A Single‐Capillary Open Microfluidic Probe for Robust and Versatile Spatial Multi‐Omics Using Ambient Mass Spectrometry Imaging

ABSTRACT

High‐resolution spatial mapping of tissue molecular architecture—from metabolites and lipid isomers to native proteins—remains a central challenge for ambient mass spectrometry imaging (MSI). Existing methods typically rely on multi‐capillary, alignment‐sensitive liquid‐extraction probes, compromising robustness and compatibility with structure‐resolved spatial omics. We mitigate this trade‐off by introducing a monolithic, knot‐shaped open microfluidic probe (Knot‐probe) fabricated from a single capillary. Knot‐probe achieves 40 µm scan‐direction spatial resolution and acquisition throughput up to 0.16 mm s ⁻¹ , yet can be fabricated in ∼30 min with standard laboratory tools and operated reliably for over one month. Without sacrificing conventional spatial lipidomics and metabolomics, Knot‐probe enables structure‐resolved spatial omics, delivering high‐resolution imaging of phosphatidylcholine sn ‐positional isomers via tandem MS and, in a pneumatically‐assisted variant, native MSI of proteins and proteoforms with higher throughput and enhanced sensitivity than with probes lacking pneumatic assistance. These capabilities arise from the integrated knot geometry, which ensures a stable micro‐liquid junction for localized extraction with a minimized sampling footprint and efficient mass transfer, while eliminating intricate probe alignment for robust operation. Readily integrated with standard electrospray ionization MS, Knot‐probe bridges conventional ambient MSI and emerging structural spatial omics, offering a broadly accessible approach across diverse biological and chemical applications.