Nature Protocols最新文献

{"title":"Quantitative imaging of dynamic processes in intestinal organoids.","authors":"Willem Kasper Spoelstra, Xuan Zheng, Rutger N U Kok, Guizela Huelsz-Prince, Max Betjes, Pascal Ender, Yvonne Goos, Kim A Kuentzelmann, Heidi E Klumpe, Daniel Krueger, Daisong Wang, Hans Clevers, Sander J Tans, Jeroen S van Zon","doi":"10.1038/s41596-026-01432-z","DOIUrl":"https://doi.org/10.1038/s41596-026-01432-z","url":null,"abstract":"<p><p>Organoids are powerful models for studying tissue dynamics across multiple cellular generations and offer key insights into organ homeostasis, cellular differentiation and disease. Live imaging of organoids is an essential tool for understanding dynamic processes on the cell level, while still in the tissue context. It allows for simultaneous quantification of gene expression and reconstruction of lineage trees, while tracking cells over space and time. Here, we present protocols for long-term imaging and quantitative analysis of intestinal organoids. Our workflow consists of sample preparation, experimental manipulations (such as drug treatments, laser ablations and fluorescence recovery after photobleaching), long-term live imaging (120 h), in situ fixation and permeabilization, multiplexed antibody staining and single-cell tracking with lineage reconstruction. The workflow is broadly applicable to studying dynamic cellular processes in both 2D and 3D organoids, at timescales from minutes to days. We outline strategies for optimizing organoid health, imaging conditions and experimental interventions to ensure efficient downstream analysis. In addition, we describe how this workflow enables real-time quantification of gene expression using fluorescent reporters and inferring cell type changes by combining multiplexed antibody staining with cell-tracking data. The skills required for following these protocols are organoid culturing and standard live-cell confocal microscopy, and the full experimental protocol takes ~2 weeks. Together, these protocols provide a comprehensive approach for studying cellular behavior, lineage trees, expression dynamics and cell-cell interactions in organoids.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148976761","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":"Author Correction: Tutorial: biomembranes in hybrid living bioelectronics.","authors":"Alice Hattar, Jawaher Alhammadi, Jeremy Treiber, Darius Hoven, Zhongmou Chao, Andreas Offenhaeusser, Susan Daniel, Roisin Owens, Alberto Salleo, Francesca Santoro, Anna-Maria Pappa","doi":"10.1038/s41596-026-01463-6","DOIUrl":"10.1038/s41596-026-01463-6","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148970279","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":"Generation of eight-cell embryo-like cells from human pluripotent stem cells.","authors":"Md Abdul Mazid, Yunpan Li, Mindie Zhao, Lixin Fu, Hao Liu, Yu Jiang, Wenqi Jia, Junjian Lai, Jinxiu Li, Haiwei Li, Babangida Jabir Saeed, Amash Manzoor, Zhiwei Luo, Yiwei Lai, Liang Wu, Yi Zou, Chaiyaboot Ariyachet, Carl Ward, Chuanyu Liu, Longqi Liu, Miguel A Esteban, Wenjuan Li","doi":"10.1038/s41596-026-01414-1","DOIUrl":"https://doi.org/10.1038/s41596-026-01414-1","url":null,"abstract":"<p><p>Mammalian embryonic development is a highly orchestrated process initiated by the fusion of the oocyte with sperm to generate the zygote. In humans, the zygote remains transcriptionally quiescent until the major wave of zygotic genome activation (ZGA) occurs around the eight-cell (8C) stage (day 3 after fertilization). These cells and the derived morula cells are totipotent: they have the capacity to form a whole individual. Our understanding of human totipotency is very limited because of ethical concerns using embryos and the scarcity of material available for research. Recently, we established a controllable transgene-free methodology to generate totipotent eight-cell embryo-like cells (8CLCs) from human pluripotent stem cells (PSCs) in vitro. These 8CLCs are produced using a novel medium, formulated by us, containing specific chemical compounds and cytokines. Here, we provide a detailed protocol for inducing, isolating and characterizing 8CLCs generated with this medium. The induction process can be done either in a stepwise manner (primed-naive-8CLC) that requires only 5 d starting from naive PSCs or directly from primed PSCs, which takes ~7 d. The resulting 8CLCs exhibit transcriptional and epigenetic features resembling those of human 8C embryo cells. On the basis of our experience, we expect that an individual with ~1 year of experience working with human PSC culture would be able to carry out this protocol. Our approach provides a valuable model for studying human early embryogenesis, particularly the molecular events surrounding ZGA.