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 research focuses on nuclear factor kappa B, or NFκB, the master transcription factor that activates immune genes in macrophages. Decades of work have established that NFκB does not simply switch on and off. Instead, it shuttles in and out of the nucleus with distinctive temporal patterns — oscillatory for some stimuli, transient for others — and these dynamic signatures, sometimes called signaling codons, carry information about which ligand triggered the response. Six such codons have been characterized: activation speed, peak amplitude, duration of signaling, total integral activity, the balance of early versus late activity, and oscillatory content. Together, they form something like a Morse code of immunity, allowing the cell to distinguish, for example, a cytokine signal from a bacterial lipopolysaccharide encounter.

The problem with most previous studies, the authors note, is that they examined one ligand at a time. Nature rarely cooperates so neatly. During an E. coli infection, a macrophage may first encounter LPS from the bacterial envelope, then CpG DNA from the ruptured genome, and simultaneously inflammatory TNF produced by neighboring cells. In the gut, immune cells confront a rolling storm of microbial and host-derived signals. The combinatorial space of possible ligand mixtures is astronomically large, making comprehensive experimental exploration essentially impossible. The UCLA team’s solution was to build a virtual laboratory: a mechanistic mathematical model of NFκB signaling, comprising 52 ordinary differential equations, 101 reactions, and 133 parameters, organized into five receptor modules corresponding to the TNF, TLR2, TLR4, TLR9, and TLR3 pathways, all converging on a shared IKK–IκBα–NFκB core.

Crucially, the model was not trained on mixture data. It was fitted to live-cell imaging trajectories of hundreds of individual macrophages responding to each of five ligands — TNF, LPS, Pam3CSK4, CpG, and poly(I:C) — at multiple doses. To extend the model into combinatorial territory, the researchers developed an elegant statistical workaround. Because each simulated cell’s parameters had only been estimated for one receptor module plus the shared core, they imputed the missing receptor parameters using nearest-neighbor hot-deck imputation, anchoring each match on the core module parameters that all pathways share. In this way, the team generated virtual cell populations capable of responding to any ligand combination — thirty-one conditions in total, spanning single ligands through the full five-ligand cocktail.

The first validation was encouraging. Experimental responder fractions, measured by live-cell microscopy of fluorescently tagged RelA, rose with the number of ligands present and plateaued much as the model predicted, suggesting that signaling from multiple ligands generally combines in an integrative fashion and that non-responding cells reflect receptor-level heterogeneity rather than cellular ill-health. But one prediction failed in a telling way. The model expected the CpG plus poly(I:C) combination to produce a stronger response than either ligand alone; the experiments showed the opposite, with the pair dampening signaling below the CpG-only level. That discrepancy became the thread the researchers pulled to unravel a previously unknown mechanism.

Both CpG and poly(I:C) are sensed by Toll-like receptors located in endosomes, and both depend on the same endosomal transport machinery to reach those receptors. The original model had treated these transport processes as independent, but reality is stingier: the cell has a finite capacity for hauling cargo into endosomes. By reformulating the transport reactions with competitive Michaelis–Menten kinetics — adding no new parameters — the researchers captured the bottleneck. The result was a negative correlation between CpG–TLR9 and pIC–TLR3 signaling complexes: because poly(I:C) signals relatively weakly, it effectively poisons the stronger CpG response by saturating their shared transport pathway. The revised model accurately reproduced the non-integrative experimental result.

With the model refined, the team asked whether ligand identity remains readable within mixtures. Using Wasserstein distance analysis and machine learning classifiers trained on the six signaling codons, they found that specificity does degrade as ligands pile up — the five-ligand condition was hardest to classify — but it never vanished. Even the five-ligand mixture was recognized well above chance. More strikingly, binary classifiers asked a biologically natural question: is this ligand present in the mixture at all? For TNF, the answer could be read from NFκB dynamics with an ROC area under the curve of 0.94; LPS and Pam followed closely, and experimental data confirmed the pattern. Macrophages, it appears, can partially detect specific threats even in a molecular crowd.

To map synergy and antagonism systematically, the team simulated roughly 360,000 single-cell trajectories across all ten ligand pairs at six doses each. Synergy emerged mainly at low doses and traced back to ultrasensitive IKK activation: in cells with low receptor abundance and constrained TAK1 signaling, two weak inputs could combine to push the ultrasensitive IKK switch over threshold, amplifying NFκB peaks. Antagonism, by contrast, arose at high doses through resource competition. For the LPS–Pam pair, the shared co-receptor CD14 proved to be the limiting resource. Pam binds and releases CD14 more rapidly, peaking early and sequestering the co-receptor before LPS signaling can fully mature, thereby diverting CD14 away from productive TLR4 signaling and weakening the downstream NFκB response.

The most consequential discovery concerned what these interactions do for stimulus-response specificity. When the researchers compared a model lacking CpG–pIC antagonism with the updated model that includes it, the antagonistic version showed significantly greater separability between conditions in Wasserstein distance analyses. Multidimensional scaling visualizations made the point vividly: without competition, the CpG–pIC condition drifted into the cluster of other mixtures, becoming just another blended signal. With competition included, it stood apart, carrying a distinctive dynamic fingerprint. Antagonism, born of a mundane biochemical bottleneck, was actively manufacturing uniqueness. Synergy, though dramatic at the single-cell level, did not confer any similar benefit — it boosted peak responses without improving the distinguishability of the informative signaling codons.

The implications reach well beyond this particular pair of ligands. Resource-limited antagonism — competition for endosomal transport, for co-receptors like CD14, or for shared kinase modules — may represent a general design principle by which immune signaling networks preserve information under combinatorial overload. The computational workflow itself is a contribution: by combining mechanistic ordinary differential equation models with nonlinear mixed-effects parameter estimation and anchor-based statistical matching, the team demonstrated a path toward virtual cell models that retain single-cell heterogeneity while extending predictions far beyond their training data. With all code and data released openly, the framework is ready to absorb additional pathways such as AP1, p38, and the interferon axis, and to explore sequential stimulation over longer timescales. For now, the message is clear: in the immune system’s crowded molecular marketplace, a little rivalry among signals may be exactly what keeps the message intelligible.

Subject of Research: How signaling pathway antagonism shapes macrophage stimulus-response specificity to mixtures of immune ligands

Article Title: Macrophage response specificity to ligand mixtures is improved by signaling pathway antagonism

Article References: Guo, X., Sen, S., Gonzalez, J., & Hoffmann, A. (2026). Macrophage response specificity to ligand mixtures is improved by signaling pathway antagonism. Molecular Systems Biology. https://doi.org/10.1038/s44320-026-00230-9

Image Credits: AI Generated

DOI: 10.1038/s44320-026-00230-9

Keywords: macrophages, NF-kB, signaling dynamics, Toll-like receptors, stimulus-response specificity, ligand mixtures, mathematical modeling, systems biology, innate immunity, antagonism, synergy, single-cell imaging