The core problem SIMworks addresses is deceptively simple to state and notoriously hard to solve. SIM works by illuminating a specimen with patterned light, typically a fine grid of stripes, and computationally extracting high-frequency information that the objective lens alone cannot capture. Because the final image is assembled by an algorithm rather than recorded directly, the technique is exquisitely sensitive to imperfections in the raw data. Misaligned illumination patterns, photobleaching during acquisition, spherical aberrations introduced by the sample itself, and noise amplification during reconstruction can all conspire to produce images that look plausible but contain artifacts, sometimes even fabricating structures that do not exist in the specimen. For a field increasingly reliant on quantitative measurements of nanoscale biology, such artifacts are not merely cosmetic; they can distort conclusions about protein clustering, chromatin organization, or organelle interactions.

SIMworks tackles this challenge by unifying four previously established tools into a single, coherent platform. The suite builds on SIMcheck, a widely used toolbox for assessing raw and reconstructed SIM data quality; Chromagnon, a program for correcting chromatic shifts between color channels; ChaiN, which addresses channel registration and related corrections; and SIMinspector, a tool for examining reconstruction quality. Rather than forcing users to juggle separate programs with incompatible interfaces and workflows, SIMworks wraps these capabilities into a modular architecture that runs within Fiji, the open-source image analysis environment built on ImageJ. Because Fiji is already a fixture in most biology laboratories, the barrier to adoption is dramatically lower than it would be for a standalone package requiring unfamiliar installation procedures or proprietary licenses.

The workflow that the protocol describes is organized around a logical progression from raw data to quantitative results. It begins with calibration and quality checks on the raw, unreconstructed images, verifying that the illumination pattern was properly modulated, that signal-to-noise ratios are adequate, and that the acquisition parameters fall within acceptable ranges. Only after the raw data pass these checks does the user proceed to reconstruction and a second round of quality assessment on the processed images. This two-stage gating is crucial: artifacts introduced during acquisition can be caught before they are amplified by reconstruction, while reconstruction-specific problems, such as residual ringing or noise-driven pseudo-structures, can be identified in the output. The protocol provides detailed guidance on interpreting each quality metric, helping users decide whether their data are suitable for processing and how to optimize parameters for the best possible results.

One of the most technically interesting aspects of SIMworks is its handling of augmentation and artifact correction. The suite includes tools for Fourier bandpass filtering, which suppresses high-frequency noise that would otherwise manifest as a characteristic hammerstroke pattern in the final image. The protocol’s extended data illustrate this vividly: in DAPI-stained chromatin imaged by three-dimensional SIM, appropriate bandpass filtering transforms a noisy frequency profile into a near-linear response that kinks cleanly into the noise floor, corresponding to an effective spatial resolution of around 108 nanometers on the tested instrument. The authors also demonstrate masking based on the modulation contrast-to-noise ratio, a metric that helps distinguish genuine spot-like signals from noise-driven pseudo-structures. Crucially, they show that the optimal threshold depends on raw data quality, with lower-quality datasets requiring more conservative settings to avoid eroding real features.

Beyond quality control, SIMworks extends into quantitative analysis, which is where the software’s ambitions become most apparent. The suite includes segmentation and classification modules capable of identifying spot-like signals, such as DNA replication foci or cohesin complexes, as well as more complex structures like Golgi apparatus, peroxisomes, lysosomes and mitochondria. Once objects are segmented, the software can quantify their spatial relationships, measuring distances and co-localization in ways that support rigorous statistical comparison across conditions. The protocol even demonstrates compatibility with images from other modalities, including Zeiss Airyscan confocal data, suggesting that the segmentation and quantification tools have value beyond SIM proper. This breadth positions SIMworks not just as a SIM utility but as a general framework for quantitative analysis of high-resolution fluorescence data.

Reproducibility and interoperability were clearly central design considerations. The authors emphasize that SIMworks is compatible with both custom-built and commercial SIM systems and supports advanced SIM modalities, not just the classical two-dimensional implementations. All code is distributed through Fiji’s update sites, ensuring that users receive updates through the same mechanism they already use for other plugins, and the peer-reviewed code is archived on Zenodo with a persistent digital object identifier. A freely available test dataset, including raw images, reconstructed images and alignment files, accompanies the project on GitHub, allowing newcomers to practice the full workflow before committing their own precious samples. The authors report that the complete workflow, from raw data to quantitative output, can be completed in approximately half a day, depending on dataset size and complexity.

The timing of this release is significant. The past few years have seen an explosion of SIM variants, including lattice SIM for large fields of view, Hessian SIM for fast live imaging, point-spread-function-engineered SIM for high fidelity, and deep-learning approaches that promise instant denoising and super-resolution. Each advance brings new capabilities but also new reconstruction algorithms, new parameter choices and new opportunities for artifacts to creep in. At the same time, institutional imaging facilities are making SIM available to researchers with no optical engineering background, meaning that the people operating the microscopes often cannot diagnose technical problems on their own. A unified, well-documented quality control pipeline arrives at precisely the moment the community needs one, providing a common language for discussing data quality across instruments and laboratories.

The biological payoff is already evident in the authors’ own research programs. The Ochs laboratory in Copenhagen, which studies genome integrity and chromatin organization, has used SIM to reveal how sister chromatid cohesion is mediated by individual cohesin complexes and how chromatin topology safeguards the genome. The Schermelleh group in Oxford has long contributed to the development and dissemination of three-dimensional SIM methods for imaging the nuclear periphery. Tools like SIMworks are what allow such discoveries to be made reliably and, just as importantly, to be reproduced by other groups. When a claimed nanoscale arrangement of chromatin or a measured distance between protein clusters can be traced through a documented, artifact-checked pipeline, the entire field gains confidence in the underlying biology.

For laboratories considering adopting SIM, or struggling to make sense of data they already have, the message from this protocol is encouraging. The technical barriers that once made SIM the province of specialist facilities are being dismantled not by making the physics simpler, but by making the software smarter and the best practices explicit. SIMworks does not eliminate the need for careful sample preparation, appropriate fluorophore choice, or thoughtful experimental design; no software can rescue poorly acquired data. What it does provide is a safety net, catching problems before they contaminate the scientific record and lowering the expertise threshold for rigorous quantitative imaging. As super-resolution microscopy continues its march from specialist tool to standard laboratory technique, platforms like this one will determine whether the resulting data can be trusted, compared and built upon. In that sense, SIMworks may prove as important for what it prevents, namely artifact-driven false discoveries, as for what it enables.

Subject of Research: Quality-controlled quantitative structured illumination microscopy software

Article Title: SIMworks—an integrated software suite for quality-controlled augmented quantitative structured illumination microscopy

Article References: Pytowski, L., Jandi, W., Henggeler, A., Cho, W., Schermelleh, L., & Ochs, F. (2026). SIMworks—an integrated software suite for quality-controlled augmented quantitative structured illumination microscopy. Nature Protocols. https://doi.org/10.1038/s41596-026-01444-9

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01444-9

Keywords: structured illumination microscopy, super-resolution microscopy, SIMworks, Fiji, image quality control, artifact correction, image segmentation, quantitative imaging, chromatin imaging, Nature Protocols, open-source software, cell biology

The historical roots of the field stretch back further than many researchers realize. As the tutorial recounts, the earliest experiments in tissue self-organization date to 1907, when Wilson demonstrated that dissociated sponge cells could reassemble into functional organisms. Decades later, work by Holtfreter, Weiss and Taylor established the concept of tissue affinity and the remarkable capacity of embryonic cells to reconstitute organized structures from single-cell suspensions. Steinberg’s differential adhesion hypothesis in 1970 provided a theoretical framework for why cells sort themselves into coherent tissues. These classical observations laid the conceptual groundwork for the modern organoid revolution, which arrived in earnest in 2008 and 2009 with two landmark achievements: Eiraku and colleagues’ self-organized formation of polarized cortical tissue from embryonic stem cells, and Sato and colleagues’ demonstration that single Lgr5-positive intestinal stem cells could build complete crypt-villus structures in vitro without any mesenchymal niche.

