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.