Nature Protocols最新文献

{"title":"Synthesis of diiron subcluster [Fe₂[(μ-SCH₂)₂NH](CN)₂(CO)₄]^2- for artificial maturation of [FeFe]-hydrogenases.","authors":"Xin Yu, Fang Wei, Thomas B Rauchfuss","doi":"10.1038/s41596-026-01418-x","DOIUrl":"https://doi.org/10.1038/s41596-026-01418-x","url":null,"abstract":"<p><p>The [FeFe]-hydrogenases are widely distributed enzymes that efficiently make and use hydrogen (H₂). They have attracted intense interest as potential biocatalysts as well as models for biomimetic catalysis. Substrate turnover occurs at an Fe-based active site featuring an azadithiolate cofactor. The biosynthesis of the active site is remarkably elaborate. The production of these enzymes is greatly simplified by replacing the biosynthesis of this active site with reconstitution of the enzyme with chemically synthesized [Fe₂[(μ-SCH₂)₂NH](CN)₂(CO)₄]^2- ([1]^2-), the subject of this protocol. Two approaches to [1]^2- are described: a 'condensation route' and an 'Fmoc route'. Each of the two routes requires ~20 h of hands-on time. The condensation route entails the reaction of Fe₂(μ-SH)₂(CO)₆ and N₄(CH₂)₆ followed by cyanation to give [1]^2-. The most demanding step in this route is the preparation of Fe₂(μ-S₂)(CO)₆. The second route focuses on fluorenylmethoxycarbonyl (Fmoc)-protected derivatives of azadithiolate cofactor. This route proceeds via Fe₂[(μ-SCH₂)₂NFmoc](CO)₆. Both the condensation and Fmoc routes allow isotopic labeling of the active site, but ⁵⁷Fe labeling is especially simple using the Fmoc route.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148760819","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ONE microscopy.","authors":"Ali H Shaib, Mohamad Mahdi Alawieh, Silvio O Rizzoli","doi":"10.1038/s41596-026-01399-x","DOIUrl":"https://doi.org/10.1038/s41596-026-01399-x","url":null,"abstract":"<p><p>The introduction of expansion microscopy (ExM), a decade ago, marked a shift in super-resolution imaging, by physically separating fluorophores to bypass the diffraction limit. Numerous ExM developments have extended the method's reach since, yet molecular-scale resolution remained inaccessible. We recently developed one-step nanoscale ExM, which combines ExM with fluctuation-based super-resolution analysis to enable the direct visualization of individual protein shapes, using conventional fluorescence microscopes, a capability that was previously limited to cryo-electron microscopy and averaging-based techniques. Here we provide detailed procedures for gel embedding, labeling, expansion, image acquisition and data analysis. We also introduce a stable, user-friendly software package for efficient fluctuation analysis. Although one-step nanoscale ExM is broadly applicable to a range of samples, including purified proteins, cells and tissues, its most distinctive contribution lies in making single-protein shape analysis accessible and reproducible. Overall, we provide a practical framework for protein imaging on conventional equipment.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148713199","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Direct printing of metasurfaces using formulated optical materials.","authors":"Hyunjung Kang, Eunji Lee, Yujin Park, Hongyoon Kim, Dong Kyo Oh, Jaekyung Kim, Junsuk Rho","doi":"10.1038/s41596-026-01407-0","DOIUrl":"https://doi.org/10.1038/s41596-026-01407-0","url":null,"abstract":"<p><p>Optical metasurfaces represent a promising technology for compact, lightweight and multifunctional optical devices; yet, their practical implementation remains challenging because of the need for high-refractive-index (high-index) materials, complex fabrication processes and limited substrate compatibility. Conventional top-down approaches relying on deposition, electron-beam lithography and etching are expensive and typically restricted to rigid substrates, hindering scalability and integration with flexible platforms. Nanoimprint lithography is a cost-effective, high-throughput alternative for metasurface fabrication; nevertheless, the low refractive index of conventional imprint resins fundamentally limits device performance. Here, we describe the fabrication of optical metasurfaces using a printable high-index composite material known as nanoparticle-embedded resin (nanoPER). By embedding high-index nanoparticles into a curable resin matrix, nanoPER achieves an effective refractive index above 1.8 at the target wavelength and enables single-step replication of functional nanostructures on a wide range of substrates, including flexible and curved surfaces. The procedure focuses on TiO₂ nanoPER and provides a reproducible and comprehensive guide covering resin formulation, nanoimprint lithography process parameters and optical characterization. By emphasizing scalability and versatility, this protocol supports the translation of metasurface research into real-world applications such as light detection and ranging, compact imaging and integrated photonics. The entire process can be completed within 1-2 days and can be performed by researchers with experience in nanofabrication and optical measurements.