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148955742","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":"Probing and controlling Cu catalyst reconstruction during CO₂ electroreduction.","authors":"Libing Zhang, Chaofeng Zheng, Liang Xu, Jiaqi Feng, Shunhan Jia, Limin Wu, Xinning Song, Meng-Di Zhang, Ruhan Wang, Xiaoyu Zhang, Zhijuan Zhao, Xiaofu Sun, Buxing Han","doi":"10.1038/s41596-026-01430-1","DOIUrl":"https://doi.org/10.1038/s41596-026-01430-1","url":null,"abstract":"<p><p>The dynamic structural reconstruction of Cu-based catalysts profoundly influences activity, selectivity and stability during electrochemical CO₂ reduction (CO₂RR). However, it lacks standardized methodologies for probing and actively managing these transformations. Here we present a modular, reproducible Protocol that integrates electrochemical control, in situ/operando spectroscopy and mechanistic analysis to systematically investigate and regulate Cu catalyst reconstruction under CO₂RR conditions. This Protocol establishes a comprehensive 'reconstruction-understanding-intervention' workflow comprising (1) the identification and taxonomy of reconstruction phenomena, (2) quantification of electrochemical and environmental factors governing reconstruction, (3) correlation of structural dynamics with catalytic behavior through complementary in situ/operando Raman, infrared, X-ray absorption spectroscopy and quasi-in situ X-ray photoelectron spectroscopy measurements, and (4) development of rational reconstruction-control strategies that steer catalysts toward desired active states. A systematic approach is delineated to correlate reconstruction dynamics with catalytic performance and actively direct the process toward desired active states via three primary strategies: catalyst structure modulation, electrochemical operation regulation and reaction microenvironment management. The methodology is validated using representative Cu-based systems, including commercial Cu, oxide-derived Cu and bimetallic Cu-X catalysts, demonstrating enhanced performance and stability of CO₂RR. This workflow also provides an adaptable framework for investigating dynamic surface evolution in other electrocatalytic reactions. This Protocol provides useful framework for the rational development of adaptive, stable and selective electrocatalysts.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148955767","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":"netneurotools: a trainee-oriented approach to network neuroscience.","authors":"Zhen-Qi Liu, Vincent Bazinet, Justine Y Hansen, Filip Milisav, Andrea I Luppi, Eric G Ceballos, Asa Farahani, Laura E Suarez, Golia Shafiei, Ross D Markello, Bratislav Misic","doi":"10.1038/s41596-026-01446-7","DOIUrl":"https://doi.org/10.1038/s41596-026-01446-7","url":null,"abstract":"<p><p>Brain imaging is an increasingly interdisciplinary field, encompassing multiple data types and multiple analytic traditions. Projects typically involve many moving parts, such as building customized preprocessing pipelines, transforming between data formats, preparing datasets for analysis and, ultimately, displaying results. The field is conventionally built on highly specialized software packages that solve these individual challenges well but are not necessarily designed to be interoperable. Trainees new to the field are therefore often left to come up with isolated heuristics and workarounds to complete a project. Here we present a set of tools to navigate the increasingly complex informatics ecosystem of brain imaging. netneurotools is a Python toolkit that has been continuously developed and maintained by this laboratory's trainees. The philosophy of the toolkit is that it should be the Swiss army knife of the laboratory: functions and routines that we often use but that are not part of any established pipeline or package. Since its inception, the toolkit has been open and welcomes contributions from neuroscientists across the globe. netneurotools presents a necessary counterweight to out-of-the-box software packages and highlights the importance of smaller, ad hoc functions for implementing projects. By opening a window into the inner workings of a laboratory, netneurotools also presents an opportunity to begin a new type of discourse among groups and establish tangible links within the community.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148920331","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":"Dopamine polymerization-mediated surface functionalization of living cells for advanced therapeutic applications.","authors":"Lu Wang, Jinyao Liu","doi":"10.1038/s41596-026-01422-1","DOIUrl":"https://doi.org/10.1038/s41596-026-01422-1","url":null,"abstract":"<p><p>The engineering of living cells represents a promising biomedical frontier that enables the design of cells with tailored functionalities for advanced therapeutic applications. Genetic manipulation serves as a primary approach in cell engineering, yet it faces inherent limitations, including the complexity of multigene editing and poor cross-species applicability, which restrict the development of cells with sophisticated functionalities. Therefore, flexible and versatile engineering strategies capable of functionalizing living cells to address diverse therapeutic requirements are highly desirable. Given its pivotal role in mediating cellular interactions, the cell surface is an attractive target for directing cell engineering. The diverse functional groups present in surface biomolecules offer abundant chemical modification sites, making them highly amenable to