Those two founding systems, intestinal and brain organoids, form the backbone of the new tutorial, and the authors deliberately chose them because they represent opposite ends of the organoid spectrum. Adult stem cell-derived intestinal organoids, sometimes called miniguts, are genetically stable, expandable over years and remarkably faithful to the epithelium they came from. Human pluripotent stem cell-derived brain organoids, by contrast, are developmental models that recapitulate embryonic neurogenesis, offering access to human brain biology that no other experimental system can provide. By walking through both, the tutorial illustrates how organoid choice depends entirely on the biological question at hand, whether that question concerns tissue homeostasis in the adult or the choreography of human development.

One of the most technically rich sections of the tutorial addresses cell fate decisions, the process by which stem cells commit to specific differentiated identities. In the intestine, regional and local signaling gradients, particularly the Wnt, BMP and Notch pathways, control where and when cells become absorptive enterocytes, mucus-secreting goblet cells, hormone-producing enteroendocrine cells, or tuft cells. The tutorial highlights how organoids allow researchers to manipulate these gradients with unprecedented precision. Experiments have shown that enteroendocrine cells switch their hormone expression profiles along the crypt-to-villus BMP signaling gradient, and that induced quiescence of Lgr5-positive stem cells enables the differentiation of hormone-producing cells. In brain organoids, timed and combinatorial treatments with extrinsic signals can specify ventral telencephalic identities, while minimizing exogenous signals drives rostral hypothalamic differentiation, demonstrating that the same self-organizing logic governs tissues as different as gut and brain.

The tutorial also devotes substantial attention to decrypting cell-cell communication, one of the most challenging problems in tissue biology. Organoids provide a contained system in which signaling networks can be measured at single-cell resolution using multiplexed single-cell analysis, and in which genetic reporters can mark specific populations in living tissue. Fluorescent gene tagging without double-strand DNA cleavage, ASCL2-responsive minigenes that label stem cell activity, and biosensors that quantify single-cell ERK dynamics have all been deployed in organoid systems. Machine learning tools such as OrganoidTracker now allow researchers to follow cell fate dynamics in space and time across entire organoids, converting what was once a static snapshot technology into a live-imaging platform capable of resolving the oscillatory signaling events that control cell fate decisions during intestinal homeostasis.

Genetic disease modeling represents perhaps the most clinically resonant application, and the tutorial lays out the strategies in detail. CRISPR-Cas9 engineering of organoids has enabled researchers to introduce precise mutations into otherwise healthy tissue, generating isogenic disease models. Sequential introduction of cancer mutations in cultured human intestinal stem cells has recapitulated colorectal cancer progression, while CRISPR-mediated engineering of patient-derived organoids has allowed systematic dissection of tumor evolution. On the repair side, functional correction of the CFTR gene by CRISPR in intestinal stem cell organoids from cystic fibrosis patients demonstrated that gene editing could restore disease-relevant function. The forskolin-induced swelling assay, which measures CFTR channel activity in patient-derived rectal organoids, has matured into a validated diagnostic tool, and high-throughput versions of the assay now support drug repurposing for cystic fibrosis.

Infectious disease research has been transformed by organoids as well. The tutorial documents how human intestinal organoids supported the first productive infection of human gut enterocytes by SARS-CoV-2, and how brain organoids became the central platform for understanding Zika virus-induced microcephaly. Multiple groups showed that Zika depletes neural progenitors through activation of the innate immune receptor TLR3, and organoid-based drug screens identified compounds that combat infection. Microinjection platforms and microfluidic gut-on-chip systems now allow controlled exposure of organoids to defined microbes, including complex anaerobic communities, opening the door to systematic study of host-microbiome interactions that were previously inaccessible in vitro.

The tutorial’s treatment of brain organoids extends into territory that borders on the philosophical. Comparative studies using organoids from human, chimpanzee and macaque cells have identified species-specific differences in progenitor behavior that contribute to brain size, and human-specific genes such as ARHGAP11B, NOTCH2NL and SRGAP2 have been shown to expand cortical neurogenesis when tested in organoid systems. Assembloids, fused organoids representing different brain regions, now model interneuron migration, thalamocortical connectivity and even the ascending sensory pathway. Single-cell brain organoid screening has revealed developmental defects in autism, and patient-derived organoids have exposed aberrant neuronal development in schizophrenia and copy number variant disorders. Chimeric models, in which human organoids are transplanted into mouse brains, have produced vascularized, innervated human tissue that establishes subcortical projections in the host animal.

The authors are candid about the field’s remaining challenges. Organoids lack vasculature, immune cells and mechanical cues from their native environment, limiting their maturation and long-term viability. Batch-to-batch variability, particularly in brain organoids, complicates reproducibility, and stress responses within cortical organoids can impair molecular subtype specification. Matrigel, the animal-derived matrix on which most organoids are grown, remains poorly defined, spurring development of synthetic alternatives and engineered materials whose stiffness can itself direct stem cell behavior. The tutorial highlights emerging innovations, including optogenetic control of gene expression, scaffold-guided morphogenesis that produces homeostatic mini-intestines, microfluidic vascularization, and bioprinting platforms that enable drug screening at single-organoid resolution, as the technologies most likely to close these gaps.

The translational trajectory of the field is already visible. Patient-derived organoid biobanks have predicted treatment responses in metastatic gastrointestinal cancers, tumor organoid and T-cell co-culture systems are being used to evaluate engineered immunotherapies, and glioblastoma organoids now serve as real-time avatars for assessing CAR-T cell responses. Organoid transplantation has repaired colitis in mouse models, alleviated traumatic brain injury and addressed short bowel syndrome in preclinical studies. What the Munich tutorial ultimately conveys is that organoids have crossed a threshold: they are no longer a promising curiosity but a mature experimental ecosystem, one whose protocols, pitfalls and possibilities are now documented well enough that any competent laboratory can begin interrogating biology with them.

Subject of Research: Organoid technologies for interrogating fundamental biology and disease

Article Title: Tutorial: interrogating biology using organoid technologies

Article References: Bugter, J. M., Schafer, S. T., & Rad, R. (2026). Tutorial: interrogating biology using organoid technologies. Nature Protocols. https://doi.org/10.1038/s41596-026-01447-6

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01447-6

Keywords: organoids, intestinal organoids, brain organoids, stem cells, CRISPR, disease modeling, self-organization, cell fate, host-microbe interactions, drug screening, assembloids, Nature Protocols

Fibrosis itself is a double-edged biological process. In response to injury, fibroblasts become activated and deposit excessive amounts of extracellular matrix, the protein scaffold that gives tissues structure. When this deposition spirals out of control, organs stiffen and progressively lose function. In the heart, scar-like tissue replaces working muscle, impairs contraction, disrupts electrical signaling and ultimately contributes to increased morbidity and death. The researchers note that this pathological remodeling is a common feature across ischemic heart failure, genetic cardiomyopathies, diabetes mellitus and the aging heart, making it one of the most consequential targets in cardiovascular medicine.

The reason no approved antifibrotic exists for the heart, the authors argue, is not a lack of biological interest but a lack of reliable, translatable discovery platforms. Traditional approaches often rely on primary fibroblasts harvested from tissue, which are difficult to obtain, do not divide indefinitely, vary between donors and are hard to scale to the tens of thousands of wells needed for modern pharmaceutical screening. Animal studies, for their part, have repeatedly failed to predict human toxicity and efficacy accurately. The new protocol was conceived as a new approach methodology, a term regulators use for non-animal testing strategies that better reflect human biology while remaining scalable.

At the heart of the platform are reporter iPS cell-derived cardiac fibroblasts. Induced pluripotent stem cells can be generated from virtually any patient and then directed to differentiate into quiescent, resting cardiac fibroblasts that closely resemble those found in healthy heart tissue. By inserting a reporter gene whose activity tracks fibrotic activation, the researchers created cells that glow measurably when they switch into the scar-forming, myofibroblast-like state. When a compound suppresses this signal, the assay flags it as a potential antifibrotic hit. Because the cells derive from a renewable stem cell line, they can be produced in the large, uniform batches that automated 384-well plate screening demands.

The workflow is organized in two stages. In the first, the reporter fibroblasts are plated into 384-well plates and exposed to large chemical libraries in a process called quantitative high-throughput screening, a titration-based approach in which every compound is tested across multiple concentrations rather than at a single dose. This design distinguishes genuine, dose-dependent biological activity from noise and artifacts. Before screening begins, the team meticulously optimizes cell density so that the reporter signal sits in a sensitive linear range. The authors report that the complete pipeline, from cell preparation through primary screening, is robust and scalable, capable of screening approximately 5,000 compounds within six to eight weeks.

