Barnes, J.-P. | bioRxiv Science

bioRxiv2025

In situ GCIB Cryo-Sectioning Enables Subcellular Cryo-ToF-SIMS Imaging of Arabidopsis Seeds

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 {micro}m. 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.

plant biology↗

bioRxiv2024

Subcellular ToF-SIMS imaging of the snow algae Sanguina nivaloides by combining high mass and high lateral resolution acquisitions

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging has demonstrated great potential for metabolic imaging, yet achieving sufficiently high lateral and mass resolution to reach the organelle scale remains challenging. We have developed an approach by combining ToF-SIMS imaging acquisitions at high lateral resolution (> 150 nm) and high mass resolution (9,000). The data were then merged and processed using multivariate analysis (MVA), allowing for the precise identification and annotation of 85% of the main contributors to the multivariate analysis components at high lateral resolution. Insights into the electron microscopy sample preparation are provided, especially as we reveal that at least three different osmium-containing complexes can be found depending on the specific chemical environment of organelles. In cells of the snow alga Sanguina nivaloides, living in a natural environment limited in nutrients such as phosphorus (P), we were able to map elements and molecules within their subcellular context, allowing for the molecular fingerprinting of organelles at a resolution of 100 nm, as confirmed by correlative electron microscopy. It was thus possible to highlight that S. nivaloides likely absorbed selectively some inorganic P forms provided by P-rich dust deposited on the snow surface. S. nivaloides cells could maintain phosphorylations in the stroma of the chloroplast, consistently with the preservation of photosynthetic activity. The presented method can thus overcome the current limitations of ToF-SIMS for subcellular imaging and contribute to the understanding of key questions such as P homeostasis and other cell physiological processes.

plant biology↗