Hg) arc lamp can help recover STED bleaching and allow for the reactivation of single molecules for SMLM imaging. We also show that Alexa Fluor 594 and the commercially available Prolong Diamond to be excellent fluorophores and imaging media for correlative STED and SMLM. [ABSTRACT FROM AUTHOR]
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- 2024
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14. Multicolor single-molecule localization microscopy: review and prospect
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Xi Chen, Xiangyu Wang, Fang Huang, and Donghan Ma
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Super-resolution microscopy ,Singe-molecule localization microscopy ,Multicolor imaging ,DNA-PAINT ,Ratiometric imaging ,Spectroscopic imaging ,Applied optics. Photonics ,TA1501-1820 - Abstract
Abstract Single-molecule localization microscopy (SMLM) surpasses the diffraction limit by randomly switching fluorophores between fluorescent and dark states, precisely pinpointing the resulted isolated emission patterns, thereby reconstructing the super-resolution images based on the accumulated locations of thousands to millions of single molecules. This technique achieves a ten-fold improvement in resolution, unveiling the intricate details of molecular activities and structures in cells and tissues. Multicolor SMLM extends this capability by imaging distinct protein species labeled with various fluorescent probes, providing insights into structural intricacies and spatial relationships among different targets. This review explores recent advancements in multicolor SMLM, evaluates the strengths and limitations of each variant, and discusses the future prospects.
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- 2024
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15. Imaging interorganelle contacts at a glance.
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Zanellati, Maria Clara, Chih-Hsuan Hsu, and Cohen, Sarah
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HIGH resolution imaging , *MICROSCOPY , *ELECTRON microscopy , *EUKARYOTIC cells , *ION exchange (Chemistry) - Abstract
Eukaryotic cells are compartmentalized into membrane-bound organelles that must coordinate their responses to stimuli. One way that organelles communicate is via membrane contact sites (MCSs), sites of close apposition between organelles used for the exchange of ions, lipids and information. In this Cell Science at a Glance article and the accompanying poster, we describe an explosion of new methods that have led to exciting progress in this area and discuss key examples of how these methods have advanced our understanding of MCSs. We discuss how diffraction-limited and super-resolution fluorescence imaging approaches have provided important insight into the biology of interorganelle communication.We also describe how the development of multiple proximity-based methods has enabled the detection ofMCSswith high accuracy and precision. Finally, we assess howrecent advances in electronmicroscopy (EM), considered the gold standard for detecting MCSs, have allowed the visualization of MCSs and associated proteins in 3D at ever greater resolution. [ABSTRACT FROM AUTHOR]
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- 2024
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16. This Microtubule Does Not Exist: Super‐Resolution Microscopy Image Generation by a Diffusion Model.
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Saguy, Alon, Nahimov, Tav, Lehrman, Maia, Gómez‐de‐Mariscal, Estibaliz, Hidalgo‐Cenalmor, Iván, Alalouf, Onit, Balakrishnan, Ashwin, Heilemann, Mike, Henriques, Ricardo, and Shechtman, Yoav
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GENERATIVE artificial intelligence , *DATA augmentation , *SINGLE molecules , *SPATIAL resolution , *DEEP learning , *RESEARCH personnel - Abstract
Generative models, such as diffusion models, have made significant advancements in recent years, enabling the synthesis of high‐quality realistic data across various domains. Here, the adaptation and training of a diffusion model on super‐resolution microscopy images are explored. It is shown that the generated images resemble experimental images, and that the generation process does not exhibit a large degree of memorization from existing images in the training set. To demonstrate the usefulness of the generative model for data augmentation, the performance of a deep learning‐based single‐image super‐resolution (SISR) method trained using generated high‐resolution data is compared against training using experimental images alone, or images generated by mathematical modeling. Using a few experimental images, the reconstruction quality and the spatial resolution of the reconstructed images are improved, showcasing the potential of diffusion model image generation for overcoming the limitations accompanying the collection and annotation of microscopy images. Finally, the pipeline is made publicly available, runnable online, and user‐friendly to enable researchers to generate their own synthetic microscopy data. This work demonstrates the potential contribution of generative diffusion models for microscopy tasks and paves the way for their future application in this field. [ABSTRACT FROM AUTHOR]
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- 2024
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17. Neural network‐assisted localization of clustered point spread functions in single‐molecule localization microscopy.
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Choudhury, Pranjal and Boruah, Bosanta R.
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CONVOLUTIONAL neural networks , *MORPHOLOGY , *MACHINE learning , *SPATIAL resolution , *FLUOROPHORES - Abstract
Single‐molecule localization microscopy (SMLM), which has revolutionized nanoscale imaging, faces challenges in densely labelled samples due to fluorophore clustering, leading to compromised localization accuracy. In this paper, we propose a novel convolutional neural network (CNN)‐assisted approach to address the issue of locating the clustered fluorophores. Our CNN is trained on a diverse data set of simulated SMLM images where it learns to predict point spread function (PSF) locations by generating Gaussian blobs as output. Through rigorous evaluation, we demonstrate significant improvements in PSF localization accuracy, especially in densely labelled samples where traditional methods struggle. In addition, we employ blob detection as a post‐processing technique to refine the predicted PSF locations and enhance localization precision. Our study underscores the efficacy of CNN in addressing clustering challenges in SMLM, thereby advancing spatial resolution and enabling deeper insights into complex biological structures. [ABSTRACT FROM AUTHOR]
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- 2024
- Full Text
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18. Multicolor single-molecule localization microscopy: review and prospect.
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Chen, Xi, Wang, Xiangyu, Huang, Fang, and Ma, Donghan
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HIGH resolution imaging ,SPECTROSCOPIC imaging ,MOLECULAR structure ,FLUORESCENT probes ,SINGLE molecules - Abstract
Single-molecule localization microscopy (SMLM) surpasses the diffraction limit by randomly switching fluorophores between fluorescent and dark states, precisely pinpointing the resulted isolated emission patterns, thereby reconstructing the super-resolution images based on the accumulated locations of thousands to millions of single molecules. This technique achieves a ten-fold improvement in resolution, unveiling the intricate details of molecular activities and structures in cells and tissues. Multicolor SMLM extends this capability by imaging distinct protein species labeled with various fluorescent probes, providing insights into structural intricacies and spatial relationships among different targets. This review explores recent advancements in multicolor SMLM, evaluates the strengths and limitations of each variant, and discusses the future prospects. [ABSTRACT FROM AUTHOR]
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- 2024
- Full Text
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19. Graph-Based Spatial Proximity of Super-Resolved Protein–Protein Interactions Predicts Cancer Drug Responses in Single Cells.
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Zhang, Nicholas, Cai, Shuangyi, Wang, Mingshuang, Hu, Thomas, Schneider, Frank, Sun, Shi-Yong, and Coskun, Ahmet F.