The second stage is the counter-screen, and it is what sets the platform apart from conventional fibroblast assays. Any compound that quiets fibroblast activation would be clinically useless, or worse, dangerous, if it simultaneously poisons cardiomyocytes, the contractile cells of the heart. To guard against this, the researchers test their initial hits on human iPS cell-derived cardiomyocytes and exclude any molecule with detectable cardiotoxicity. The importance of this step is well documented: earlier studies from the same field showed that iPS cell-derived cardiomyocytes can recapitulate patient-specific susceptibilities to drug-induced heart injury, such as the sensitivity of some breast cancer patients to doxorubicin, and that high-throughput screens can detect electrophysiological hazards like QT interval prolongation that are missed in other preclinical models.

The protocol is not merely theoretical. It distills lessons from the group’s own discovery work, including a 2024 Cell study in which a multiscale version of this screening strategy identified MD2, an innate immune adaptor protein, as a therapeutic target for cardiac fibrosis. That campaign moved from fibroblast-level screening to validation in engineered heart tissues and animal models, illustrating how hits emerging from the platform can be triaged toward deeper mechanistic and preclinical studies. The team has also published methods for generating quiescent cardiac fibroblasts from iPS cells and for building three-dimensional cardiac organoids for antifibrotic screening, creating a family of complementary human-cell models around the same core technology.

What makes the approach broadly exciting is its adaptability. Because iPS cells can be differentiated into fibroblast-like and mesenchymal populations for many organs, including lung, liver and skin, the same screening logic could be repurposed to hunt for antifibrotic drugs for idiopathic pulmonary fibrosis, liver cirrhosis or systemic sclerosis. Indeed, related protocols for generating hepatic stellate cells and lung mesenchyme from pluripotent stem cells have already appeared in the literature. The Stanford team frames their protocol as a versatile template for phenotypic drug discovery, in which compounds are selected for their effect on a disease-relevant cellular behavior rather than against a single molecular target, an industry strategy increasingly valued for its ability to surface unexpected mechanisms.

There are practical caveats for laboratories hoping to adopt the system. Generating and authenticating a reporter iPS cell line requires genetic engineering expertise, and the differentiation protocol to produce quiescent cardiac fibroblasts demands careful timing of developmental signaling cues. Quantitative high-throughput screening also requires access to robotic liquid handling, plate readers and curated compound libraries, resources typically found at dedicated screening centers. The published protocol addresses many of these hurdles with detailed guidance on plate layout, control compounds, data normalization and hit selection criteria, and its figures walk readers through differentiation, optimization and representative screening results step by step.

For patients, the stakes could hardly be higher. Heart failure remains a leading cause of death worldwide, and fibrosis is the common pathway through which many different injuries destroy cardiac function. A reproducible, human-cell-based platform that can rapidly separate genuine antifibrotic candidates from cardiotoxic impostors offers the pharmaceutical industry a translatable bridge between bench chemistry and clinical trials, one that reduces reliance on animal experiments while keeping human biology at the center of the search. As new approach methodologies gain regulatory momentum, protocols like this one may help ensure that the next generation of fibrosis drugs is discovered not in a petri dish of rodent cells, but in human cells reprogrammed to mirror the patients they are meant to save.

Subject of Research: A high-throughput drug screening protocol using human iPS cell-derived cardiac fibroblasts and cardiomyocytes to identify non-cardiotoxic antifibrotic compounds.

Article Title: New approach methodologies: drug screening platform to identify antifibrotic compounds

Article References: Zhang, H., Ren, L., Huang, R., Tan, R., Solow-Cordero, D. E., Mukherjee, S., & Wu, J. C. (2026). New approach methodologies: drug screening platform to identify antifibrotic compounds. Nature Protocols. https://doi.org/10.1038/s41596-026-01445-8

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01445-8

Keywords: cardiac fibrosis, drug screening, induced pluripotent stem cells, cardiac fibroblasts, cardiotoxicity, new approach methodologies, high-throughput screening, phenotypic drug discovery, heart failure, cardiomyocytes, fibrosis, Nature Protocols

Mass spectrometry imaging has become one of the most powerful tools in modern biology because it can map hundreds of molecules directly within a tissue slice, revealing where lipids, metabolites, proteins and other biomolecules reside without labels or stains. Yet the technique has long been constrained by its spatial resolution. Conventional instruments raster a laser across a tissue section with a defined step size, and features smaller than that step blur together. Boosting resolution usually demands costly instrumentation such as specialized lasers, transmission-mode optics or next-generation ion optics, placing subcellular molecular imaging out of reach for many laboratories.

TEMI takes a different path. Instead of refining the instrument, the team enlarges the tissue itself. Drawing on principles from expansion microscopy, a technique introduced more than a decade ago, the researchers embed tissue in a water-rich hydrogel polymer network. When the gel swells, it carries the tissue along with it, spreading the molecules apart and effectively magnifying the sample before any mass spectrometry takes place. A laser raster that would have been too coarse for the original tissue becomes fine enough to resolve individual cells in the expanded version, yielding more than a 3.5-fold improvement in effective imaging resolution with standard equipment.

The critical challenge was chemistry. Traditional expansion protocols often rely on harsh denaturation conditions, including high heat and strong detergents, to homogenize tissue and allow gels to stretch uniformly. Those conditions would destroy the very molecules mass spectrometry aims to detect. The TEMI workflow solves this by using a re-embedding strategy that expands tissue under mild, harsh-condition-free conditions, preserving the chemical integrity of lipids, metabolites, N-glycans, peptides and proteins. The result is a sample that is both physically enlarged and chemically faithful to its original molecular composition.

The published protocol walks readers through every stage of the process. It begins with constructing a gelation chamber, then describes hydrogel-based tissue gelation and expansion, followed by cryosectioning of the expanded tissue-hydrogel composite. Because expanded samples are soft and hydrated, cutting them into thin sections requires careful handling, and the protocol provides the specific parameters that make reproducible sectioning possible. Detailed guidance covers matrix application, data acquisition and visualization pipelines, along with troubleshooting tips accumulated through the team’s extensive experimentation.

One of the most striking capabilities of TEMI is multiomics mapping on a single tissue section. The protocol describes a sequential workflow in which N-glycans are released in situ by treatment with the enzyme PNGase F, after which proteins are digested with trypsin and imaged, all following lipid and metabolite analysis on the same expanded section. This means a single slice of brain tissue can yield spatially resolved maps of multiple molecular classes, each anchored to the same anatomical context, an efficiency that conventional workflows struggle to match.

Quality control receives particular attention. The protocol includes a dedicated workflow for measuring deformation maps and expansion factors, which quantify how uniformly the gel has expanded. Uneven expansion would distort molecular maps, so the researchers provide computational tools, released through a public code repository, that quantify tissue expansion non-uniformity. They also demonstrate that analyte delocalization during sample preparation is minimal: lipid and small-metabolite signals were detected exclusively in tissue regions, with no corresponding signals in adjacent blank hydrogel areas, confirming that molecules stay where they belong during the swelling process.

The demonstration experiments showcase the method’s power on the mouse cerebellum, a tissue with exquisitely organized layers of cells. Comparing an unexpanded control cerebellum imaged with a 50-micrometer laser raster against a double-embedded, expanded sample at the same step size reveals a dramatic difference in molecular detail. Pushing further, the team performed TEMI after three cycles of gel embedding and expansion with a 10-micrometer raster step, resolving biomolecular heterogeneity that remains invisible in unexpanded tissue. The protocol also demonstrates mapping of small metabolites, N-glycans and proteins across cerebellar structures, illustrating the breadth of the multiomics capability.

The work builds on the team’s primary research paper published in Nature Methods, which introduced TEMI and established its foundational performance, and on a rich history of expansion microscopy methods developed by collaborators at the Howard Hughes Medical Institute’s Janelia Research Campus, including protein-retention expansion microscopy and ten-fold robust expansion. Raw datasets from the protocol’s figures are publicly available in the MassIVE repository, and the deformation-measurement code is hosted on GitHub, lowering barriers for laboratories that want to adopt or adapt the approach.

