OrbiSIMS spatially resolves isomeric molecules on surfaces

OrbiSIMS spatially resolves isomeric molecules on surfaces | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article OrbiSIMS spatially resolves isomeric molecules on surfaces Gustavo Trindade, Richard Paterson, Bin Yan, Jean-Luc Vorng, Alexander Pirkl, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6558145/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Orbitrap secondary ion mass spectrometry (OrbiSIMS) is growing in popularity in a wide range of applications in life sciences and materials science, with the ability to provide high confidence in molecular identification simultaneously with high confidence in localisation (spatially at the microscale and at the nanoscale within a layer). In OrbiSIMS, transmission of secondary ions depends on two key parameters, the target voltage and helium gas pressure in a cooling cell. Here, we show that controlling these parameters gives OrbiSIMS the capability to couple phenomena that enable the separation of molecules based on their dissociation patterns and transmission after gas-phase collisions, which are related to molecular structure. We show that these phenomena can be leveraged and used analytically to take OrbiSIMS beyond the limitations of the mass scale and spatially resolve the signal of mixtures of structural isomeric trisaccharides (raffinose, maltotriose and melezitose). We benchmarked our results with ion mobility spectrometry and showed a correspondence between the methods to distinguish structural isomers. The ability to spatially resolve isomeric molecules in mixtures and on surfaces using OrbiSIMS can be transformative as these types of molecules play important biological roles. Physical sciences/Chemistry/Analytical chemistry/Mass spectrometry Biological sciences/Chemical biology/Small molecules Physical sciences/Nanoscience and technology/Techniques and instrumentation/Mass spectrometry Physical sciences/Physics/Techniques and instrumentation/Characterization and analytical techniques Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Mass spectrometry imaging (MSI) has been a transformative suite of methods over the past 50 years with current technology being able to detect and spatially map various kinds of molecules in parallel and without the use of labels. However, MSI is only able to detect mass-to-charge ratios ( m/z ), which carries no direct information of a molecules higher order structure. Indirect information of structure in MSI can be inferred from charge state detected in desorption electrospray ionisation (DESI). 1 , 2 When no accurate spatial information is needed and samples can be prepared in solution, molecular structure can be inferred by coupling MS with liquid chromatography for physical separation 3 or by adding additional reaction steps for fragmenting precursor molecules (tandem MS also known as MS/MS 4,5 ) and infer structural information from fragmentation patterns of molecules in collision-induced, surface-induced dissociation or hydrogen-deuterium exchange. 6 – 8 Ion mobility spectrometry (IMS) is another established method to study molecular structure, where ions of the same m/z but different shapes are separated depending on how they transit a gas-filled collision cell. 9 IMS is suitable to be coupled with the imaging capability of MSI as previously shown with matrix-assisted laser desorption/ionization (MALDI), DESI and liquid extraction surface analysis (LESA) sources, allowing parallel imaging and structural information. 10 – 15 MALDI and on-tissue chemical derivatisation have also been used to separate isomers using MSI. 16 Secondary ion mass spectrometry (SIMS) is an MSI method based on the detection of ionised species resulting from the impact of a primary ion beam on a sample surface, with the ability to map in three dimensions. 17 Over the last decade, SIMS has had significant impact for biological imaging, with constant developments towards improved ion beams and analysers for biomolecules, 18 , 19 higher spatial resolution, better accuracy in molecular identification and preserving the native state with cryogenic analysis. Orbitrap SIMS (OrbiSIMS) is a recent technique that is growing in popularity in a wide range of applications in life sciences and materials science. 20 An OrbiSIMS (also known as Hybrid SIMS) instrument features a dual analyser configuration with a time-of-flight (ToF) analyser and an Orbitrap™ analyser (Fig. 1 a). As a consequence this instrument has the ability to combine the mass spectrometry performance usually found in state-of-the-art proteomics and metabolomics with fast 3D imaging at the microscale and from nanolayers of less than 1 nm of material, which enables molecules to be studied directly from surfaces and tissues, unlocking new capability in important applications such as proteomics 21 , 22 , lipidomics 23 – 25 , metabolomics, 26 – 30 and drug delivery. 29 Secondary ion emission is a heavily fragmenting process, therefore fragmentation patterns can be used to differentiate signals of structural isomers but may carry a lot of ambiguities in complex samples. Recently, fragmentation patterns in SIMS have been combined with the high accuracy of the Orbitrap analyser to infer structure of proteins, 21 , 22 biomarkers 31 and nucleotides. 32 However, there are no established methods using a SIMS platform for the direct detection, separation and mapping of structural isomers on solid surfaces and interfaces (via 3D analysis). In OrbiSIMS, for secondary ions to be efficiently transferred to the Orbitrap analyser, their potential energy is matched to the Orbitrap acceptance window by biasing the sample with a target potential (V T ) and their kinetic energy distribution from the SIMS collision process is reduced through elastic collisions with a helium gas at a pressure P He (Fig. 1 a). We previously conducted a systematic assessment of V T and P He on the transmitted secondary ion intensities of inorganic and organic molecules and revealed interesting patterns, indicating the possibility for additional information from secondary ion molecules. 