Samples were transferred to the OrbiSIMS instrument under liquid nitrogen after TEM imaging for molecular depth profiling under cryogenic conditions (-170°C). An Ar3000 + gas cluster source was used both as a primary ion analysis and depth profiling beam. Data was acquired from an ensemble of particles simultaneously over a 400 µm x 400 µm area. Since the lateral resolution of the analysis beam is not sufficient to resolve individual LNPs, this approach aimed to maximise the secondary ion count from the LNPs available from such a limited amount of material. Secondary ions diagnostic of lipids were observed in the first spectrum acquired from each sample, consistent with the view of frozen particles supported by the 10 nm thick amorphous carbon support film, rather than particles embedded in ice (Figure ). Water clusters from the aqueous suspension are not seen in the OrbiSIMS spectra, a phenomenon common across all frozen hydrated analysis, rationalised as the instability of the water clusters prior to Orbitrap analysis. A ToF-SIMS analysis from the surface of the sample before etching (Figure ) shows water clusters, confirming the hydration of the LNPs, consistent with the model of frozen hydrated LNPs sitting on the carbon support, rather than the embedded alternative shown in Figure . Secondary ion assignments were guided by the individual component reference spectra (Figure ), although in the case of this LNP formulation, all observed fragments were unique to one component and definitively linked to the lipid molecular structures.