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

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.