Bio-analysis by SIMS is not new. There are nice examples in the literature dating back 50 years!

However, that does not mean there has not been significant progress that has expanded our capabilities for extracting information from these impressively complicated samples.

This presentation details some of the journey that bio-SIMS has made and looks forward to exciting prospects, and challenges, that lie ahead.

Environmental stresses such as nutrient restriction or hypoxia can lead to fetal growth restriction. It is well established, however, that growth decreases less in the CNS than in other organs, an effect known as brain sparing (Gruenwald, 1963 PMID: 14081642). The molecular mechanisms underlying brain sparing are not yet fully understood. We previously demonstrated that the Drosophila larval brain recapitulates may of the features of mammalian brain sparing (Cheng et al., 2011 PMID: 21816278; Bailey et al., 2015 PMID: 26451484; Lanet et al., 2013 PMID:23478023). To investigate how brain sparing changes metabolism in the larval brain, we have been developing ambient temperature and cryogenic workflows for mass spectrometry imaging using our recently developed OrbiSIMS instrument (Parsarelli et al., 2017 PMID: 29131162; Newell et al., 2020 PMID: 32603009). OrbiSIMS provides high lateral and mass resolution simultaneously, enabling metabolite imaging at a near-cellular level. In this study, we use OrbiSIMS to map the localizations of more than 100 polar and apolar metabolites in the larval CNS. We also conduct a spatial lipidomics survey of the effects of environmental stresses upon larval CNS metabolism.

OrbiSIMS combines high-resolution imaging using a focused gas cluster ion beam with an Orbitrap mass spectrometer to enable sub-cellular resolution imaging with high mass-resolving power^[1]. The ability to perform cryogenic measurements is valuable, as it enables imaging in the native state^[2,3] and of volatile molecules in ultra-high vacuum^[2].

High-resolution imaging creates the challenge of detecting sufficient ions within a pixel. To enable improvements in spatial imaging capability, the secondary ion yield must improve concomitantly. Previous studies have shown improved ion yield using water cluster beams^[4] or hydrated samples^{[3, 5]}. Additionally, it has been shown that the positive ion yield decreases with lipophilicity (Log P) of a compound^[6], thus degrading the detection sensitivity for polar tumour metabolites as well as drugs designed for improved solubility.

Here, we present targeted and untargeted assessments of the secondary ion yield enhancement of a range of endogenous biomolecules within frozen-hydrated mouse liver tissue when compared to in situ freeze-dried tissue. To ensure equivalence of molar amounts the secondary ion signal was integrated over a fixed area for the entire thickness of tissue. In positive polarity, we show an enhancement up to three orders of magnitude, and the relative enhancement increases for polar molecules (low Log P). In negative polarity, no enhancement is observed supporting the hypothesis that increased protonation aids ionisation. We provide an assessment of the secondary ion yield enhancement with respect to classes of biomolecule and to mass.

We demonstrate the benefits of cryo imaging for biological tissues in terms of signal and structural integrity. This supports future work to improve OrbiSIMS spatial resolution.

[1] M. K. Passarelli, A. Pirkl, R. Moellers, D. Grinfeld, F. Kollmer, R. Havelund, C. F. Newman, P. S. Marshall, H. Arlinghaus, M. R. Alexander, A. West, S. Horning, E. Niehuis, A. Makarov, C. T. Dollery, I. S. Gilmore, Nat Methods 2017, 14, 1175-+.

[2] C. L. Newell, J. L. Vorng, J. I. MacRae, I. S. Gilmore, A. P. Gould, Angew Chem Int Edit 2020, 59, 18194-18200;

[3] J. T. Zhang, J. Brown, D. J. Scurr, A. Bullen, K. MacLellan-Gibson, P. Williams, M. R. Alexander, K. R. Hardie, I. S. Gilmore, P. D. Rakowska, Anal Chem 2020, 92, 9008-9015.

[4] K. D. Nilsson, A. Karagianni, I. Kaya, M. Henricsson, J. S. Fletcher, Anal Bioanal Chem 2021, 413, 4181-4194.

[5] X. A. Conlan, N. P. Lockyer, J. C. Vickerman, Rapid Commun Mass Sp 2006, 20, 1327-1334.