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GRAPH neural networks , *COMBINATION drug therapy , *DRUG interactions , *PROTEIN-protein interactions , *DRUG resistance , *DEEP learning - Abstract
Purpose: Current bulk molecular assays fail to capture spatial signaling activities in cancers, limiting our understanding of drug resistance mechanisms. We developed a graph-based super-resolution protein-protein interaction (GSR-PPI) technique to spatially resolve single-cell signaling networks and evaluate whether higher resolution microscopy enhances the biological study of PPIs using deep learning classification models. Methods: Single-cell spatial proximity ligation assays (PLA, ≤ 9 PPI pairs) were conducted on EGFR mutant (EGFRm) PC9 and HCC827 cells (>10,000 cells) treated with 100 nM Osimertinib. Multiplexed PPI images were obtained using wide-field and super-resolution microscopy (Zeiss Airyscan, SRRF). Graph-based deep learning models analyzed subcellular protein interactions to classify drug treatment states and test GSR-PPI on clinical tissue samples. GSR-PPI triangulated PPI nodes into 3D relationships, predicting drug treatment labels. Biological discriminative ability (BDA) was evaluated using accuracy, AUC, and F1 scores. The method was also applied to 3D spatial proteomic molecular pixelation (PixelGen) data from T cells. Results: GSR-PPI outperformed baseline models in predicting drug responses from multiplexed PPI imaging in EGFRm cells. Super-resolution data significantly improved accuracy over localized wide-field imaging. GSR-PPI classified drug treatment states in cancer cells and human lung tissues, with performance improving as imaging resolution increased. It differentiated single and combination drug therapies in HCC827 cells and human tissues. Additionally, GSR-PPI accurately distinguished T-cell stimulation states, identifying key nodes such as CD44, CD45, and CD54. Conclusion: The GSR-PPI framework provides valuable insights into spatial protein interactions and drug responses, enhancing the study of signaling biology and drug resistance. [ABSTRACT FROM AUTHOR]
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- 2024
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20. Expanding Insights: Harnessing Expansion Microscopy for Super-Resolution Analysis of HIV-1–Cell Interactions.
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Petrich, Annett, Hwang, Gyu Min, La Rocca, Laetitia, Hassan, Mariam, Anders-Össwein, Maria, Sonntag-Buck, Vera, Heuser, Anke-Mareil, Laketa, Vibor, Müller, Barbara, Kräusslich, Hans-Georg, and Klaus, Severina
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EXPANSION microscopy , *LIFE cycles (Biology) , *MORPHOLOGY , *CELL nuclei , *HIV - Abstract
Expansion microscopy has recently emerged as an alternative technique for achieving high-resolution imaging of biological structures. Improvements in resolution are achieved by physically expanding samples through embedding in a swellable hydrogel before microscopy. However, expansion microscopy has been rarely used in the field of virology. Here, we evaluate and characterize the ultrastructure expansion microscopy (U-ExM) protocol, which facilitates approximately four-fold sample expansion, enabling the visualization of different post-entry stages of the HIV-1 life cycle, focusing on nuclear events. Our findings demonstrate that U-ExM provides robust sample expansion and preservation across different cell types, including cell-culture-adapted and primary CD4+ T-cells as well as monocyte-derived macrophages, which are known HIV-1 reservoirs. Notably, cellular targets such as nuclear bodies and the chromatin landscape remain well preserved after expansion, allowing for detailed investigation of HIV-1–cell interactions at high resolution. Our data indicate that morphologically distinct HIV-1 capsid assemblies can be differentiated within the nuclei of infected cells and that U-ExM enables detection of targets that are masked in commonly used immunofluorescence protocols. In conclusion, we advocate for U-ExM as a valuable new tool for studying virus–host interactions with enhanced spatial resolution. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
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21. GPCR Inhibitors Have Antiviral Properties against JC Polyomavirus Infection.
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Sandberg, Amanda L., Bond, Avery C. S., Bennett, Lucas J., Craig, Sophie E., Winski, David P., Kirkby, Lara C., Kraemer, Abby R., Kelly, Kristina G., Hess, Samuel T., and Maginnis, Melissa S.
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G protein coupled receptors , *PROGRESSIVE multifocal leukoencephalopathy , *POLYOMAVIRUS diseases , *SEROTONIN receptors , *NEURODEGENERATION , *ANTIVIRAL agents - Abstract
JC polyomavirus (JCPyV) infects the majority of the population and initially establishes a persistent but asymptomatic infection of the kidneys. In healthy individuals, the infection remains controlled by the host immune system, but for individuals experiencing prolonged immunosuppression, the infection can reactivate and spread to the brain, where it causes progressive multifocal leukoencephalopathy (PML), which is a fatal neurodegenerative disease. Currently, there are no approved therapies to treat PML, and affected individuals suffer rapid motor weakness and cognitive deterioration. To identify novel therapeutic treatments for JCPyV infection, receptor agonists/antagonists identified in a previously published drug screen were evaluated for their antiviral properties. Seven drugs were selected and validated using infectivity assays, and the mechanism of inhibition was further explored for G protein coupled receptor (GPCR)-associated inhibitors due to the role of the GPCR 5-hydroxytryptamine 2 receptors (5-HT2Rs) in JCPyV entry. The inhibitors cetirizine and paroxetine both reduced infection early in the JCPyV infectious cycle. Paroxetine specifically reduced viral internalization through altering the receptor density of 5-HT2CR, inhibiting β-arrestin recruitment to the receptor, and reducing MAPK signaling through ERK. These findings highlight the potential of receptor signaling and viral entry mechanisms as possible targets for antiviral drug development. Further, this research suggests that FDA-approved receptor agonists/antagonists currently used to treat other medical conditions could be repurposed into antivirals for the possible treatment of JCPyV infection and the fatal disease PML. [ABSTRACT FROM AUTHOR]
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- 2024
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22. Unveiling intracellular phase separation: advances in optical imaging of biomolecular condensates.
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Guo, Yinfeng and Zhang, Xin
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PHASE separation , *MICROSCOPY , *OPTICAL images , *CELL physiology , *MOLECULES - Abstract
Advanced optical imaging techniques enhance our ability to investigate various intracellular biomolecular condensates that form through phase separation. Biomolecular condensates demonstrate diverse organizations and dynamics, serving as the physical infrastructure that enables the execution of biological processes. The physicochemical properties of condensates dictate the rules governing their multilayered ultrastructure. Intracellular biomolecular condensates, which form via phase separation, display a highly organized ultrastructure and complex properties. Recent advances in optical imaging techniques, including super-resolution microscopy and innovative microscopic methods that leverage the intrinsic properties of the molecules observed, have transcended the limitations of conventional microscopies. These advances facilitate the exploration of condensates at finer scales and in greater detail. The deployment of these emerging but sophisticated imaging tools allows for precise observations of the multiphasic organization and physicochemical properties of these condensates, shedding light on their functions in cellular processes. In this review, we highlight recent progress in methodological innovations and their profound implications for understanding the organization and dynamics of intracellular biomolecular condensates. [ABSTRACT FROM AUTHOR]
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- 2024
- Full Text
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23. A novel super-resolution microscopy platform for cutaneous alpha-synuclein detection in Parkinson's disease.
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Sade, Ofir, Fischel, Daphna, Barak-Broner, Noa, Halevi, Shir, Gottfried, Irit, Bar-On, Dana, Sachs, Stefan, Mirelman, Anat, Thaler, Avner, Gour, Aviv, Kestenbaum, Meir, Weisz, Mali Gana, Anis, Saar, Soto, Claudio, Roitman, Melanie Shanie, Shahar, Shimon, Doppler, Kathrin, Sauer, Markus, Giladi, Nir, and Lev, Nirit
- Subjects
PARKINSON'S disease ,SINGLE molecules ,CENTRAL nervous system ,NEURONS ,SKIN biopsy - Abstract
Alpha-synuclein (aSyn) aggregates in the central nervous system are the main pathological hallmark of Parkinson's disease (PD). ASyn aggregates have also been detected in many peripheral tissues, including the skin, thus providing a novel and accessible target tissue for the detection of PD pathology. Still, a wellestablished validated quantitative biomarker for early diagnosis of PD that also allows for tracking of disease progression remains lacking. The main goal of this research was to characterize aSyn aggregates in skin biopsies as a comparative and quantitative measure for PD pathology. Using direct stochastic optical reconstruction microscopy (dSTORM) and computational tools, we imaged total and phosphorylated-aSyn at the single molecule level in sweat glands and nerve bundles of skin biopsies from healthy controls (HCs) and PD patients. We developed a user-friendly analysis platform that offers a comprehensive toolkit for researchers that combines analysis algorithms and applies a series of cluster analysis algorithms (i.e., DBSCAN and FOCAL) onto dSTORM images. Using this platform, we found a significant decrease in the ratio of the numbers of neuronal marker molecules to phosphorylated-aSyn molecules, suggesting the existence of damaged nerve cells in fibers highly enriched with phosphorylatedaSyn molecules. Furthermore, our analysis found a higher number of aSyn aggregates in PD subjects than in HC subjects, with differences in aggregate size, density, and number of molecules per aggregate. On average, aSyn aggregate radii ranged between 40 and 200 nm and presented an average density of 0.001-0.1 molecules/nm2. Our dSTORM analysis thus highlights the potential of our platform for identifying quantitative characteristics of aSyn distribution in skin biopsies not previously described for PD patients while offering valuable insight into PD pathology by elucidating patient aSyn aggregation status. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