The implications extend across basic biology and medicine. Single-cell spatial resolution for untargeted molecular imaging could illuminate how metabolic gradients shape tissue function, how lipid compositions vary between neighboring cells in the nervous system, and how disease states such as cancer or neurodegeneration alter molecular architecture at scales previously accessible only to antibody-based imaging. Because TEMI requires no instrument modifications, it promises broad accessibility: laboratories with standard mass spectrometry imaging systems can now reach a resolution regime once reserved for a handful of specialized facilities. The protocol’s authors, spanning the University of Wisconsin-Madison and HHMI Janelia, and supported in part by the National Institutes of Health, position the method as a practical bridge between the spatial-omics revolution and the everyday mass spectrometry lab, inviting a wide research community to stretch its view of tissue, quite literally, into sharper focus.

Subject of Research: Tissue expansion combined with mass spectrometry imaging for high-spatial-resolution multiomics molecular mapping

Article Title: Tissue expansion mass spectrometry imaging (TEMI) for high-spatial-resolution multiomics molecular mapping

Article References: Tissue expansion mass spectrometry imaging (TEMI) for high-spatial-resolution multiomics molecular mapping. (n.d.). https://doi.org/10.1038/s41596-026-01427-w

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01427-w

Keywords: mass spectrometry imaging, tissue expansion, TEMI, spatial omics, single-cell imaging, lipidomics, metabolomics, N-glycans, proteomics, hydrogel expansion, Nature Protocols, multiomics

The core idea is elegantly simple. A fluorescent tracer dye called Fast Blue is applied to the organ of interest. Neurons whose axons terminate in that organ take up the dye at their nerve endings and transport it backward along their axons to their cell bodies, a process known as retrograde axonal transport. A few days later, the relevant ganglia are dissected, dissociated into single cells, and passed through a fluorescence-activated cell sorter. Only the fluorescently labeled neurons, those with a proven physical connection to the target tissue, are retained. Each captured cell is then subjected to single-cell RNA sequencing, generating a full transcriptome for every traced neuron.

The crucial advance over existing approaches lies in selectivity. Sequencing a whole ganglion without labeling captures everything: sensory neurons serving skin and muscle, autonomic neurons innervating unrelated organs, satellite glial cells and other stromal populations. That background can completely swamp the small subset of neurons connected to the tissue a researcher actually cares about. Bulk sequencing is even blunter, averaging signals across thousands of cells and erasing neuronal subtype identity altogether. Trace-n-Seq eliminates this noise by design, allowing researchers to characterize organ-specific neurons at single-cell resolution and to detect even rare tissue-innervating subsets that would otherwise be invisible.

The complete workflow, published in Nature Protocols by Vera Thiel, Manuel Mastel, Simon Renders and colleagues in the laboratories of Martin Sprick and Andreas Trumpp, can be completed in about two weeks. It encompasses tracer application, ganglion dissection, enzymatic tissue digestion, careful neuronal validation, fluorescence-based sorting, SMART-seq2 library preparation and a dedicated bioinformatic pipeline. The published protocol walks readers through each stage with schematic illustrations, dissection guidance, quality-control checkpoints and computational scripts. The authors note that the method suits researchers experienced in molecular biology, neuronal tissue handling, sequencing techniques and bioinformatics, and they have deposited their complete Seurat-based analysis workflow, quality-control scripts and annotation code openly on GitHub. The underlying sequencing data are accessible through the ArrayExpress repository under accession E-MTAB-12940.

Why does this matter biologically? Neuronal innervation is now recognized as a master regulator of tissue function well beyond classical sensation and motor control. Nerves shape organ function during homeostasis and regeneration, and they are increasingly implicated in pathological settings including inflammation, autoimmune disease, fibrosis and cancer. The emerging field of cancer neuroscience, highlighted in the protocol’s own key reference by Monje and Winkler, argues that tumors are not just passive targets of nerve growth but active participants in a dialogue with the nervous system. Tumor-associated nerves can be reprogrammed by the tumor microenvironment, and in some contexts these remodelled neurons appear to support malignancy itself.

Trace-n-Seq was developed and validated in exactly such a context. The protocol builds on the team’s earlier research published in Nature in 2025, in which they characterized single neurons reprogrammed by pancreatic cancer. Pancreatic ductal adenocarcinoma is one of the most densely innervated human tumors, and the earlier study showed that individual neurons connected to the tumor acquire distinct transcriptional states compared with neurons innervating healthy tissue. By tracing neurons from the tumor back to their ganglia and sequencing them one by one, the researchers could separate genuine disease-associated neuronal states from the bulk signature of the ganglion. The new protocol packages that experimental logic into a step-by-step recipe that any qualified laboratory can follow.

The versatility of the method is a major selling point. Because it depends only on functional connectivity, Trace-n-Seq is in principle applicable to any healthy, inflamed or diseased tissue innervated by the peripheral nervous system. Researchers can compare traced neurons from a healthy pancreas with those from a pancreatitis model or a tumor-bearing organ, directly quantifying how disease remodels neuronal subtype composition and gene expression. They can capture rare tissue-specific neuronal subsets, distinguish disease-associated states from background ganglia signatures, and follow neuronal plasticity across physiological and pathological conditions at single-cell resolution. This functional, connectivity-based profiling contrasts with purely anatomical or genetic labeling strategies and provides an orthogonal view of peripheral neuron diversity.

The technical details matter for anyone hoping to reproduce the approach. Fast Blue is a retrograde tracer well established in neuroanatomy, valued for its intense fluorescence and compatibility with downstream flow cytometry. After allowing sufficient time for axonal transport, ganglia are harvested and must be gently digested to yield viable, intact neurons, a step the authors emphasize with dedicated validation procedures to confirm that sorted cells are indeed neurons rather than debris or contaminating glia. Fluorescence-activated cell sorting then enriches the labeled population before library construction. The bioinformatic workflow includes quality control, filtering and annotation steps tailored to traced neurons, including reference-based neuronal annotation, so that researchers can confidently classify traced cells into known sensory and autonomic subtypes and discover novel disease-associated clusters.

The publication arrives at a moment of rapid growth for the neuro-immune and neuro-oncology fields. Other studies have revealed, for example, that lymph nodes are innervated by a unique population of sensory neurons with immunomodulatory potential, and barcoded viral tracing approaches have begun to map single-cell interactions during central nervous system inflammation. Large-scale atlases from single-cell sequencing efforts have catalogued the molecular architecture of the mouse nervous system, classifying sensory and visceral motor neuron types in unprecedented detail. What these atlases lack, however, is information about where each neuron actually projects. Trace-n-Seq adds that anatomical dimension, linking molecular identity to functional tissue connectivity in a single experiment.

For clinical translation, the implications are considerable. If neurons innervating tumors can be identified and molecularly profiled, they become potential therapeutic targets or biomarkers. Clinically actionable strategies for studying neural influences in cancer have been a subject of active discussion, and tools like Trace-n-Seq provide the granular data needed to move from observation to intervention. Understanding which neuronal subprograms a tumor co-opts, and how inflammatory or fibrotic diseases rewire peripheral innervation, could eventually inform drugs that interrupt harmful neuro-tissue signaling while sparing essential nerve function. The open release of the protocol, the analysis code and the source datasets lowers the barrier for laboratories worldwide to adopt the technique, and the authors declare no competing interests. As neuroscience and organ biology continue to converge, methods that reveal which neurons talk to which tissues, and what they say at the transcriptomic level, are likely to become standard equipment in the effort to understand how the nervous system shapes health and disease.

Subject of Research: A single-cell sequencing protocol combining retrograde fluorescent tracing and cell sorting to molecularly profile peripheral neurons innervating specific tissues.

