Scientists Stretch Tissues to Reveal Single-Cell Molecular Maps Without New Hardware

Researchers have unveiled a detailed step-by-step protocol for a technique that physically enlarges tissue samples before imaging them with mass spectrometry, achieving single-cell molecular resolution on instruments found in laboratories around the world. The method, known as tissue expansion mass spectrometry imaging, or TEMI, sidesteps the need for expensive hardware upgrades by making the sample bigger rather than making the instrument’s laser smaller. Published in Nature Protocols, the guide distills years of development into a practical workflow that any reasonably equipped mass spectrometry imaging lab can follow.

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