24. Dissecting the role of SMN multimerization in its dissociation from the Cajal body using harmine as a tool compound.
- Author
-
Saki Ohazama, Akiko Fujimoto, Daisuke Konda, Ryota Yokoyama, Shinichi Nakagawa, and Hiroshi Maita
- Subjects
- *
SPINAL muscular atrophy , *MOLECULAR motor proteins , *MOTOR neurons , *ALKALOIDS , *MICROSCOPY - Abstract
Survival motor neuron protein (SMN), which is linked to spinal muscular atrophy, is a key component of the Gemin complex, which is essential for the assembly of small nuclear RNA-protein complexes (snRNPs). After initial snRNP assembly in the cytoplasm, both snRNPs and SMN migrate to the nucleus and associate with Cajal bodies, where final snRNP maturation occurs. It is assumed that SMN must be free from the Cajal bodies for continuous snRNP biogenesis. Previous observation of the SMN granules docked in the Cajal bodies suggests the existence of a separation mechanism. However, the precise processes that regulate the spatial separation of SMN complexes from Cajal bodies remain unclear. Here, we have employed a super-resolution microscope alongside the β-carboline alkaloid harmine, which disrupts the Cajal body in a reversible manner. Upon removal of harmine, SMN and Coilin first appear as small interconnected condensates. The SMN condensates mature into spheroidal structures encircled by Coilin, eventually segregating into distinct condensates. Expression of a multimerization-deficient SMN mutant leads to enlarged, atypical Cajal bodies in which SMN is unable to segregate into separate condensates. These findings underscore the importance of multimerization in facilitating the segregation of SMN from Coilin within Cajal bodies. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
25. Photoactivatable Xanthone (PaX) Dyes Enable Quantitative, Dual Color, and Live‐Cell MINFLUX Nanoscopy.
- Author
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Remmel, Michael, Matthias, Jessica, Lincoln, Richard, Keller‐Findeisen, Jan, Butkevich, Alexey N., Bossi, Mariano L., and Hell, Stefan W.
- Subjects
- *
NUCLEAR pore complex , *FLUORESCENT dyes , *MOLECULAR switches , *FLUORESCENT proteins , *SPECTRAL imaging - Abstract
The single‐molecule localization concept MINFLUX has triggered a reevaluation of the features of fluorophores for attaining nanometer‐scale resolution. MINFLUX nanoscopy benefits from temporally controlled fluorescence ("on"/"off") photoswitching. Combined with an irreversible switching behavior, the localization process is expected to turn highly efficient and quantitative data analysis simple. The potential in the recently reported photoactivable xanthone (PaX) dyes is recognized to extend the list of molecular switches used for MINFLUX with 561 nm excitation beyond the fluorescent protein mMaple. The MINFLUX localization success rates of PaX560, PaX+560, and mMaple are quantitatively compared by analyzing the effective labeling efficiency of endogenously tagged nuclear pore complexes. The PaX dyes prove to be superior to mMaple and on par with the best reversible molecular switches routinely used in single‐molecule localization microscopy. Moreover, the rationally designed PaX595 is introduced for complementing PaX560 in dual color 561 nm MINFLUX imaging based on spectral classification and the deterministic, irreversible, and additive‐independent nature of PaX photoactivation is showcased in fast live‐cell MINFLUX imaging. The PaX dyes meet the demands of MINFLUX for a robust readout of each label position and fill the void of reliable fluorophores dedicated to 561 nm MINFLUX imaging. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
26. SV2A controls the surface nanoclustering and endocytic recruitment of Syt1 during synaptic vesicle recycling.
- Author
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Small, Christopher, Harper, Callista, Jiang, Anmin, Kontaxi, Christiana, Pronot, Marie, Yak, Nyakuoy, Malapaka, Anusha, Davenport, Elizabeth C., Wallis, Tristan P., Gormal, Rachel S., Joensuu, Merja, Martínez‐Mármol, Ramón, Cousin, Michael A., and Meunier, Frédéric A.
- Subjects
- *
SYNAPTIC vesicles , *ENDOCYTOSIS , *CELL membranes , *EXOCYTOSIS , *NEURAL transmission - Abstract
Following exocytosis, the recapture of plasma membrane‐stranded vesicular proteins into recycling synaptic vesicles (SVs) is essential for sustaining neurotransmission. Surface clustering of vesicular proteins has been proposed to act as a 'pre‐assembly' mechanism for endocytosis that ensures high‐fidelity retrieval of SV cargo. Here, we used single‐molecule imaging to examine the nanoclustering of synaptotagmin‐1 (Syt1) and synaptic vesicle protein 2A (SV2A) in hippocampal neurons. Syt1 forms surface nanoclusters through the interaction of its C2B domain with SV2A, which are sensitive to mutations in this domain (Syt1K326A/K328A) and SV2A knockdown. SV2A co‐clustering with Syt1 is reduced by blocking SV2A's cognate interaction with Syt1 (SV2AT84A). Surprisingly, impairing SV2A‐Syt1 nanoclustering enhanced the plasma membrane recruitment of key endocytic protein dynamin‐1, causing accelerated Syt1 endocytosis, altered intracellular sorting and decreased trafficking of Syt1 to Rab5‐positive endocytic compartments. Therefore, SV2A and Syt1 are segregated from the endocytic machinery in surface nanoclusters, limiting dynamin recruitment and negatively regulating Syt1 entry into recycling SVs. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
27. Identification of z‐axis filopodia in growth cones using super‐resolution microscopy.
- Author
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Nozumi, Motohiro, Sato, Yuta, Nishiyama‐Usuda, Miyako, and Igarashi, Michihiro
- Subjects
- *
OPTICAL diffraction , *LIPID rafts , *CYTOSKELETON , *MICROSCOPY , *FILOPODIA , *EPHRIN receptors - Abstract
A growth cone is a highly motile tip of an extending axon that is crucial for neural network formation. Three‐dimensional‐structured illumination microscopy, a type of super‐resolution light microscopy with a resolution that overcomes the optical diffraction limitation (ca. 200 nm) of conventional light microscopy, is well suited for studying the molecular dynamics of intracellular events. Using this technique, we discovered a novel type of filopodia distributed along the z‐axis ("z‐filopodia") within the growth cone. Z‐filopodia were typically oriented in the direction of axon growth, not attached to the substratum, protruded spontaneously without microtubule invasion, and had a lifetime that was considerably shorter than that of conventional filopodia. Z‐filopodia formation and dynamics were regulated by actin‐regulatory proteins, such as vasodilator‐stimulated phosphoprotein, fascin, and cofilin. Chromophore‐assisted laser inactivation of cofilin induced the rapid turnover of z‐filopodia. An axon guidance receptor, neuropilin‐1, was concentrated in z‐filopodia and was transported together with them, whereas its ligand, semaphorin‐3A, was selectively bound to them. Membrane domains associated with z‐filopodia were also specialized and resembled those of lipid rafts, and their behaviors were closely related to those of neuropilin‐1. The results suggest that z‐filopodia have unique turnover properties, and unlike xy‐filopodia, do not function as force‐generating structures for axon extension. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
28. Enhanced Performance of the Optimized Dye CF583R in Direct Stochastic Optical Reconstruction Microscopy of Active Zones in Drosophila Melanogaster.