Article Title: Trace-n-Seq combines retrograde fluorescent tracing with sorting and single-cell sequencing of innervating peripheral neurons

Article References: Thiel, V., Mastel, M., Renders, S., Panten, J., Bauer, K., Jackstadt, R., Sprick, M. R., & Trumpp, A. (2026). Trace-n-Seq combines retrograde fluorescent tracing with sorting and single-cell sequencing of innervating peripheral neurons. Nature Protocols. https://doi.org/10.1038/s41596-026-01416-z

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01416-z

Keywords: Trace-n-Seq, retrograde tracing, single-cell RNA sequencing, peripheral neurons, fluorescence-activated cell sorting, ganglia, cancer neuroscience, neuronal innervation, Fast Blue, pancreatic cancer, Nature Protocols, neuron-tissue interactions

The core innovation is deceptively simple. Rather than relying on invasive skin biopsies, blood draws or urine collection, the protocol uses hairs plucked with intact follicles, ideally in the anagen or growth phase, when the follicle is rich in proliferative keratinocytes. Once plucked, the hairs are placed in a kit and can be mailed over long distances without refrigeration. In the laboratory, keratinocytes are released from the follicles by trypsinization, an enzymatic digestion that separates the cells from the hair shaft, and are then cultured under conditions that preserve their proliferative capacity. The authors report that samples remain stable for days at ambient temperature, provided standard biosafety precautions are observed, which makes ordinary postal and courier services viable conduits for human biological material.

Why does the choice of starting cell matter so much? Induced pluripotent stem cells, first generated by Shinya Yamanaka and colleagues in 2007 through the forced expression of defined transcription factors, can differentiate into all three embryonic lineages, including the neural lineage that gives rise to neurons and glia. But the quality and efficiency of reprogramming depend heavily on the source cell. Dermal fibroblasts require a punch biopsy, an uncomfortable procedure that typically must be performed by a clinician. Peripheral blood mononuclear cells require venipuncture and careful handling, and although blood held at room temperature has been used successfully, the window is limited. Renal epithelial cells from urine are noninvasive but yield variable numbers of cells and are not suitable for every donor. Keratinocytes, by contrast, reprogram efficiently, and hair plucking is essentially painless.

The Yale team’s protocol lowers the technical barrier even further by requiring fewer follicles than previous hair-based approaches. Earlier methods for isolating keratinocytes from plucked hair existed, including protocols published by Aasen and colleagues in 2008 and 2010, but they generally demanded either fresh local collection or specialized handling. The new kit-based workflow explicitly anticipates the realities of remote participation: a donor, a family member or a healthcare provider can perform the collection after watching a short instructional video that accompanies the protocol, and the resulting sample tolerates the delays of long-distance shipping. This matters enormously for rare disease research, where patients are geographically dispersed and where systematic reviews have documented substantial inequities in access to clinical genetic services.

Once the keratinocytes arrive in the laboratory, the workflow follows a well-trodden but carefully optimized path. The cells are expanded in culture, with the Rho kinase inhibitor Y-27632 playing a supporting role in improving survival, a trick borrowed from the keratinocyte literature where ROCK inhibition prolongs the lifespan of adult cells in vitro. Reprogramming then converts the keratinocytes into iPS cells, a process the protocol completes within roughly two months of receiving the hair samples. Notably, the authors emphasize that the procedure requires only basic familiarity with mammalian cell culture techniques and no specialized equipment beyond what a standard cell biology laboratory already possesses. That accessibility is a deliberate design choice: the protocol is written to be executable by labs that have never worked with human iPS cells before.

Quality control is built into the workflow. The published protocol includes characterization steps confirming that the resulting iPS cells express canonical pluripotency markers, retain a normal karyotype, and can differentiate into all three germ layers, including neural lineages. The authors demonstrate the full pipeline by generating cerebral organoids, three-dimensional self-organizing cultures that recapitulate key features of early human brain development. Organoid generation from the iPS cells takes 30 to 40 days and requires one piece of specialized equipment, an orbital shaker, which keeps the growing organoids suspended and nourished in culture. Whole-mount imaging and immunostaining confirm that the organoids contain the expected neural cell populations, establishing that hair-derived iPS cells are fully competent for demanding three-dimensional differentiation protocols.

The protocol did not emerge in a vacuum. It was developed and refined in the course of a primary research study, published in Nature in 2025, in which Zhang and colleagues showed that dysregulation of mTOR signalling is a converging mechanism in lissencephaly, a severe malformation of cortical development. For that study, the team needed iPS cells and brain organoids from patients with rare neurogenetic conditions, many of whom lived far from any research center. The kit-based hair collection method proved to be the practical answer, and the new Nature Protocols article distills that hard-won experience into a form other laboratories can adopt directly. The authors acknowledge the patients and families who contributed samples, underscoring that the method was shaped by the needs of the very people it is meant to serve.

The broader implications reach into drug development, disease modeling and eventually cell therapy. Human iPS cell-derived models allow researchers to study cellular and molecular mechanisms of disease in genuinely human tissue, something animal models often fail to capture, and cerebral organoids in particular have transformed the study of neurodevelopmental disorders since Lancaster and colleagues first described them in 2013. By making the front end of that pipeline, patient sample acquisition, dramatically easier, the Yale protocol could expand the diversity of genetic backgrounds represented in organoid studies, a long-standing concern in a field where most cell lines derive from patients already connected to major academic medical centers. Populations in low-resource settings, pediatric patients for whom blood draws are difficult, and elderly donors with fragile veins all stand to benefit from a collection method that requires nothing more than a pair of tweezers and a mailing envelope.

There are, of course, practical considerations. The protocol specifies that hairs must be plucked with follicles intact, since the follicle bulb contains the keratinocyte population of interest, and the accompanying video walks collectors through identifying suitable anagen-phase hairs. Shipping times must remain within the window during which the keratinocytes stay viable at ambient temperature, and laboratories must handle all human material under appropriate biosafety procedures. Reprogramming efficiency, while generally high for keratinocytes, still varies between donors, as it does for all somatic cell sources. Yet the authors argue that the advantages outweigh these constraints: the method is noninvasive, the samples are robust, the timeline is competitive, and the equipment requirements are minimal. As personalized medicine pushes toward models built from each patient’s own genome, protocols like this one may determine who gets to participate. A technology that turns a handful of plucked hairs into a patient-specific mini brain, mailed across continents in an ordinary package, is a striking reminder that sometimes the most transformative tools in biomedicine are also the most humble.

Subject of Research: A kit-based protocol for remote collection of hair follicle keratinocytes and their reprogramming into induced pluripotent stem cells for cerebral organoid generation

Article Title: Kit-based remote collection and isolation of human reprogrammable somatic cells for generation of induced pluripotent stem cells and cerebral organoids

Article References: Cheng, I. Q., Ruiz, J. F., Casalino, E. K., Zhang, C., & Louvi, A. (2026). Kit-based remote collection and isolation of human reprogrammable somatic cells for generation of induced pluripotent stem cells and cerebral organoids. Nature Protocols. https://doi.org/10.1038/s41596-026-01440-z

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01440-z

Keywords: induced pluripotent stem cells, keratinocytes, hair follicles, cerebral organoids, reprogramming, remote sample collection, disease modeling, personalized medicine, neurodevelopmental disorders, Nature Protocols, cell culture, lissencephaly

The significance of self-administration models in addiction neuroscience is difficult to overstate. Unlike passive drug exposure experiments, in which the investigator simply injects an animal with a drug and observes the consequences, self-administration paradigms allow the animal itself to control when and how much drug it receives, typically by pressing a lever or poking its nose into a port. This voluntary element matters enormously. It allows investigators to dissociate the behavioral and neural mechanisms that motivate drug seeking and taking from the pharmacological effects of drug exposure itself, a distinction that lies at the heart of modern addiction research. Compulsive drug taking, relapse after abstinence, and the escalation of intake over time are all phenomena that only emerge when the animal has agency over its own drug consumption.

The intellectual lineage of this approach stretches back more than eight decades. As the authors note in their News and Views commentary accompanying the protocol, the earliest demonstrations that animals will work to obtain drugs appeared in the comparative psychology literature of 1940, when Spragg reported that chimpanzees would manipulate mechanisms to gain access to morphine. In the mid-1950s, Headlee, Coppock, and Hichols described intravenous morphine administration procedures in the Journal of Pharmaceutical Sciences, laying groundwork for controlled delivery of drugs into the bloodstream of laboratory animals. These early efforts culminated in a landmark 1962 study by James Weeks, published in Science, which established the operant intravenous drug self-administration procedure in rats—a technique that remains the workhorse of preclinical addiction research to this day.

