Rapid Cold Fixation of Mouse Colon (Col’RFix) Enables High-Resolution Mass Spectrometry Imaging

Cellular-scale MALDI mass spectrometry imaging (MSI) requires tissue preparation strategies that preserve both tissue morphology and native molecular integrity. Mouse colon poses a particular challenge as fresh-frozen sections are mechanically fragile and frequently lose architectural definition, whereas formalin-fixed paraffin-embedded-based approaches require retrieval steps that can compromise molecular signals. Here, we present Col'RFix (Colon Rapid Fixation), a rapid, liquid-based fixation workflow optimized for transverse mouse colon tissue. Col'RFix employs short-duration cold fixation (4% paraformaldehyde at 5 °C for 10 min) combined with low-melting agarose embedding to provide mechanical support during cryosectioning. Protocol validation assessed morphology preservation, analyte diffusion, molecular coverage, and spatial localization using replicate colon sections from two high-fat diet mice analyzed across multiple MSI runs at 5, 10, and 20 μm pixel sizes, three MALDI-MSI modalities, and in positive and negative ion modes. Feasibility of biological comparison was evaluated in an independent cohort of lean (n = 3) and high-fat diet (n = 3) mice. Col'RFix enabled reproducible imaging down to 5 μm pixel size, supporting spatial lipidomics of fatty acids and multiple lipid classes with minimal delocalization. Compared to unfixed tissue sections sampled within 1 cm of the same region, Col'RFix preserved fine colon morphology, as confirmed by H&E staining, allowing precise correlation of molecular features with histological details. Spectral comparison of fixed and unfixed tissues indicated largely conserved molecular profiles, with compound-dependent signal attenuation. Together, these results establish Col'RFix as a reproducible workflow, enabling compartment-resolved molecular maps of mouse colon at 5 μm pixel size.