- Author
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Noß, Marvin, Ljaschenko, Dmitrij, and Mrestani, Achmed
- Subjects
- *
DROSOPHILA melanogaster , *MICROSCOPY , *SCAFFOLD proteins , *NEUROPLASTICITY , *FLUORITE - Abstract
Super-resolution single-molecule localization microscopy (SMLM) of presynaptic active zones (AZs) and postsynaptic densities contributed to the observation of protein nanoclusters that are involved in defining functional characteristics and in plasticity of synaptic connections. Among SMLM techniques, direct stochastic optical reconstruction microscopy (dSTORM) depends on organic fluorophores that exert high brightness and reliable photoswitching. While multicolor imaging is highly desirable, the requirements necessary for high-quality dSTORM make it challenging to identify combinations of equally performing, spectrally separated dyes. Red-excited carbocyanine dyes, e.g., Alexa Fluor 647 (AF647) or Cy5, are currently regarded as "gold standard" fluorophores for dSTORM imaging. However, a recent study introduced a set of chemically modified rhodamine dyes, including CF583R, that promise to display similar performance in dSTORM. In this study, we defined CF583R's performance compared to AF647 and CF568 based on a nanoscopic analysis of Bruchpilot (Brp), a nanotopologically well-characterized scaffold protein at Drosophila melanogaster AZs. We demonstrate equal suitability of AF647, CF568 and CF583R for basal AZ morphometry, while in Brp subcluster analysis CF583R outperforms CF568 and is on par with AF647. Thus, the AF647/CF583R combination will be useful in future dSTORM-based analyses of AZs and other subcellularly located marker molecules and their role in physiological and pathophysiological contexts. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
29. Advancing cell biology with nanoscale fluorescence imaging: essential practical considerations.
- Author
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DʼEste, Elisa, Lukinavičius, Gražvydas, Lincoln, Richard, Opazo, Felipe, and Fornasiero, Eugenio F.
- Subjects
- *
CELL imaging , *STEREOLOGY , *CELL anatomy , *CYTOLOGY , *MICROSCOPY , *FLUORESCENT dyes - Abstract
Here, we provide an overview of the key factors to consider when using fluorescence nanoscopy (FN), including biological questions that can be addressed and aspects that might improve the reliability and effectiveness of FN experiments. We cover the main aspects related to sample preparation, including the selection of appropriate fixation, affinity-based labels, and fluorescent dyes. We discuss current limitations and possible future developments in the field that would facilitate a broader application of FN. We discuss multiplexing possibilities (allowing the simultaneous detection of multiple targets in a single experiment), live cell imaging for the study of cellular and molecular dynamic processes, and quantitative workflows. Recently, biologists have gained access to several far-field fluorescence nanoscopy (FN) technologies that allow the observation of cellular components with ~20 nm resolution. FN is revolutionizing cell biology by enabling the visualization of previously inaccessible subcellular details. While technological advances in microscopy are critical to the field, optimal sample preparation and labeling are equally important and often overlooked in FN experiments. In this review, we provide an overview of the methodological and experimental factors that must be considered when performing FN. We present key concepts related to the selection of affinity-based labels, dyes, multiplexing, live cell imaging approaches, and quantitative microscopy. Consideration of these factors greatly enhances the effectiveness of FN, making it an exquisite tool for numerous biological applications. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
30. A framework for the simulation of individual glycan coordinates to analyze spatial relationships within the glycocalyx
- Author
-
Sarah Fritsche and Leonhard Möckl
- Subjects
glycocalyx ,super-resolution microscopy ,simulation ,computational methods ,sialic acids ,ocular glycocalyx ,Biology (General) ,QH301-705.5 - Abstract
The glycocalyx is a dense and dynamic layer of glycosylated species that covers every cell in the human body. It plays crucial roles in various cellular processes in health and disease, such as cancer immune evasion, cancer immune therapy, blastocyst implantation, and functional attenuation of membrane protein diffusion. In addition, alterations in glycocalyx structure may play an important role in ocular surface diseases, e.g., dry eye disease. Despite the emerging importance of the glycocalyx, various aspects of its functional organization remain elusive to date. A central reason for this elusiveness is the nanoscale dimension of the glycocalyx in conjunction with its high structural complexity, which is not accessible to observation with conventional light microscopy. Recent advances in super-resolution microscopy have enabled resolutions down to the single-digit nanometer range. In order to fully leverage the potential of these novel methods, computational frameworks that allow for contextualization of the resulting experimental data are required. Here, we present a simulation-based approach to analyze spatial relationships of glycan components on the cell membrane based on known geometrical parameters. We focus on sialic acids in this work, but the technique can be adapted to any glycan component of interest. By integrating data from mass spectrometry and quantitative biological studies, these simulations aim to model possible experimental outcomes, which can then be used for further analysis, such as spatial point statistics. Importantly, we include various experimental considerations, such as labeling and detection efficiency. This approach may contribute to establishing a new standard of connection between geometrical and molecular-resolution data in service of advancing our understanding of the functional role of the glycocalyx in biology as well as its clinical potential.
- Published
- 2025
- Full Text
- View/download PDF
31. Bilateral regulation of EGFR activity and local PI(4,5)P2 dynamics in mammalian cells observed with superresolution microscopy
- Author
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Mitsuhiro Abe, Masataka Yanagawa, Michio Hiroshima, Toshihide Kobayashi, and Yasushi Sako
- Subjects
EGFR ,epidermal growth factor receptor ,membrane lipid ,phosphatidylinositol-4,5-bisphosphate ,super-resolution microscopy ,single-molecule imaging ,Medicine ,Science ,Biology (General) ,QH301-705.5 - Abstract
Anionic lipid molecules, including phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), are implicated in the regulation of epidermal growth factor receptor (EGFR). However, the role of the spatiotemporal dynamics of PI(4,5)P2 in the regulation of EGFR activity in living cells is not fully understood, as it is difficult to visualize the local lipid domains around EGFR. Here, we visualized both EGFR and PI(4,5)P2 nanodomains in the plasma membrane of HeLa cells using super-resolution single-molecule microscopy. The EGFR and PI(4,5)P2 nanodomains aggregated before stimulation with epidermal growth factor (EGF) through transient visits of EGFR to the PI(4,5)P2 nanodomains. The degree of coaggregation decreased after EGF stimulation and depended on phospholipase Cγ, the EGFR effector hydrolyzing PI(4,5)P2. Artificial reduction in the PI(4,5)P2 content of the plasma membrane reduced both the dimerization and autophosphorylation of EGFR after stimulation with EGF. Inhibition of PI(4,5)P2 hydrolysis after EGF stimulation decreased phosphorylation of EGFR-Thr654. Thus, EGFR kinase activity and the density of PI(4,5)P2 around EGFR molecules were found to be mutually regulated.