The field built rapidly on that foundation. Thompson and Schuster demonstrated in 1964 that the principles of operant conditioning could be applied to morphine self-administration, and Risner and Jones extended the approach in 1975 to compare the reinforcing effects of different drugs. By 1991, Carney and colleagues were using self-administration procedures to study stimulant pharmacology, and the paradigm had become a standard screen for the abuse liability of novel compounds. A 2020 review by Venniro, Banks, Heilig, Epstein, and Shaham in Nature Reviews Neuroscience synthesized how decades of self-administration research had refined the field’s understanding of the neural circuits of drug reward, relapse, and the economic and social factors that influence drug taking. The historical arc is clear: each generation of methodological refinement has allowed sharper questions to be asked about why drugs are so compelling.

What the new protocol adds is the head-fixed dimension. In a conventional self-administration chamber, a mouse moves freely, presses levers, and receives intravenous infusions through a catheter implanted in its jugular vein. This arrangement is behaviorally rich but experimentally constrained. The animal’s head moves unpredictably, which degrades optical recordings, complicates the delivery of precisely timed sensory stimuli, and makes it nearly impossible to hold a microscope objective or a stimulus display in a fixed relationship to the animal’s eyes and whiskers. Head fixation solves these problems. With the skull rigidly stabilized, researchers can perform stable two-photon imaging of the same population of neurons across many sessions, deliver visual or auditory cues with millisecond precision, and apply optogenetic or pharmacological manipulations to genetically identified circuits while the animal is actively working for drug.

Combining head fixation with intravenous self-administration, however, is far from trivial, and this is precisely where the protocol makes its contribution. The authors provide step-by-step instructions for assembling the necessary hardware, integrating the head-fixation apparatus with the operant response devices and the infusion system that delivers drug through the implanted catheter. The technical challenges are considerable: the catheter must remain patent across repeated sessions while the animal is restrained; the operant manipulandum must be positioned so that a head-fixed mouse can respond comfortably; and the infusion line must be routed so that drug delivery is synchronized with the animal’s responses without introducing movement artifacts or leaks. By documenting these details, the protocol lowers the barrier for laboratories that want to adopt the technique without months of trial and error.

The timing of this methodological advance reflects a broader convergence in the field. A companion protocol by Doncheck and colleagues, also published in Nature Protocols in 2026 and cited in the commentary, indicates that head-fixed drug self-administration is emerging as a coherent methodological platform rather than the idiosyncratic setup of a single laboratory. Meanwhile, recent work published in Neuron in 2024 by Paniccia and colleagues demonstrated the scientific payoff of head-fixed approaches, and a 2021 study by Vollmer and colleagues in Frontiers in Behavioral Neuroscience explored related territory in freely moving animals. Together, these publications sketch a research landscape in which the voluntary taking of intravenous drugs can finally be observed with the full arsenal of modern circuit neuroscience.

For researchers weighing whether to adopt the technique, the trade-offs are worth understanding. Freely moving self-administration preserves the naturalistic ethology of drug seeking—the animal navigates its environment, approaches the drug source, and integrates spatial and contextual cues into its behavior. Head-fixed preparations sacrifice some of that naturalism in exchange for experimental control and optical stability. The authors’ framing suggests that the two approaches should be viewed as complementary rather than competing: freely moving studies establish the validity of a behavioral phenomenon, while head-fixed preparations dissect its circuit-level mechanisms with cellular precision. A protocol that makes the head-fixed variant accessible and reproducible therefore expands the toolkit available to the field rather than replacing the established one.

The potential applications span the major questions of addiction neuroscience. With a head-fixed mouse pressing a port for intravenous cocaine, opioid, or other drug infusions, investigators could track how neurons in the prefrontal cortex, nucleus accumbens, dorsal striatum, or amygdala change their activity as drug seeking becomes habitual, as intake escalates, or as cues that predict drug availability come to drive behavior. They could test whether manipulating specific projections alters the propensity to seek drug, and they could do so across many consecutive sessions with imaging planes held perfectly stable. Because the animals are mice rather than rats, the full power of mouse genetics—cell-type-specific reporters, conditional knockouts, and activity-dependent labeling—becomes available to self-administration research in ways that were previously cumbersome.

As with any methodological innovation, the ultimate test will be how widely the technique is adopted and what it reveals. The authors declare no competing interests, and the protocol, published as a U.S. Government work, is accompanied by supplementary information including an additional figure and references to support implementation. For a field that has spent more than sixty years refining the operant self-administration paradigm since Weeks first described it in Science, the arrival of a validated head-fixed variant marks a genuine expansion of the frontier. If it delivers on its promise, the coming years should see drug self-administration experiments in which every lever press is paired with a window into the living brain—bringing addiction neuroscience closer to the mechanistic depth that the problem of addiction demands.

Subject of Research: A protocol for performing intravenous drug self-administration in head-fixed mice to study the neural mechanisms of addiction.

Article Title: Head-fixed intravenous drug self-administration: a new frontier in addiction neuroscience

Article References: Negishi, K., D’Ottavio, G., & Shaham, Y. (2026). Head-fixed intravenous drug self-administration: a new frontier in addiction neuroscience. Nature Protocols. https://doi.org/10.1038/s41596-026-01438-7

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01438-7

Keywords: addiction neuroscience, drug self-administration, head-fixed mice, intravenous drug delivery, Nature Protocols, behavioral neuroscience, two-photon imaging, operant conditioning, drug reward, relapse, NIDA, mouse models

That bottleneck may finally be easing. A team led by Wei Zhai, Zhenyu Shi, Rui Tao, Zijian Li, Zhuangchai Lai and Hua Zhang, working across the City University of Hong Kong, The Hong Kong Polytechnic University and partner institutions, has published a detailed protocol in Nature Protocols describing a salt-assisted method that reliably converts ordinary 2H-phase crystals into phase-pure, highly crystalline 1T′-phase materials. The procedure covers a broad compositional palette, including MoS2, MoSe2, WS2, WSe2 and the mixed-chalcogen alloys MoS2xSe2(1−x) and WS2xSe2(1−x), and it does so at a scale that previous approaches could only dream of: gram quantities per batch, with individual crystals reaching hundreds of micrometers across.

The elegance of the method lies in its simplicity. Rather than building 1T′ crystals from scratch under exotic conditions, the protocol starts with commercially available 2H-phase transition metal dichalcogenides and co-anneals them with elemental chalcogen powders, such as sulfur or selenium, together with common alkali metal salts such as potassium oxalate hydrate or potassium carbonate. The reaction is carried out in a sliding tube furnace under a hydrogen-argon atmosphere. When the mixture is heated, the alkali metal salts act as the trigger for a remarkable structural transformation, coaxing the atomic layers of the stable 2H phase to slide and distort into the metastable 1T′ configuration. The chalcogen vapor maintains the correct chemical environment, preventing decomposition while the phase change proceeds.

Understanding why this works requires a brief tour of the underlying crystallography. In the 2H phase, each metal atom sits in a trigonal prismatic coordination environment, and the layers stack in a specific sequence that renders the material a semiconductor. The 1T′ phase, by contrast, features octahedral coordination with a periodic lateral displacement of the metal atoms, breaking the symmetry in a way that produces metallic conductivity. Because the 1T′ arrangement is metastable, it does not form spontaneously under ordinary synthesis conditions; it must be kinetically trapped. Alkali metal ions appear to facilitate this trapping by intercalating between the layers, weakening the interlayer bonding and lowering the energetic barrier for the intralayer atomic rearrangement. Subsequent deintercalation of these ions leaves behind the transformed lattice, now locked into its new metastable geometry.

The published protocol walks researchers through every stage of this process with unusual care. It specifies the setup of the sliding tube furnace, a configuration that allows the reactants to be rapidly moved into and out of the hot zone, giving precise control over reaction timing. It details the loading of the 2H-phase starting materials, the chalcogen powders and the salt, the purging and establishment of the hydrogen-argon atmosphere, the heating profile and the post-reaction washing steps that remove residual salts and recover the transformed crystals. The entire procedure takes approximately 37 hours and 20 minutes from start to finish, a time investment that is modest compared with the weeks of trial and error that often accompany attempts to grow metastable phases by conventional routes.