[6] J. L. Vorng, A. M. Kotowska, M. K. Passarelli, A. West, P. S. Marshall, R. Havelund, M. P. Seah, C. T. Dollery, P. D. Rakowska, I. S. Gilmore, Anal Chem 2016, 88, 11028-11036.

The estimated size of the global agrochemicals market in 2022 amounted to USD 227.9 billion with a projected increase to USD 234.27 billion in 2023 [Market Analysis Report 2018-2022]. The notable increase in agrochemical usage observed worldwide can be attributed to the considerable economic benefits that accrue to farmers through the safeguarding of crops against invasive species, including the improvement in quality and quantity of harvests. However, limited knowledge of the in-situ chemical composition of wheat leaves and the permeation mechanisms of pesticides into skin and leaf tissues restricts research and development of new products.

The 3D OrbiSIMS has been recently demonstrated as a powerful tool for skin research, providing label-free insight into the 3D permeation profiles of endogenous and exogenous compounds [Starr et. al., 2022] characterizing the molecular composition of the stratum corneum and tracking the permeation of a Pal-GHK peptide. Building upon these advancements, our study extends the application of 3D OrbiSIMS to explore the native chemistry of wheat leaves, with a focus on the plant cuticle as the primary diffusion barrier. The study also illustrates the distribution of a fungicide formulation across wheat leaves and skin, providing insights into the formulation's diffusion in two relevant biological matrices.

The molecular architecture of wheat leaves was first probed, with a focus on the cuticle. In-situ analysis provided novel insights into the localisation of endogenous species, including fatty acids, amino acids, phospholipids, flavones and vitamins. Depth profiling revealed non-homogeneity of the leaf as a function of depth where the distribution profiles of fatty acids and aldehydes associated with the cuticle and epicuticular waxes displayed a pronounced abundance at the surface of the leaf. Conversely, flavones and vitamins were predominantly localised in the epidermis.

Exogenous compounds were successfully identified in both skin and wheat leaves, in addition to the endogenous species. The investigation focused on evaluating the impact of exposure time and concentration on the permeation of a fungicide formulation across skin and wheat leaves. Utilizing in situ analysis, the entire formulation was concurrently detected and tracked, even at 100 ppm. The active, cyproconazol, displayed increased permeation with prolonged exposure time, and higher concentrations resulted in a higher relative quantity in both matrices. Co-formulants showed diverse localization patterns, with carrier solvents resembling the permeation of the active ingredient, while emulsifiers remained primarily at the surface, as expected.

Our findings highlight the 3D OrbiSIMS' potential to detect native and exogenously delivered chemistries in skin and leaf samples, enabling comprehensive observation of all components of the investigated formulation. The molecular elucidation and permeation insights obtained from this approach could be implemented in designing agrochemical formulations with targeted delivery and reduced associated issues.

Various techniques are available to measure mechanical properties such as material hardness, for example, nanoindentation. However, certain materials or structures provide challenges to measuring actual hardness, such as when an underlying material is much softer than the one above it (e.g., an ice cube sitting on water).

Water Cluster SIMS is a technique with the potential to simultaneously obtain information on both molecular and mechanical properties of materials. Since water clusters are more robust than other popular clusters such as Ar clusters, it has been observed in Water Cluster SIMS spectra that water cluster ions colliding with a surface dissociate into smaller ions with the formula [(H₂O)_n+H]⁺ or [(H₂O)_n+OH]^-, where 2 ≤ n.

The ion intensity ratios for the ion yield (e.g. [(H₂O)₂+H]⁺/ Σ[(H₂O)_n+H]⁺) are highly sensitive to the mechanical properties of materials. The intensity ratio called ‘dissociation ratio’ appears to depend on the surface's mechanical properties and the energy of the ion beam. In other words, we see high signals of back-scattered water cluster ions at a low ion beam energy for the dissociation ratio as well as enhanced sensitivities of high-mass compounds.

A previous study demonstrated a good relationship between Young's modulus and the dissociation ratio using observed ion intensities on substrates including metals and polymers. The ability to measure the mechanical properties of a surface in situ whilst performing SIMS measurements would be beneficial for materials where other measurements have failed. We examine whether the use of water cluster SIMS may also be used to measure rigidity on more complex biological structures and whether the parameters of the cluster should be similar to those used for harder materials.