- Published
- 2024
- Full Text
- View/download PDF
32. High-sensitivity whole-mount in situ Hybridization of Mouse Oocytes and Embryos Visualizes the Super-resolution Structures and Distributions of mRNA Molecules
- Author
-
Takahiro Sanada and Tomoya Kotani
- Subjects
Mammal ,Oocyte ,Embryo ,Maternal mRNA ,in situ hybridization ,Super-resolution microscopy ,Medicine (General) ,R5-920 ,Biology (General) ,QH301-705.5 - Abstract
Abstract Mammalian oocytes accumulate more than ten thousand mRNAs, of which three to four thousand mRNAs are translationally repressed. The timings and sites of translational activation of these dormant mRNAs are crucial for promoting oocyte maturation and embryonic development. How these mRNAs are accumulated and distributed in oocytes is therefore a fundamental issue to be explored. A method that enables visualization of mRNA molecules with high resolution in a simple manner would be valuable for understanding how oocytes accumulate and regulate the dormant mRNAs. We have developed a highly sensitive whole-mount in situ hybridization method using in vitro-synthesized RNA probes and the tyramide signal amplification (TSA) system optimized for mouse oocytes and embryos. By using this method, Pou5f1/Oct4, Emi2, and cyclin B1 mRNAs were detected in immature oocytes and 2-cell stage embryos. Confocal microscopy showed that these mRNAs formed granular structures in the oocyte cytoplasm. The structures of Pou5f1/Oct4 and cyclin B1 mRNAs persisted in 2-cell stage embryos. Pou5f1/Oct4 RNA granules exhibited a solid-like property in immature oocytes and became liquid-like droplets in 2-cell stage embryos. Double-staining of cyclin B1 mRNA with Emi2 or Pou5f1/Oct4 mRNA revealed that these mRNAs were distributed as different RNA granules without overlapping each other and that the size of cyclin B1 RNA granules tended to be larger than that of Emi2 RNA granules. The structures and distribution patterns of these mRNAs were further analyzed by N-SIM super-resolution microscopy. This analysis revealed that the large-sized RNA granules consist of many small-sized granules, suggesting the accumulation and regulation of dormant mRNAs as basal-sized RNA granules. The method established in this study can easily visualize the structure and distribution of mRNAs accumulated in mammalian oocytes and embryos with high sensitivity and super-resolution. This method is useful for investigating the cellular and molecular mechanisms of translational control of mRNAs by which maturation and early developmental processes are promoted.
- Published
- 2024
- Full Text
- View/download PDF
33. A non-toxic equinatoxin-II reveals the dynamics and distribution of sphingomyelin in the cytosolic leaflet of the plasma membrane
- Author
-
Toshiki Mori, Takahiro Niki, Yasunori Uchida, Kojiro Mukai, Yoshihiko Kuchitsu, Takuma Kishimoto, Shota Sakai, Asami Makino, Toshihide Kobayashi, Hiroyuki Arai, Yasunari Yokota, Tomohiko Taguchi, and Kenichi G. N. Suzuki
- Subjects
Nontoxic equinatoxin-II variant ,Sphingomyelin ,Rafts ,Inner leaflet ,Super-resolution microscopy ,Single molecule imaging ,Medicine ,Science - Abstract
Abstract Sphingomyelin (SM) is a major sphingolipid in mammalian cells. SM is enriched in the extracellular leaflet of the plasma membrane (PM). Besides this localization, recent electron microscopic and biochemical studies suggest the presence of SM in the cytosolic leaflet of the PM. In the present study, we generated a non-toxic SM-binding variant (NT-EqtII) based on equinatoxin-II (EqtII) from the sea anemone Actinia equina, and examined the dynamics of SM in the cytosolic leaflet of living cell PMs. NT-EqtII with two point mutations (Leu26Ala and Pro81Ala) had essentially the same specificity and affinity to SM as wild-type EqtII. NT-EqtII expressed in the cytosol was recruited to the PM in various cell lines. Super-resolution microscopic observation revealed that NT-EqtII formed tiny domains that were significantly colocalized with cholesterol and N-terminal Lyn. Meanwhile, single molecule observation at high resolutions down to 1 ms revealed that all the examined lipid probes including NT-EqtII underwent apparent fast simple Brownian diffusion, exhibiting that SM and other lipids in the cytosolic leaflet rapidly moved in and out of domains. Thus, the novel SM-binding probe demonstrated the presence of the raft-like domain in the cytosolic leaflet of living cell PMs.
- Published
- 2024
- Full Text
- View/download PDF
34. Nanoscale insights into hematology: super-resolved imaging on blood cell structure, function, and pathology
- Author
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Jinghan Liu, Yuping Yolanda Tan, Wen Zheng, Yao Wang, Lining Arnold Ju, and Qian Peter Su
- Subjects
Fluorescence nanoscopy ,Super-resolution microscopy ,Blood cells ,Platelets ,Red blood cells ,White blood cells ,Biotechnology ,TP248.13-248.65 ,Medical technology ,R855-855.5 - Abstract
Abstract Fluorescence nanoscopy, also known as super-resolution microscopy, has transcended the conventional resolution barriers and enabled visualization of biological samples at nanometric resolutions. A series of super-resolution techniques have been developed and applied to investigate the molecular distribution, organization, and interactions in blood cells, as well as the underlying mechanisms of blood-cell-associated diseases. In this review, we provide an overview of various fluorescence nanoscopy technologies, outlining their current development stage and the challenges they are facing in terms of functionality and practicality. We specifically explore how these innovations have propelled forward the analysis of thrombocytes (platelets), erythrocytes (red blood cells) and leukocytes (white blood cells), shedding light on the nanoscale arrangement of subcellular components and molecular interactions. We spotlight novel biomarkers uncovered by fluorescence nanoscopy for disease diagnosis, such as thrombocytopathies, malignancies, and infectious diseases. Furthermore, we discuss the technological hurdles and chart out prospective avenues for future research directions. This review aims to underscore the significant contributions of fluorescence nanoscopy to the field of blood cell analysis and disease diagnosis, poised to revolutionize our approach to exploring, understanding, and managing disease at the molecular level. Graphical Abstract
- Published
- 2024
- Full Text
- View/download PDF
35. Integrating bioengineering, super-resolution microscopy and mechanobiology in autophagy research: addendum to the guidelines (4th edition)
- Author
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Ravasio, Andrea, Klionsky, Daniel J., and Bertocchi, Cristina
- Subjects
- *
AUTOPHAGY , *MICROSCOPY , *DISRUPTIVE innovations , *MICROFABRICATION , *BIOENGINEERING , *SYNTHETIC biology - Abstract
Recent key technological developments, such as super-resolution microscopy and microfabrication, enabled investigation of biological processes, including macroautophagy/autophagy, with unprecedented spatiotemporal resolution and control over experimental conditions. Such disruptive innovations deepened our capability to provide mechanistic understandings of the autophagic process and its causes. This addendum aims to expand the guidelines on autophagy in three key directions: optical methods enabling visualization of autophagic machinery beyond the diffraction-limited resolution; bioengineering enabling accurate designs and control over experimental conditions; and theoretical advances in mechanobiology connecting autophagy and mechanical processes of the cell.
Abbreviation: 3D: three-dimensional; SIM: structured illumination microscopy; STORM: stochastic optical reconstruction microscopy. [ABSTRACT FROM AUTHOR]- Published
- 2024
- Full Text
- View/download PDF
36. A non-toxic equinatoxin-II reveals the dynamics and distribution of sphingomyelin in the cytosolic leaflet of the plasma membrane.