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148713267","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Standardized assays for evaluating superoxide dismutase-like and catalase-like activities of nanozymes.","authors":"Ruofei Zhang, Hanqing Zhao, Sijie Zhang, Xiyun Yan, Kelong Fan","doi":"10.1038/s41596-026-01417-y","DOIUrl":"https://doi.org/10.1038/s41596-026-01417-y","url":null,"abstract":"<p><p>Antioxidant nanozymes are nanomaterials with superoxide dismutase-like or catalase-like activities. They have emerged as promising therapeutics for oxidative stress-related disorders. However, the lack of standardized quantitative assays for activity characterization has limited cross-study reproducibility and direct comparison of reported performance. Here this protocol provides step-by-step procedures for the quantitative characterization of the superoxide dismutase-like and catalase-like activities of nanozymes. Superoxide dismutase-like activity is quantified via a self-prepared xanthine/xanthine oxidase/water-soluble tetrazolium salt-1 system, in which nanozymes catalyze the dismutation of superoxide anions (O₂^•-), thereby suppressing formazan formation. By tuning O₂^•- generation through controlled xanthine oxidase concentrations, the protocol enables the determination of Michaelis-Menten kinetics for the short-lived O₂^•-. Catalase-like activity is quantified by monitoring hydrogen peroxide (H₂O₂) decomposition at 240 nm via ultraviolet-visible spectrophotometry, which yields specific activity and kinetic constants, whereas a complementary 3,3',5,5'-tetramethylbenzidine oxidation assay excludes interference from peroxidase-like reactions. Representative nanozymes spanning carbon-based, metal oxide, noble metal and single-atom systems are used as case studies to demonstrate the robustness and versatility of this protocol. Functional validation in cellular models using flow cytometry further demonstrates the practical applicability of reactive oxygen species scavenging. This protocol enables reliable and reproducible evaluation of antioxidant nanozymes, facilitating direct comparison across materials, elucidation of structure-activity relationships and rational optimization for biomedical applications. The complete procedure can be performed by researchers with standard training in biochemistry and flow cytometric analysis within 10-12 h.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148707122","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Drug self-administration in head-fixed mice.","authors":"Elizabeth M Doncheck, Rachel E Clarke, Adam G Gordon, Josh Boquiren, Logan M Manusky, Jade Baek, Jacqueline E Paniccia, Isabella E Dinu, Eric B Dereschewitz, Roger I Grant, Garret D Stuber, James M Otis","doi":"10.1038/s41596-026-01406-1","DOIUrl":"10.1038/s41596-026-01406-1","url":null,"abstract":"<p><p>Drug self-administration has the greatest construct and predictive validity of the preclinical models for substance use disorder, providing landmark insights into the neurobiology of addiction. However, these experiments have traditionally been performed in freely moving animals, which can prohibit the incorporation of emerging neurotechnologies that require or are greatly facilitated by head restraint. Recently, we developed and validated a head-restrained approach in mice for intravenous and oral self-administration of drug and nondrug rewards. Here we present a step-by-step protocol for these experiments, including custom equipment construction, open-source software implementation and adaptation, catheter implantation, and unique considerations for conducting head-fixed self-administration experiments. To ensure that each component can be implemented by a wide range of audiences, detailed descriptions are provided so that this Protocol may serve as a standalone guide for researchers with varying levels of experience.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148707087","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A general approach for the synthesis of nanoisland catalysts.","authors":"Xu Li, Di Wu, Jingyi Zhang, Yuan Ji, Junyi He, Chuan Xia","doi":"10.1038/s41596-026-01431-0","DOIUrl":"https://doi.org/10.1038/s41596-026-01431-0","url":null,"abstract":"<p><p>The stabilization of metal catalysts remains a major challenge in heterogeneous catalysis, particularly for single atoms and clusters that operate under demanding industrial conditions where sintering and agglomeration often lead to substantial deactivation. Here, to address the issue, this protocol presents a general method for synthesizing nanoisland catalysts in which nanoscale oxide islands are isolated on high-surface-area substrates to confine and stabilize metal species. The method uses strong electrostatic adsorption to deposit oxides such as CeO_x, LaO_x and InO_x onto substrates such as SiO₂ and Al₂O₃, resulting in small-sized, high-density and uniformly distributed nanoislands. Then, transition-metal precursors, including those of Pt, Pd and Ru, are introduced in a controlled manner such that single atoms or clusters are positioned preferentially on the nanoislands. These nanoislands have stronger interactions with metals than the substrates do, confining metal species within well-defined regions and preventing their migration and agglomeration. This confinement effect is crucial for maintaining the dispersion and activity of metal catalysts, especially at elevated temperatures. Compared with conventional impregnation or deposition-precipitation methods, this approach achieves more precise spatial control over metal deposition and creates strong confinement environments that effectively suppress sintering. This protocol enables the preparation of thermally stable small-sized metal catalysts suitable for fuel processing, exhaust treatment, chemical manufacturing and other uses. The complete workflow, which includes oxide deposition, metal loading and material characterization, typically requires about 4 d, and catalytic testing requires an additional 5-20 h depending on the system. The protocol can be implemented in laboratories equipped with standard wet-chemistry facilities and with prior experience in synthesizing inorganic nanomaterials.