Equally important is the protocol’s emphasis on verification. Phase purity is not assumed; it is demonstrated. The authors prescribe a characterization workflow built on three complementary techniques. Raman spectroscopy probes the vibrational fingerprints of the lattice, which differ distinctly between the 2H and 1T′ arrangements. X-ray photoelectron spectroscopy examines the chemical states of the metal and chalcogen atoms, confirming that the transformation is complete and that no residual intercalants or decomposition products remain. X-ray diffraction provides the definitive structural verdict, revealing the characteristic peak positions of the 1T′ lattice and the absence of any detectable 2H-phase remnants. Together, these measurements give researchers the confidence that the material they are studying is genuinely the phase they intended to make.

The significance of gram-scale availability is difficult to overstate. Much of the early literature on metastable transition metal dichalcogenides relied on chemically exfoliated nanosheets produced through lithium intercalation, a method dating back to the 1980s that yields small, often defective flakes with variable phase content. Colloidal synthesis routes later offered better control but typically produced nanometer-scale particles rather than the large, well-ordered crystals needed for fundamental physics experiments and device fabrication. Hydrothermal and solution-phase approaches to 1T′ WSe2 and related compounds expanded the toolkit further, yet crystal size and phase purity remained persistent limitations. With crystals hundreds of micrometers wide available in gram batches, experiments that were previously impractical, such as building van der Waals heterostructures from 1T′ layers, performing detailed transport measurements, or loading industrial-scale electrocatalyst electrodes, become routine possibilities.

The applications that stand to benefit span an impressive range. In electrocatalysis, the metallic character of 1T′-phase materials makes them natural candidates for the hydrogen evolution reaction, the cathodic half of water splitting that underpins green hydrogen production. Previous work by members of the same team demonstrated that the phase of the support material dramatically influences how platinum catalysts grow and perform on molybdenum disulfide, achieving highly efficient hydrogen evolution when the metal deposited on metallic-phase surfaces. In electronics, 1T′ phases offer low-resistance contacts to semiconducting 2H layers, a strategy known as phase engineering that has been used to build better transistors. In photonics and sensing, 1T′ monolayers stabilized on gold nanowires have enabled ultrasensitive surface-enhanced Raman scattering detection. And in condensed matter physics, the metallic 1T′ phases of certain dichalcogenides host superconductivity, making high-purity crystals essential for studying emergent quantum phenomena.

The protocol also reflects a maturing view of what its authors call phase engineering of nanomaterials, a discipline that treats crystal phase as a design parameter on par with composition and dimensionality. Over the past decade, reviews in Chemical Reviews and Nature Reviews Chemistry have charted the rapid growth of this field, cataloguing unconventional phases across two-dimensional materials and the synthetic strategies developed to access them. The salt-assisted approach described here joins other recent innovations, including photoredox phase transformation driven by light, as part of a growing arsenal for navigating the energy landscapes of metastable matter. By codifying the method into a reproducible, step-by-step protocol, the team has effectively lowered the barrier to entry for laboratories worldwide.

For the broader materials community, the message is one of cautious optimism. Metastable phases have long been viewed as fragile curiosities, accessible only to specialists with finely tuned equipment and generous patience. A protocol that transforms shelf-stable commercial powders into gram quantities of phase-pure 1T′ crystals using a standard tube furnace and inexpensive salts suggests that these materials are ready to move from the margins of feasibility into the mainstream of research practice. Whether the next breakthrough comes in hydrogen catalysis, quantum devices or entirely unforeseen directions, the raw ingredients are now, quite literally, available by the gram.

Subject of Research: Salt-assisted synthesis of high phase-purity metastable 1T′-phase group VIB transition metal dichalcogenide crystals

Article Title: Salt-assisted synthesis of high phase-purity metastable 1T′-phase group VIB transition metal dichalcogenides

Article References: Zhai, W., Shi, Z., Tao, R., Li, Z., Dong, D., Tang, J., Wang, W., Wang, L., Yang, H., Zhai, L., Ruan, X., Wu, Y., Wang, J., Yao, Y., Zhao, C., He, Q., Lai, Z., & Zhang, H. (2026). Salt-assisted synthesis of high phase-purity metastable 1T′-phase group VIB transition metal dichalcogenides. Nature Protocols. https://doi.org/10.1038/s41596-026-01429-8

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01429-8

Keywords: transition metal dichalcogenides, 1T′ phase, phase engineering, salt-assisted synthesis, metastable materials, two-dimensional materials, hydrogen evolution, electrocatalysis, MoS2, WS2, crystal growth, Nature Protocols

The challenge that the method, called VINE-seq, was designed to solve is fundamentally mechanical. Most single-nucleus sequencing workflows begin by gently breaking brain tissue apart and releasing nuclei from individual cells. That approach works reasonably well for neurons and glia, but vascular cells are a different story. They are encased within a tough basement membrane, a dense meshwork of proteins that resists standard dissociation methods. As a result, when researchers homogenize brain tissue for single-nucleus profiling, vascular cells are systematically lost, depleted, or damaged. The consequence has been a major blind spot in the single-cell atlases of the human brain that now underpin much of modern neuroscience: the very cells that form the blood-brain barrier and regulate cerebral blood flow are dramatically underrepresented in the datasets used to study health and disease.

VINE-seq, short for vessel isolation and nucleus extraction for sequencing, tackles the problem head-on by isolating the vessels themselves before any attempt is made to extract their genetic material. The workflow begins with fresh or frozen brain tissue, either human or mouse, which is homogenized under conditions that preserve vascular integrity. The homogenate is then subjected to dextran-based density-gradient centrifugation, a technique that exploits differences in buoyant density to separate intact vessels from the surrounding myelin and the parenchymal fraction containing neurons and glia. The vessels recovered by this step are predominantly capillaries and small arterioles and venules, roughly 100 micrometers in diameter or smaller, which represent the functional workhorses of the cerebral circulation.

Once the vascular pellet has been collected, the protocol calls for rigorous washing of the isolated vessels over a cell strainer. This deceptively simple step removes trapped contaminants, including parenchymal cells and debris that can cling to the outside of vessel fragments and muddy downstream molecular profiles. The critical innovation, however, comes in the third stage: the optimized extraction of nuclei from the purified vessels using enzymatic digestion, specifically with collagenase III. The authors report that the precise conditions of this digestion step were tuned to liberate intact nuclei from endothelial, mural, and perivascular cells without compromising their quality, a balance that had defeated earlier attempts to profile vascular cells from frozen tissue.

After extraction, the protocol employs fluorescence-activated cell sorting, or FACS, to ensure that only high-purity nuclei proceed to sequencing. This sorting step is what makes the method compatible with the droplet-based platforms that dominate the field, including the widely used 10x Genomics single cell 3′ gene expression assay and the 10x multiome platform, which captures both gene expression and chromatin accessibility from the same nucleus. In parallel with the vascular nuclei, the protocol recovers parenchymal nuclei from the density gradient, meaning that a single dissection can yield both a vascular and a non-vascular view of the same tissue sample. Both fractions are purified by FACS, ensuring compatibility with single-nucleus RNA sequencing, single-nucleus ATAC sequencing, and combined multiomic workflows.

The practical demands of the protocol are modest by the standards of modern genomics. The authors estimate that the full workflow, from homogenized tissue to sorted nuclei ready for sequencing, requires approximately four to five hours to complete. Researchers with training in single-cell techniques and flow cytometry should be able to carry it out, which lowers the barrier to entry considerably compared with bespoke methods that demand specialized instrumentation or rare expertise. The protocol also accommodates both fresh and frozen tissue, a crucial feature for human research, where most brain samples arrive from brain banks as frozen material. Detailed guidance is provided for interpreting quality-control checkpoints along the way, including the use of Agilent BioAnalyzer traces during downstream library preparation, so that laboratories can diagnose and correct problems before committing precious samples to sequencing.

The significance of the method is best understood through the discoveries it has already enabled. VINE-seq formed the technical backbone of a human brain vascular atlas published in Nature in 2022, which revealed diverse mediators of Alzheimer’s disease risk expressed in specific vascular cell populations. Its multiome successor, MultiVINE-seq, extended the approach to paired measurements of gene expression and chromatin accessibility, and underpinned a 2025 study in Neuron demonstrating that human brain vascular multi-omics can elucidate disease-risk associations that transcriptomics alone cannot resolve. By publishing the complete protocol, the authors are effectively handing the wider research community the keys to a molecular map of the cerebrovasculature that was previously accessible only to a handful of specialist laboratories.