- Author
-
Mori, Toshiki, Niki, Takahiro, Uchida, Yasunori, Mukai, Kojiro, Kuchitsu, Yoshihiko, Kishimoto, Takuma, Sakai, Shota, Makino, Asami, Kobayashi, Toshihide, Arai, Hiroyuki, Yokota, Yasunari, Taguchi, Tomohiko, and Suzuki, Kenichi G. N.
- Subjects
CELL membranes ,SPHINGOMYELIN ,PAMPHLETS ,SINGLE molecules ,SEA anemones ,MOLECULAR probes - Abstract
Sphingomyelin (SM) is a major sphingolipid in mammalian cells. SM is enriched in the extracellular leaflet of the plasma membrane (PM). Besides this localization, recent electron microscopic and biochemical studies suggest the presence of SM in the cytosolic leaflet of the PM. In the present study, we generated a non-toxic SM-binding variant (NT-EqtII) based on equinatoxin-II (EqtII) from the sea anemone Actinia equina, and examined the dynamics of SM in the cytosolic leaflet of living cell PMs. NT-EqtII with two point mutations (Leu26Ala and Pro81Ala) had essentially the same specificity and affinity to SM as wild-type EqtII. NT-EqtII expressed in the cytosol was recruited to the PM in various cell lines. Super-resolution microscopic observation revealed that NT-EqtII formed tiny domains that were significantly colocalized with cholesterol and N-terminal Lyn. Meanwhile, single molecule observation at high resolutions down to 1 ms revealed that all the examined lipid probes including NT-EqtII underwent apparent fast simple Brownian diffusion, exhibiting that SM and other lipids in the cytosolic leaflet rapidly moved in and out of domains. Thus, the novel SM-binding probe demonstrated the presence of the raft-like domain in the cytosolic leaflet of living cell PMs. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
37. High-sensitivity whole-mount in situ Hybridization of Mouse Oocytes and Embryos Visualizes the Super-resolution Structures and Distributions of mRNA Molecules.
- Author
-
Sanada, Takahiro and Kotani, Tomoya
- Subjects
IN situ hybridization ,OVUM ,MESSENGER RNA ,EMBRYOLOGY ,EMBRYOS - Abstract
Mammalian oocytes accumulate more than ten thousand mRNAs, of which three to four thousand mRNAs are translationally repressed. The timings and sites of translational activation of these dormant mRNAs are crucial for promoting oocyte maturation and embryonic development. How these mRNAs are accumulated and distributed in oocytes is therefore a fundamental issue to be explored. A method that enables visualization of mRNA molecules with high resolution in a simple manner would be valuable for understanding how oocytes accumulate and regulate the dormant mRNAs. We have developed a highly sensitive whole-mount in situ hybridization method using in vitro-synthesized RNA probes and the tyramide signal amplification (TSA) system optimized for mouse oocytes and embryos. By using this method, Pou5f1/Oct4, Emi2, and cyclin B1 mRNAs were detected in immature oocytes and 2-cell stage embryos. Confocal microscopy showed that these mRNAs formed granular structures in the oocyte cytoplasm. The structures of Pou5f1/Oct4 and cyclin B1 mRNAs persisted in 2-cell stage embryos. Pou5f1/Oct4 RNA granules exhibited a solid-like property in immature oocytes and became liquid-like droplets in 2-cell stage embryos. Double-staining of cyclin B1 mRNA with Emi2 or Pou5f1/Oct4 mRNA revealed that these mRNAs were distributed as different RNA granules without overlapping each other and that the size of cyclin B1 RNA granules tended to be larger than that of Emi2 RNA granules. The structures and distribution patterns of these mRNAs were further analyzed by N-SIM super-resolution microscopy. This analysis revealed that the large-sized RNA granules consist of many small-sized granules, suggesting the accumulation and regulation of dormant mRNAs as basal-sized RNA granules. The method established in this study can easily visualize the structure and distribution of mRNAs accumulated in mammalian oocytes and embryos with high sensitivity and super-resolution. This method is useful for investigating the cellular and molecular mechanisms of translational control of mRNAs by which maturation and early developmental processes are promoted. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
38. Function of nodal cilia in left-right determination: Mechanical regulation in initiation of symmetry breaking.
- Author
-
Katoh, Takanobu A.
- Subjects
- *
OPTICAL tweezers , *SENSE of direction , *DEVELOPMENTAL biology , *CILIA & ciliary motion , *SYMMETRY breaking - Abstract
Visceral organs in vertebrates are arranged with left-right asymmetry; for example, the heart is located on the left side of the body. Cilia at the node of mouse early embryos play an essential role in determining this left-right asymmetry. Using information from the anteroposterior axis, motile cilia at the central region of the node generate leftward nodal flow. Immotile cilia at the periphery of the node mechanically sense the direction of leftward nodal flow in a manner dependent on the polarized localization of Pkd2, which is localized on the dorsal side of cilia. Therefore, only left-side cilia are activated by leftward nodal flow. This activation results in frequent calcium transients in the cilia via the Pkd2 channel, which leads to the degradation of Dand5 mRNA only at the left-side crown-cells. This process is the mechanism of initial determination of the left-side-specific signal. In this review, we provide an overview of initial left-right symmetry breaking that occurs at the node, focusing mainly on a recent biophysical study that revealed the function of nodal immotile cilia using advanced microscopic techniques, such as optical tweezers and super-resolution microscopy. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
39. Photoswitching Reagent for Super‐Resolution Fluorescence Microscopy.
- Author
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Go, Ga‐eun, Jeong, Uidon, Park, Hyunbum, Go, Seokran, and Kim, Doory
- Subjects
- *
FLUORESCENCE microscopy , *MATERIALS science , *HIGH resolution imaging , *MOLECULAR structure , *MICROSCOPY , *LASER-induced fluorescence , *COLLISION induced dissociation - Abstract
Single‐molecule localization microscopy (SMLM) has revolutionized optical microscopy by exceeding the diffraction limit and revealing previously unattainable nanoscale details of cellular structures and molecular dynamics. This super‐resolution imaging capability relies on fluorophore photoswitching, which is crucial for optimizing the imaging conditions and accurately determining the fluorophore positions. To understand the general on and off photoswitching mechanisms of single dye molecules, various photoswitching reagents were evaluated. Systematic measurement of the single‐molecule‐level fluorescence on and off rates (kon and koff) in the presence of various photoswitching reagents and theoretical calculation of the structure of the photoswitching reagent‐fluorophore pair indicated that the switch‐off mechanism is mainly determined by the nucleophilicity of the photoswitching reagent, and the switch‐on mechanism is a two‐photon‐induced dissociation process, which is related to the power of the illuminating laser and bond dissociation energy of this pair. This study contributes to a broader understanding of the molecular photoswitching mechanism in SMLM imaging and provides a basis for designing improved photoswitching reagents with potential applications extending to materials science and chemistry. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
40. DNA choreography: correlating mobility and organization of DNA across different resolutions from loops to chromosomes.
