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.

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.

Afterglow imaging eliminates the need for real-time excitation light, thereby greatly reducing tissue autofluorescence to achieve highly sensitive bioimaging. However, the clinical translation of existing photoafterglow and sonoafterglow probes developed so far is hampered by their limited tissue penetration. To address this, we recently developed radioafterglow nanoprobes (RANPs) on the basis of a cascade X-ray energy-converting mechanism for cancer-specific imaging and image-guided surgery. RANPs are constructed by co-assembling radioabsorbers, radiosensitizers and radioafterglow substrates into the amphiphilic polymers using the film rehydration method. This approach is rapid (<10 min), offers precise control over the concentration of encapsulated compounds and is highly scalable. Upon X-ray irradiation, RANPs exhibit near-infrared radioafterglow emission (peaked at 788 nm), with long half-life (~4.8 min) and high brightness. These properties enable an excitation depth of the RANPs up to 15 cm, three times deeper than that of photoafterglow. Here we further develop a tumor-specific RANP for precision molecular imaging that activates its radioafterglow signal specifically in the presence of hydrogen peroxide, a key biomarker of the tumor microenvironment. This design enables the ultrasensitive detection (signal-to-background ratio of 169) and surgical resection of small tumors (1 mm[3]) under an X-ray dosage 20 times lower than that required for inorganic materials. This protocol is suitable for users with expertise in chemistry, biology and materials science. The typical timeline is 1-2 weeks for nanoprobe construction and characterization, 1-2 weeks for cell assays and 3-4 weeks for animal experiments.

Modern water treatment and resource recovery demand materials that combine high performance with real-world durability. Traditional remediation approaches (e.g., precipitation and coagulation) have drawbacks (e.g., poor selectivity) that can be overcome using adsorption-based strategies. Metal-organic frameworks (MOFs) offer high tunability, high uptake capacities and the potential for regeneration. Translating MOF adsorbents into scalable, reliable technologies requires consistent method reporting and improved mechanistic insight, toward improving long-term stability in realistic water matrices. In this protocol, we describe how to deploy and assess the performance of MOF-based adsorbents for simultaneous heavy-metal sequestration (e.g., Pb(II), Cd(II), Ni(II) and Mn(II)) and rare-earth element recovery (e.g., Nd(III), Y(III) and Dy(III)) from complex water matrices. The workflow is broadly applicable across MOF chemistries and is illustrated using Cu(II)-based frameworks as representative model systems, synthesized at gram scale using commercially available precursors. We stabilize these frameworks through controlled defect engineering (e.g., partial metal substitution) to mitigate hydrolytic degradation and prolong operation time. We further tune morphology (e.g., nanosheets) to enhance surface accessibility and enable recyclability. For industrial applicability, we shape the MOFs into macrobeads via a green process. The procedure comprises: (i) MOF synthesis; (ii) comprehensive pre-adsorption characterization to assess crystallinity, porosity, morphology and composition using powder X-ray diffraction, nitrogen adsorption-desorption, scanning electron microscopy and inductively coupled plasma optical emission spectrometry; (iii) mechanistic adsorption assessment with kinetic, isotherm, thermodynamic, pH and selectivity analyses; (iv) regeneration and recovery workflows; and (v) deployment considerations in complex aqueous matrices, including industrial effluents, saline waters and e-waste leachates. The protocol provides a reproducible framework for implementing MOF-based adsorption technologies in water remediation and circular resource applications.

Here we present the pathology workflow of the Mutographs project, a 7-year, multicancer genomics initiative led by the Sanger Institute and funded by Cancer Research UK. Mutographs aims to elucidate global differences in cancer incidence through whole-genome sequencing and mutational signature analysis of selected cancers with unexplained variation of incidence rate worldwide. The Protocol was developed by the International Agency for Research on Cancer, where pathology assessment and paired tumor-blood DNA extraction were performed. We provide details for handling frozen cancer tissues for multistep pathology evaluation and quality control, tumor-enrichment methods (such as macrodissecion and laser-capture microdissection) for certain tumor types, and manual and automated DNA extraction. This Protocol has achieved an overall success rate of 88% in providing high-quality DNA for whole-genome sequencing, aiding in the decision to exclude low-quality or noneligible tissues. This Protocol provides a reproducible, fully integrated pathology-molecular pipeline in challenging conditions to ensure high-quality DNA and reliable data interpretation at an unprecedented multinational scale and could be considered a model for high-quality tissue processing in international genomics initiatives. It is based on routine practice of pathology and requires practical experience of processing frozen tissues and morphological assessment of frozen tissues by digital pathology.

Exposomics is an emerging field of research that aims to comprehensively investigate individuals' environmental exposures and how these exposures relate to health outcomes. Liquid chromatography-tandem mass spectrometry is widely used in exposomics studies. MetaboAnalyst (https://www.metaboanalyst.ca/) is a widely used platform for statistical and functional analysis of metabolomics data. The current MetaboAnalyst 6.0 release incorporates updates to meet the needs of exposomics studies, including improved support for tandem mass spectrometry compound identification, exposome annotation, dose-response analysis and linking to genetics and functions. Here we extend our 2022 Nature Protocol by providing step-by-step instructions on how to use MetaboAnalyst 6.0 for exposomics data analysis, including: liquid chromatography-tandem mass spectrometry spectra processing and compound identification (Stage 1), exposomics data processing and exploratory analysis (Stage 2), dose-response modeling to study metabolic responses to exposure levels (Stage 3) and leveraging known genetic associations for causal inference (Stage 4). We demonstrate Stages 1-3 using data from a recent blood exposomics study concerning electronic waste exposure. Stage 4 is illustrated through an investigation of the potential causal link between ʟ-isoleucine and type 2 diabetes. Stage 1 may take ~2 h to complete depending on server load, and the remaining stages may be executed in a total of ~90 min.

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 [57]Fe labeling is especially simple using the Fmoc route.

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.

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.

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.

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.

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 CeOx, LaOx and InOx 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.