functionalization. Leveraging this inherent chemical accessibility, we have recently developed a flexible and versatile platform for surface functionalization of living cells through in situ dopamine polymerization that allows us to design personalized living cells with customizable functions by tuning the surface components. Here we provide a detailed protocol describing two distinct methods for bacterial functionalization. The first method uses dopamine polymerization-mediated mono-functionalization to construct mucus-penetrating bacteria that can reinforce intestinal mucosal barrier to prevent colitis. The second method uses dopamine polymerization-mediated dual-functionalization to generate synergy-immunoactivation bacteria that can simultaneously induce anticancer and antiviral immunity to treat cancer and prevent infection. Excluding bacterial culture, preparation of mucus-penetrating bacteria and synergy-immunoactivation bacteria takes ~3 h and 1 h, respectively. We anticipate that this protocol can offer valuable guidance for the engineering of living cells with designable and tailorable functionalities for innovative cell-based therapy.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148891960","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":"VINE-seq and MultiVINE-seq for single-nucleus and multiome profiling of the brain vasculature.","authors":"Jane Oberhauser, Bella Ding, Madigan M Reid, William H Xie, Andrew C Yang","doi":"10.1038/s41596-026-01434-x","DOIUrl":"https://doi.org/10.1038/s41596-026-01434-x","url":null,"abstract":"<p><p>The human cerebrovasculature is a critical yet historically understudied component of neurological health. Dysfunction of the diverse endothelial, mural, and perivascular cells that comprise cerebral vessels is central to diseases ranging from stroke to Alzheimer's disease. However, characterizing these cell populations at a molecular level has proven exceptionally challenging. Encased within a robust basement membrane, vascular cells resist standard dissociation methods, leading to their systematic depletion and underrepresentation in existing single-nucleus genomic atlases. This has created a major blind spot in neuroscience. To overcome this barrier, we developed vessel isolation and nucleus extraction for sequencing (VINE-seq) and its advanced iteration, MultiVINE-seq. The protocol provides a robust, reproducible workflow for the enrichment and high-resolution profiling of vascular, perivascular, and immune cells from fresh or frozen human and mouse brain tissue. First, intact vessels (predominantly capillaries and small arterioles/venules, 100 µm in diameter) are isolated from homogenized brain tissue via dextran-based density-gradient centrifugation, separating the vascular pellet from myelin and the parenchymal fraction. Second, the collected vessels are rigorously washed over a cell strainer to remove trapped contaminants. A critical innovation lies in the third stage: the optimized extraction of nuclei from purified vessels using enzymatic digestion. After extraction, the protocol uses fluorescence-activated cell sorting (FACS) to ensure collection of high-purity nuclei suitable for widely used droplet-based sequencing platforms (e.g., 10x Genomics single cell 3' or multiome). This protocol requires 4-5 h to complete and can be carried out by researchers with single-cell and flow cytometry training.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148888055","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":"Salt-assisted synthesis of high phase-purity metastable 1T'-phase group VIB transition metal dichalcogenides.","authors":"Wei Zhai, Zhenyu Shi, Rui Tao, Zijian Li, Dan Dong, Jiabei Tang, Wenbin Wang, Lixin Wang, Hua Yang, Li Zhai, Xinyang Ruan, Yuxuan Wu, Jie Wang, Yao Yao, Chen Zhao, Qiyuan He, Zhuangchai Lai, Hua Zhang","doi":"10.1038/s41596-026-01429-8","DOIUrl":"https://doi.org/10.1038/s41596-026-01429-8","url":null,"abstract":"<p><p>With the increasing progress in the emerging field of phase engineering of nanomaterials, transition metal dichalcogenides (TMDs) with metastable phases have attracted extensive research interest owing to their unique physicochemical properties and promising potential for various applications. Unlike their semiconducting counterparts, metastable 1T'-phase group VIB TMDs exhibit distinctive metallic behavior, making them highly promising for clean energy electrocatalysis (for example, hydrogen evolution), high-performance electronics and superconducting devices. However, conventional synthetic strategies usually suffer from low yields, poor phase purity, small crystal size and harsh experimental conditions, hindering their fundamental research and practical applications. Here we describe a salt-assisted method for the controlled preparation of various metastable 1T'-phase group VIB TMDs with high crystalline quality and high phase purity. In this context, 'salt-assisted' refers to the co-annealing of commercially available 2H-phase TMDs, chalcogen powders (for example, S or Se) and alkali metal salts (for example, K₂C₂O₄·H₂O or K₂CO₃). When heated in a tube furnace under an H₂/Ar atmosphere, these alkali metal salts trigger a phase transformation of TMDs from the thermodynamically stable 2H phase to the metastable 1T' phase. This Protocol describes the details of experimental procedures to prepare a series of 1T'-TMDs, that is, MoS₂, MoSe₂, WS₂, WSe₂, MoS_2xSe_2(1-x) and WS_2xSe_2(1-x). It takes ~37 h 20 min and yields robust 1T'-TMD crystals with their size of up to hundreds of micrometers at a gram scale (up to ~2 g per batch), overcoming the longstanding synthetic bottlenecks and paving the way for advanced fundamental studies and the exploration of practical applications.