- Author
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Pabba, Maruthi K., Meyer, Janis, Celikay, Kerem, Schermelleh, Lothar, Rohr, Karl, and Cardoso, M. Cristina
- Subjects
- *
CHROMOSOMES , *CELL nuclei , *IMAGE registration , *CELL cycle , *ORGANIZATIONAL structure , *DNA replication - Abstract
The dynamics of DNA in the cell nucleus plays a role in cellular processes and fates but the interplay of DNA mobility with the hierarchical levels of DNA organization is still underexplored. Here, we made use of DNA replication to directly label genomic DNA in an unbiased genome-wide manner. This was followed by live-cell time-lapse microscopy of the labeled DNA combining imaging at different resolutions levels simultaneously and allowing one to trace DNA motion across organization levels within the same cells. Quantification of the labeled DNA segments at different microscopic resolution levels revealed sizes comparable to the ones reported for DNA loops using 3D super-resolution microscopy, topologically associated domains (TAD) using 3D widefield microscopy, and also entire chromosomes. By employing advanced chromatin tracking and image registration, we discovered that DNA exhibited higher mobility at the individual loop level compared to the TAD level and even less at the chromosome level. Additionally, our findings indicate that chromatin movement, regardless of the resolution, slowed down during the S phase of the cell cycle compared to the G1/G2 phases. Furthermore, we found that a fraction of DNA loops and TADs exhibited directed movement with the majority depicting constrained movement. Our data also indicated spatial mobility differences with DNA loops and TADs at the nuclear periphery and the nuclear interior exhibiting lower velocity and radius of gyration than the intermediate locations. On the basis of these insights, we propose that there is a link between DNA mobility and its organizational structure including spatial distribution, which impacts cellular processes. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
41. Mild Hyperthermia-Induced Thermogenesis in the Endoplasmic Reticulum Defines Stress Response Mechanisms.
- Author
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Dukic, Barbara, Ruppert, Zsófia, Tóth, Melinda E., Hunya, Ákos, Czibula, Ágnes, Bíró, Péter, Tiszlavicz, Ádám, Péter, Mária, Balogh, Gábor, Erdélyi, Miklós, Timinszky, Gyula, Vígh, László, Gombos, Imre, and Török, Zsolt
- Subjects
- *
UNFOLDED protein response , *FLUORESCENCE microscopy , *FLUORESCENT probes , *BODY temperature regulation , *HIGH temperatures , *ENDOPLASMIC reticulum , *FEVER - Abstract
Previous studies reported that a mild, non-protein-denaturing, fever-like temperature increase induced the unfolded protein response (UPR) in mammalian cells. Our dSTORM super-resolution microscopy experiments revealed that the master regulator of the UPR, the IRE1 (inositol-requiring enzyme 1) protein, is clustered as a result of UPR activation in a human osteosarcoma cell line (U2OS) upon mild heat stress. Using ER thermo yellow, a temperature-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we detected significant intracellular thermogenesis in mouse embryonic fibroblast (MEF) cells. Temperatures reached at least 8 °C higher than the external environment (40 °C), resulting in exceptionally high ER temperatures similar to those previously described for mitochondria. Mild heat-induced thermogenesis in the ER of MEF cells was likely due to the uncoupling of the Ca2+/ATPase (SERCA) pump. The high ER temperatures initiated a pronounced cytosolic heat-shock response in MEF cells, which was significantly lower in U2OS cells in which both the ER thermogenesis and SERCA pump uncoupling were absent. Our results suggest that depending on intrinsic cellular properties, mild hyperthermia-induced intracellular thermogenesis defines the cellular response mechanism and determines the outcome of hyperthermic stress. [ABSTRACT FROM AUTHOR]
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- 2024
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42. The Alphabet Soup of Microscopy: An Introduction to Advanced Imaging Techniques. Part I: Super-Resolution's STED, SIM, SMI, and SMLM.
- Author
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Worden, Austin N
- Subjects
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STIMULATED emission , *MICROSCOPY , *ACRONYMS , *TERMS & phrases , *LIGHTING - Abstract
As technology advances, the field of microscopy offers unique opportunities for scientific exploration. However, the complex terminology used to describe these cutting-edge techniques often appears as a jumble of letters, akin to alphabet soup. Among the plethora of acronyms, some of the most prevalent are associated with super-resolution microscopy, including Stimulated Emission Depletion Microscopy (STED), Structured Illumination Microscopy (SIM), Spatially Modulated Illumination (SMI), and Single-Molecule Localization Microscopy (SMLM). These techniques push past the limitations of conventional light microscopy, achieving resolutions down to the tens of nanometers. This article aims to decode these common super-resolution techniques, fostering a deeper understanding of microscopy and igniting interest in their application. [ABSTRACT FROM AUTHOR]
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- 2024
- Full Text
- View/download PDF
43. Nanoscale insights into hematology: super-resolved imaging on blood cell structure, function, and pathology.
- Author
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Liu, Jinghan, Tan, Yuping Yolanda, Zheng, Wen, Wang, Yao, Ju, Lining Arnold, and Su, Qian Peter
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BLOOD cells ,CELL anatomy ,LEUCOCYTES ,ERYTHROCYTES ,HEMATOLOGY - Abstract
Fluorescence nanoscopy, also known as super-resolution microscopy, has transcended the conventional resolution barriers and enabled visualization of biological samples at nanometric resolutions. A series of super-resolution techniques have been developed and applied to investigate the molecular distribution, organization, and interactions in blood cells, as well as the underlying mechanisms of blood-cell-associated diseases. In this review, we provide an overview of various fluorescence nanoscopy technologies, outlining their current development stage and the challenges they are facing in terms of functionality and practicality. We specifically explore how these innovations have propelled forward the analysis of thrombocytes (platelets), erythrocytes (red blood cells) and leukocytes (white blood cells), shedding light on the nanoscale arrangement of subcellular components and molecular interactions. We spotlight novel biomarkers uncovered by fluorescence nanoscopy for disease diagnosis, such as thrombocytopathies, malignancies, and infectious diseases. Furthermore, we discuss the technological hurdles and chart out prospective avenues for future research directions. This review aims to underscore the significant contributions of fluorescence nanoscopy to the field of blood cell analysis and disease diagnosis, poised to revolutionize our approach to exploring, understanding, and managing disease at the molecular level. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
44. Synapse-Specific Trapping of SNARE Machinery Proteins in the Anesthetized Drosophila Brain.
- Author
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Hines, Adam D., Kewin, Amber B., Van De Poll, Matthew N., Anggono, Victor, Bademosi, Adekunle T., and van Swinderen, Bruno
- Subjects
- *
SNARE proteins , *SYNAPSES , *DROSOPHILA , *LARGE-scale brain networks , *ION channels , *GENERAL anesthesia - Abstract
General anesthetics disrupt brain network dynamics through multiple pathways, in part through postsynaptic potentiation of inhibitory ion channels as well as presynaptic inhibition of neuroexocytosis. Common clinical general anesthetic drugs, such as propofol and isoflurane, have been shown to interact and interfere with core components of the exocytic release machinery to cause impaired neurotransmitter release. Recent studies however suggest that these drugs do not affect all synapse subtypes equally. We investigated the role of the presynaptic release machinery in multiple neurotransmitter systems under isoflurane general anesthesia in the adult female Drosophila brain using live-cell super–resolution microscopy and optogenetic readouts of exocytosis and neural excitability. We activated neurotransmitter-specific mushroom body output neurons and imaged presynaptic function under isoflurane anesthesia. We found that isoflurane impaired synaptic release and presynaptic protein dynamics in excitatory cholinergic synapses. In contrast, isoflurane had little to no effect on inhibitory GABAergic or glutamatergic synapses. These results present a distinct inhibitory mechanism for general anesthesia, whereby neuroexocytosis is selectively impaired at excitatory synapses, while inhibitory synapses remain functional. This suggests a presynaptic inhibitory mechanism that complements the other inhibitory effects of these drugs. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