The broader implications reach into nearly every corner of neuroscience and neurology. The blood-brain barrier, formed by tightly joined endothelial cells and supported by pericytes and astrocyte endfeet, controls what enters the brain from the bloodstream and is implicated in systemic infection, inflammation, and the failure of countless drug candidates to reach their targets. Perivascular macrophages, which the protocol captures alongside endothelial and mural cells, are increasingly recognized as immunological sentinels with roles in neurodegeneration. Recent work has linked glycocalyx dysregulation to blood-brain barrier failure in aging, identified angiopoietin signaling as a central axis of amyloid-driven vascular dysfunction, and shown that depletion of the RNA-binding protein TDP-43 in endothelial cells disrupts core barrier pathways in neurodegeneration. Each of these lines of inquiry depends on the ability to molecularly profile vascular cells from real human tissue, precisely what VINE-seq and MultiVINE-seq make routine.

The authors have also made the underlying data openly available, with raw sequencing data for VINE-seq deposited in the NCBI Gene Expression Omnibus under accession GSE163577 and MultiVINE-seq data available in the Sequence Read Archive under BioProject PRJNA1182356. For a field that has spent more than half a century developing methods to isolate brain microvessels, dating back to pioneering capillary isolation work in the 1970s, the arrival of a standardized, hours-long workflow that feeds directly into single-nucleus and multiome sequencing marks a genuine inflection point. As laboratories around the world adopt the protocol, the vascular blind spot in brain atlases is likely to shrink rapidly, and with it, the gaps in our understanding of how the brain’s plumbing shapes neurological health, aging, and disease.

Subject of Research: Single-nucleus and multiome sequencing protocols for profiling cells of the brain vasculature

Article Title: VINE-seq and MultiVINE-seq for single-nucleus and multiome profiling of the brain vasculature

Article References: Oberhauser, J., Ding, B., Reid, M. M., Xie, W. H., & Yang, A. C. (2026). VINE-seq and MultiVINE-seq for single-nucleus and multiome profiling of the brain vasculature. Nature Protocols. https://doi.org/10.1038/s41596-026-01434-x

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01434-x

Keywords: VINE-seq, MultiVINE-seq, brain vasculature, single-nucleus sequencing, blood-brain barrier, endothelial cells, pericytes, perivascular macrophages, FACS, Alzheimer's disease, multiome profiling, Nature Protocols

A detailed protocol published in Nature Protocols by Chaojun Zhang, Zhewei Yan, Jing Li, and Mingjie Liu of Beihang University now lays out a practical, step-by-step route around that bottleneck. The method, which the authors call nanosheet superspreading alignment, exploits shear-flow forces generated at the interface between two immiscible phases to drive long-range, high-order alignment of two-dimensional nanosheets. Once the sheets are oriented, in situ interfacial crystallization or cross-linking locks the configuration in place, and subsequent solvent dewetting spreads the material into continuous films over large areas without destroying the carefully engineered microstructure. The full procedure, from precursor preparation through film fabrication and characterization, can be completed in twenty-three days or less.

The physics at the heart of the technique is deceptively simple. When a nanosheet-laden droplet contacts an immiscible phase, it spreads rapidly across the interface, and the resulting flow field subjects the platelets to intense shear. Because nanosheets are extremely anisotropic—atomically thin but laterally large—shear flow torques them until their planes align with the flow direction. The protocol reports an orientation order parameter exceeding 0.85, a figure that indicates a degree of registry approaching the idealized laminated structures of biological materials. Crucially, the alignment is not transient: interfacial crystallization or cross-linking immediately after spreading freezes the oriented configuration before thermal motion or capillary forces can scramble it.

The authors describe two complementary implementation routes. In the first, gelation-assisted superspreading, the nanosheet dispersion spreads across a gel surface where polymerization or gelation locks the aligned sheets into a solid film. In the second, alignment occurs on hydrophilic solid substrates through crystallization-driven confinement, a variant the team highlights as suitable for assembling components of magnetoelectric sensors, where crystalline polymer-inorganic interfaces couple mechanical strain to electrical signals. Both routes share the same core principle—shear first, lock second—and both are compatible with a broad palette of nanosheet chemistries, including graphene oxide, MXenes, transition-metal dichalcogenides, and layered clays.

The mechanical results are striking. Nanocomposite films built from graphene oxide and clay nanosheets reach a tensile strength of up to 1,215 ± 80 megapascals, with a Young’s modulus of 198.8 ± 6.5 gigapascals—figures that place these bioinspired films among the strongest synthetic layered materials reported. Clay-based nanocomposite films achieve a toughness of 36.7 ± 3.0 megajoules per cubic meter, demonstrating that the method does not simply trade ductility for stiffness. In aligned lamellar architectures, load transfers efficiently along the stiff nanosheet planes while the polymer matrix and interlayer interfaces deflect cracks, dissipate energy, and prevent catastrophic failure, echoing the design logic of nacre and mineralized collagen.

What distinguishes this protocol from earlier demonstrations is its explicit bridge between structural precision and scalability. Vacuum filtration produces well-ordered films but only slowly and in limited areas; layer-by-layer assembly offers exquisite control but at impractical throughput for bulk applications; solution casting is fast but yields poorly oriented structures. The superspreading approach sidesteps these trade-offs and, importantly, can be scaled using a multi-nozzle extrusion setup compatible with commercial heating and film-collection components. Schematics in the protocol illustrate how adjacent superspreading droplets coalesce during continuous fabrication, allowing large-area films to form seamlessly while preserving the aligned microstructure across the entire web of material.

The protocol is written as a working laboratory manual rather than a conceptual overview. It covers nanosheet precursor preparation—including considerations for exfoliation quality and dispersion stability—followed by continuous-film fabrication and microstructural characterization. The authors emphasize troubleshooting-oriented detail: controlling spreading kinetics, tuning the viscosity of the immiscible phases, selecting cross-linking chemistries that cure on the timescale of the alignment process, and managing dewetting so that films remain continuous rather than fragmenting into islands. Characterization guidance covers the tools needed to verify orientation order and lamellar spacing, the parameters that ultimately govern mechanical performance.

The versatility of the approach extends well beyond structural mechanics. Because aligned nanosheet films can also serve as membranes, conductors, sensors, and energy-storage components, the protocol positions superspreading alignment as a general platform for functional nanocomposites. Prior work by the same community showed that shear-flow-induced alignment could produce layered nanocomposites with exceptional properties, and more recent studies demonstrated strain-coupled crystalline polymer-inorganic interfaces for efficient magnetoelectric sensing. By codifying those advances into a reproducible procedure, the new protocol lowers the barrier for laboratories worldwide to adopt the technique and adapt it to their own material systems.

The broader significance lies in what scalable, high-order nanosheet alignment makes possible. Lightweight composites approaching the specific strength of advanced structural materials could transform aerospace panels, protective equipment, and flexible electronics. Aligned MXene and graphene oxide films could serve as electromagnetic shielding, thermal management layers, or ion-selective membranes with precisely confined nanochannels. Magnetoelectric composites built on crystalline interfacial coupling could enable ultrasensitive, room-temperature magnetic field sensors for biomedical diagnostics. In each case, the limiting factor has been the same: turning atomically thin, intrinsically strong building blocks into macroscopic materials whose architecture preserves that strength. The superspreading protocol offers a concrete, tested answer, and its publication in a methods journal signals that shear-flow-induced assembly is moving from laboratory curiosity toward a manufacturing-ready tool for the next generation of bioinspired materials.

Subject of Research: Shear-flow-induced alignment of two-dimensional nanosheets for fabricating high-strength bioinspired nanocomposite films

Article Title: Shear-flow-induced assembly of 2D nanosheets for the fabrication of composite films with high tensile strength

Article References: Zhang, C., Yan, Z., Li, J., & Liu, M. (2026). Shear-flow-induced assembly of 2D nanosheets for the fabrication of composite films with high tensile strength. Nature Protocols. https://doi.org/10.1038/s41596-026-01442-x

Image Credits: AI Generated

DOI: 10.1038/s41596-026-01442-x

Keywords: 2D nanosheets, shear flow alignment, superspreading, nanocomposite films, graphene oxide, MXenes, layered clays, tensile strength, bioinspired materials, nacre mimetics, Nature Protocols, materials science