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148679557","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reply to: Overestimations in the cyclization efficiency of small, restriction enzyme-digested DNA fragments.","authors":"Roman Teo Oliynyk, George M Church","doi":"10.1038/s41596-026-01424-z","DOIUrl":"https://doi.org/10.1038/s41596-026-01424-z","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148630895","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Overestimations in the cyclization efficiency of small, restriction enzyme-digested DNA fragments.","authors":"Fenfei Leng","doi":"10.1038/s41596-026-01423-0","DOIUrl":"10.1038/s41596-026-01423-0","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148630892","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Core2Edge: a human glioblastoma organoid-brain slice model capturing infiltration and transcriptional heterogeneity from core to single-cell dispersion.","authors":"Ahmad Melhem, Barbara E F Pregler, Juan Eduardo Rodriguez-Gatica, Lea L Friker, Jake Thomas, Marieta I Toma, Mike-Andrew Westhoff, Vidhya M Ravi, Valeri Borger, Dieter Henrik Heiland, Julian P Layer, Andreas Schlitzer, Torsten Pietsch, Michael Hölzel, Andreas Waha, Hartmut Vatter, Martin K Schwarz, Ulrich Herrlinger, Ulrich Kubitscheck, Anna-Laura Potthoff, Matthias Schneider","doi":"10.1038/s41596-026-01412-3","DOIUrl":"https://doi.org/10.1038/s41596-026-01412-3","url":null,"abstract":"<p><p>Glioblastomas function as intricate cellular networks that extend into the surrounding brain tissue, facilitating long-distance communication. This malignant connectivity spans from the tumor core to remote infiltration zones, in support of the concept of glioblastoma as a whole-brain disease. With growing ethical concerns in biomedical research and the inherent limitations of animal models in recapitulating human glioblastoma biology, there is an increasing demand for human ex vivo platforms capable of capturing the full infiltration spectrum from the tumor core to single-cell dispersion. Here we present a 3D, fully human ex vivo glioblastoma model (Core2Edge) that replicates this extensive infiltration range while preserving the intratumoral heterogeneity of the original tumor. This model involves implanting fluorescently labeled human glioblastoma organoids (GBOs) into organotypic human brain slices, maintaining the genetic integrity and cytoarchitecture of both brain and tumor. By combining tissue expansion with light-sheet fluorescence microscopy, we achieve high-resolution, 3D imaging of the entire GBO-brain slice model. This approach allows the study of initial infiltration steps, in-depth analysis of the invasive front, and exploration of cell-cell interactions between tumor cells and the tumor microenvironment, and offers a platform for drug screening and testing, reducing the need for animal models. Once GBOs are prepared, the protocol takes ~7-12 d. Key steps include brain slice preparation (~4-6 h, depending on quantity), 1 d for initial culture before GBO staining and transplantation, a variable culture period (≤10 d), and fixation (~8 h). The protocol requires experience with human brain slice and organoid culture.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148605436","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Three-dimensional tracking of dynamic structures in living cells using single-plane imaging with focus feedback.","authors":"Simona V Antonova, Wim Pomp, Joseph V W Meeussen, Tineke L Lenstra","doi":"10.1038/s41596-026-01419-w","DOIUrl":"https://doi.org/10.1038/s41596-026-01419-w","url":null,"abstract":"<p><p>Tracking dynamic subcellular processes in live cells presents a major challenge in biological research, as it often requires high precision in three-dimensional imaging. Here this protocol introduces a focus-feedback microscopy algorithm that enables the accurate tracking of structures within a single optical plane over time, thereby overcoming the limitations of traditional z-stack imaging and reducing imaging intervals and light exposure. This results in prolonged imaging sessions with minimal phototoxicity, making it ideal for studying dynamic molecular processes in living cells. The protocol provides step-by-step guidance for integrating the focus-feedback algorithm within Zeiss Zen or custom microscope software, incorporating cylindrical lenses for z-position detection, performing bead-based calibration and analyzing time-lapse data using both standard and custom tools. It is designed to be accessible to researchers with varying levels of experience and, depending on research question, can be completed within a single day. Focus-feedback microscopy has been successfully applied to track single gene loci within the nucleus and has potential extensions to cytoplasmic structures such as organelles or vesicles. Its compatibility with multichannel and single-molecule imaging makes it a powerful tool for studying dynamic cellular processes with precise spatial and temporal resolution.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-07-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148605418","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}