45. Deep learning-based spectroscopic single-molecule localization microscopy.
- Author
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Gaire, Sunil Kumar, Daneshkhah, Ali, Flowerday, Ethan, Ruyi Gong, Frederick, Jane, and Backman, Vadim
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MACHINE learning , *MICROSCOPY , *ARTIFICIAL chromosomes , *DEEP learning , *HIGH resolution imaging - Abstract
Significance: Spectroscopic single-molecule localization microscopy (sSMLM) takes advantage of nanoscopy and spectroscopy, enabling sub-10 nm resolution as well as simultaneous multicolor imaging of multi-labeled samples. Reconstruction of raw sSMLM data using deep learning is a promising approach for visualizing the subcellular structures at the nanoscale. Aim: Develop a novel computational approach leveraging deep learning to reconstruct both label-free and fluorescence-labeled sSMLM imaging data. Approach: We developed a two-network-model based deep learning algorithm, termed DsSMLM, to reconstruct sSMLM data. The effectiveness of DsSMLM was assessed by conducting imaging experiments on diverse samples, including label-free single-stranded DNA (ssDNA) fiber, fluorescence-labeled histone markers on COS-7 and U2OS cells, and simultaneous multicolor imaging of synthetic DNA origami nanoruler. Results: For label-free imaging, a spatial resolution of 6.22 nm was achieved on ssDNA fiber; for fluorescence-labeled imaging, DsSMLM revealed the distribution of chromatin-rich and chromatin-poor regions defined by histone markers on the cell nucleus and also offered simultaneous multicolor imaging of nanoruler samples, distinguishing two dyes labeled in three emitting points with a separation distance of 40 nm. With DsSMLM, we observed enhanced spectral profiles with 8.8% higher localization detection for single-color imaging and up to 5.05% higher localization detection for simultaneous two-color imaging. Conclusions: We demonstrate the feasibility of deep learning-based reconstruction for sSMLM imaging applicable to label-free and fluorescence-labeled sSMLM imaging data. We anticipate our technique will be a valuable tool for high-quality super-resolution imaging for a deeper understanding of DNA molecules' photophysics and will facilitate the investigation of multiple nanoscopic cellular structures and their interactions. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
46. PvdL Orchestrates the Assembly of the Nonribosomal Peptide Synthetases Involved in Pyoverdine Biosynthesis in Pseudomonas aeruginosa.
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Manko, Hanna, Steffan, Tania, Gasser, Véronique, Mély, Yves, Schalk, Isabelle, and Godet, Julien
- Subjects
- *
NONRIBOSOMAL peptide synthetases , *PSEUDOMONAS aeruginosa , *BIOSYNTHESIS , *SPATIAL arrangement , *PEPTIDES , *RHAMNOLIPIDS - Abstract
The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
47. Mitochondrial Dynamics at Different Levels: From Cristae Dynamics to Interorganellar Cross Talk.
- Author
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Kondadi, Arun Kumar and Reichert, Andreas S.
- Abstract
Mitochondria are essential organelles performing important cellular functions ranging from bioenergetics and metabolism to apoptotic signaling and immune responses. They are highly dynamic at different structural and functional levels. Mitochondria have been shown to constantly undergo fusion and fission processes and dynamically interact with other organelles such as the endoplasmic reticulum, peroxisomes, and lipid droplets. The field of mitochondrial dynamics has evolved hand in hand with technological achievements including advanced fluorescence super-resolution nanoscopy. Dynamic remodeling of the cristae membrane within individual mitochondria, discovered very recently, opens up a further exciting layer of mitochondrial dynamics. In this review, we discuss mitochondrial dynamics at the following levels: (a) within an individual mitochondrion, (b) among mitochondria, and (c) between mitochondria and other organelles. Although the three tiers of mitochondrial dynamics have in the past been classified in a hierarchical manner, they are functionally connected and must act in a coordinated manner to maintain cellular functions and thus prevent various human diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
48. Next-Generation Genetically Encoded Fluorescent Biosensors Illuminate Cell Signaling and Metabolism.
- Author
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Frei, Michelle S., Mehta, Sohum, and Zhang, Jin
- Abstract
Genetically encoded fluorescent biosensors have revolutionized the study of cell signaling and metabolism, as they allow for live-cell measurements with high spatiotemporal resolution. This success has spurred the development of tailor-made biosensors that enable the study of dynamic phenomena on different timescales and length scales. In this review, we discuss different approaches to enhancing and developing new biosensors. We summarize the technologies used to gain structural insights into biosensor design and comment on useful screening technologies. Furthermore, we give an overview of different applications where biosensors have led to key advances over recent years. Finally, we give our perspective on where future work is bound to make a large impact. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
49. From Cell Populations to Molecular Complexes: Multiplexed Multimodal Microscopy to Explore p53-53BP1 Molecular Interaction.
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Pelicci, Simone, Furia, Laura, Pelicci, Pier Giuseppe, and Faretta, Mario
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CELL populations , *MOLECULAR interactions , *P53 antioncogene , *MICROSCOPY , *FLUORESCENCE microscopy , *DNA repair - Abstract
Surpassing the diffraction barrier revolutionized modern fluorescence microscopy. However, intrinsic limitations in statistical sampling, the number of simultaneously analyzable channels, hardware requirements, and sample preparation procedures still represent an obstacle to its widespread diffusion in applicative biomedical research. Here, we present a novel pipeline based on automated multimodal microscopy and super-resolution techniques employing easily available materials and instruments and completed with open-source image-analysis software developed in our laboratory. The results show the potential impact of single-molecule localization microscopy (SMLM) on the study of biomolecules' interactions and the localization of macromolecular complexes. As a demonstrative application, we explored the basis of p53-53BP1 interactions, showing the formation of a putative macromolecular complex between the two proteins and the basal transcription machinery in situ, thus providing visual proof of the direct role of 53BP1 in sustaining p53 transactivation function. Moreover, high-content SMLM provided evidence of the presence of a 53BP1 complex on the cell cytoskeleton and in the mitochondrial space, thus suggesting the existence of novel alternative 53BP1 functions to support p53 activity. [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
50. OneFlowTraX: a user-friendly software for super-resolution analysis of single-molecule dynamics and nanoscale organization.
- Author
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Rohr, Leander, Ehinger, Alexandra, Rausch, Luiselotte, Burmeister, Nina Glöckner, Meixner, Alfred J., Gronnier, Julien, Harter, Klaus, Kemmerling, Birgit, and Oven-Krockhaus, Sven zur
- Subjects
GRAPHICAL user interfaces ,SINGLE molecules ,MACROMOLECULAR dynamics ,BLOOD proteins ,CLUSTER analysis (Statistics) - Abstract
Super-resolution microscopy (SRM) approaches revolutionize cell biology by providing insights into the nanoscale organization and dynamics of macromolecular assemblies and single molecules in living cells. A major hurdle limiting SRM democratization is post-acquisition data analysis which is often complex and time-consuming. Here, we present OneFlowTraX, a user-friendly and open-source software dedicated to the analysis of single-molecule localization microscopy (SMLM) approaches such as single-particle tracking photoactivated localization microscopy (sptPALM). Through an intuitive graphical user interface, OneFlowTraX provides an automated all-in-one solution for single-molecule localization, tracking, as well as mobility and clustering analyses. OneFlowTraX allows the extraction of diffusion and clustering parameters of millions of molecules in a few minutes. Finally, OneFlowTraX greatly simplifies data management following the FAIR (Findable, Accessible, Interoperable, Reusable) principles. We provide a detailed step-bystep manual and guidelines to assess the quality of single-molecule analyses. Applying different fluorophores including mEos3.2, PA-GFP, and PATagRFP, we exemplarily used OneFlowTraX to analyze the dynamics of plant plasma membrane-localized proteins including an aquaporin, the brassinosteroid receptor Brassinosteroid Insensitive 1 (BRI1) and the Receptor-Like Protein 44 (RLP44). [ABSTRACT FROM AUTHOR]
- Published
- 2024
- Full Text
- View/download PDF
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