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148874812","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":"Shear-flow-induced assembly of 2D nanosheets for the fabrication of composite films with high tensile strength.","authors":"Chaojun Zhang, Zhewei Yan, Jing Li, Mingjie Liu","doi":"10.1038/s41596-026-01442-x","DOIUrl":"https://doi.org/10.1038/s41596-026-01442-x","url":null,"abstract":"<p><p>High-performance bioinspired composite materials require the precise alignment of synthetic nanosheets to mimic the mechanical properties of highly ordered laminated microstructures found in nature. Here, we detail a nanosheet superspreading alignment strategy for fabricating nanocomposite films that uses shear-flow forces at the interface between two immiscible phases to induce long-range, high-order alignment of the 2D nanosheets that enhances the films' mechanical properties. In situ interface crystallization or cross-linking follows the alignment and effectively locks the oriented configuration (the resulting orientation order parameter is >0.85). Subsequent solvent dewetting enables continuous film formation over large areas while maintaining well-defined microstructural integrity. This process overcomes the misorientation and aggregation typical of conventional alignment methods and can be scaled using a multi-nozzle extrusion setup compatible with commercial heating and film-collection components. The step-by-step procedures cover the nanosheet precursor preparation, the continuous-film fabrication and their microstructural characterization and require ≤23 days to complete. The procedures are applicable to a broad range of nanosheet materials, including graphene oxide, MXenes, transition-metal dichalcogenides and layered clays, and the resulting composite films exhibit enhanced mechanical strength, toughness and multifunctionality. Nanocomposites based on graphene oxide and clay nanosheets exhibit a tensile strength of up to 1,215 ± 80 MPa (mean ± s.d) and a Young's modulus of 198.8 ± 6.5 GPa, while clay-based nanocomposite films reach a toughness of 36.7 ± 3.0 MJ m^-3. Superspreading alignment is a versatile and robust approach for the scalable fabrication of high-performance composites for materials science.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148851041","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":"Preparation of polycaprolactone-based 2D platelets via living crystallization-driven self-assembly.","authors":"Tianlai Xia, Laihui Xiao, Neha Yadav, Andrew P Dove, Rachel K O'Reilly","doi":"10.1038/s41596-026-01428-9","DOIUrl":"https://doi.org/10.1038/s41596-026-01428-9","url":null,"abstract":"<p><p>Here we describe the preparation of two-dimensional (2D) polymer platelets based on poly(ε-caprolactone) (PCL) using living crystallization-driven self-assembly (CDSA), a seeded growth strategy that uses crystallization as a directional driving force to achieve epitaxial growth with controlled size and shape. The method integrates ring-opening polymerization for preparation of PCL segments, reversible addition-fragmentation chain-transfer polymerization for corona-forming blocks, sonication fragmentation or the flash-freezing method to generate seed particles and living CDSA to produce uniform and monodisperse 2D nanostructures. These 2D platelets are prepared from mixtures of PCL-based homopolymers and PCL-b-PDMA block copolymers, where PDMA refers to poly(N,N-dimethylacrylamide); similar solvophilic blocks with comparable properties could also be employed in place of PDMA. In this system, the semicrystalline PCL block provides the driving force for self-assembly, while the amorphous solvophilic corona block imparts colloidal stability and tunes interfacial properties. Owing to the living nature of CDSA, the platelet dimensions scale linearly with the unimer-to-seed ratio, enabling reproducible control over platelet area, layered structures and overall morphology. The Protocol is compatible with a broad range of PCL-based polymers bearing diverse functional groups and corona chemistries, and it can be readily adapted to incorporate responsive, supramolecular or fluorescent components. The resulting 2D platelets serve as versatile model systems for probing CDSA mechanisms and as platforms for optical and stimuli-responsive materials. The complete workflow-from polymer synthesis to structural characterization-can be completed within ~2 weeks and can be performed by researchers with basic experience in polymer synthesis and self-assembly.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148819102","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}