In situ GCIB Cryo-Sectioning Enables Subcellular Cryo-ToF-SIMS Imaging of <i>Arabidopsis</i> Seeds

ABSTRACT Time-of-flight secondary ion mass spectrometry (ToF-SIMS) enables label-free molecular imaging at submicrometric resolution, but its application to biological samples remains limited due to sample preparation challenges. Conventional fixation or dehydration alters morphology and induces analyte relocation, while cryo-transfer systems are costly and technically demanding. We present an in situ cryo-etching approach using a gas cluster ion beam (GCIB) and a new sample holder with a flat titanium ridge mask, enabling the sectioning of frozen specimens directly inside the ToF-SIMS instrument. Using Arabidopsis thaliana seeds as a model, this method produced flat, artefact-free surfaces suitable for subcellular imaging without chemical treatment or cryo-transfer. Mass spectra showed intact molecular profiles up to 1000 Da, and ToF-SIMS ion maps revealed preserved tissue architecture and distinct subcellular compartments at a resolution of ∼1 µm. Compared to air-dried cryosections, cryo-etching eliminated structural collapse and analyte delocalization. This workflow provides a practical and accessible route for cryo-ToF-SIMS analysis of hydrated biological materials, combining structural fidelity with molecular integrity. It offers a simple alternative to conventional cryo-transfer methods for high-resolution chemical imaging.