33 Here, we explore OrbiSIMS V T spectra and use them analytically to detect and spatially resolve the signal of mixtures of structural isomeric trisacharides. Identifying and separating molecules by their higher order structure, or shape, combined with their spatial localisation can bring new biological insight, for example understanding the function of glycans in cell walls, 34 unambiguous determination of lipids distribution within a tissue 35 and better understanding of protein and nucleotide function. Results Varying target potential selects dissociated ions We acquired OrbiSIMS mass spectra of a reference silver sample with a scanned V T potential (Fig. 1 b) to produce V T spectra of various clusters (Fig. 1 c). Experimental details are in Supplementary Note 1 . The standard target potential for OrbiSIMS analysis of conducting materials is V T1 = 57.5 V which is suitable to detect all stable Ag n + clusters from a reference silver sample. 33 However, if V T values are increased up to 400 V, a more complex distribution pattern is revealed in the silver cluster intensities, with changing maxima in transmission which vary with cluster size (Fig. 1 b-c and Supplementary Figs. 2.1-2 ). The V T values for the multiple maxima are inversely proportional to m/z (Fig. 1 d) in agreement with our model ( Supplementary Note 2) that suggests Ag n + clusters are dissociating into smaller clusters at some point before they are decelerated to enter the collision cell and the ones that dissociate in an electric field-free region are successfully transmitted to the Orbitrap. For singly charged molecules ( z = 1), Eq. 1 predicts the position of all maxima, V T2 , for daughter ions with mass m 2 where the maximum for the parent ion with mass m 1 = m 2 + Δ is given by V T1 : $$\:{V}_{T2}^{max}=\left({V}_{T1}+\:{V}_{E}\right)\:\frac{{m}_{2}}{{(m}_{2}+\varDelta\:)}-{V}_{E}$$ 1 Where V E is the extraction potential. For silver clusters, Δ can only be multiples of the masses of the two Ag isotopes ( 107 Ag and 109 Ag) and V T1 is the optimised potential of 57.5 V. This is sufficient to estimate all possible dissociations and the experimental data has great agreement with the model (Fig. 1 d and Supplementary Figs. 2.1-2 ). To aid the interpretation of the results on secondary ion dissociation, we established an ion optics simulation model (Fig. 1 e-g, details in Supplementary Note 3 ). The number of simulated Ag 11 + ions transmitted to the Orbitrap as a function of V T shows good agreement with the experimental data for the same secondary ions (Fig. 1 f). The small differences are likely due to second-order effects such as kinetic energy of formation, decays inside electric field gradients (which may also lead to background signal in V T spectra) and sub-optimal transmission and restrictions on the simulations. 36 , 37 This shall be investigated in future work. To assess whether the model can predict dissociation of more complex organic molecules, we have acquired V T spectra of a cytochrome C sample (Fig. 2 a experimental details in Supplementary Note 1 ). The OrbiSIMS mass spectrum of cytochrome C in the m/z range between 100 and 700 has several peptide fragments as well as multiple peaks of higher intensity relating to the heme C molecule (Fig. 2 b). Many of these ions will have more than one peak in their V T spectra and by identifying the voltage for those maxima, the m/z of parent ions and neutral losses can be estimated using Eq. 1 (Fig. 2 c and Supplementary Fig. 2.4 ). For the heme C molecule, the predicted losses are multiples of HS, CH, CH 2 , CH 3 and COOH, which based on their putative assignments (Fig. 2 c), all relate to dissociation of the functional side chains on the four pyrrole rings. Molecules with different structures may have different side chain stability and present different V T spectra. Therefore, the fact that we can separate dissociated fragments in V T spectra, together with our previous observations on the dependency transmission of secondary ions on V T and the collision cell pressure, 33 reveal that there is potential for the study of further molecular properties using OrbiSIMS that extend beyond the m/z scale to separate molecules based on their structure. Structural isomers on surfaces To investigate the potential for OrbiSIMS to separate molecules by shape using V T spectra, we studied three trisaccharide structural isomers, raffinose, maltotriose and melezitose. These have the same molecular formula C 18 H 32 O 16 and are detected as the [M-H] − ion but have different structures (Fig. 3 a). Thin films of each sugar were produced on top of a silicon wafer using a spin coater to avoid electric charge build up during the analysis and to allow for the preparation of mixtures (further details in Supplementary Note 1 ). The mass spectrum intensity of the [M-H] − is not useful to distinguish between these structural isomers. V T spectra of the [M-H] − ion at standard He cell pressure (0.04 mbar) for organic materials for each sample show that optimal transmission occurs at approximately − 30 V, however, both raffinose and melezitose show multiple maxima in their V T spectra with an oscillating pattern with clear peaks at -207 V, -262 V, -320 V, -375 V and − 435 V, which have a mean difference of 59 V. Those features are absent for maltotriose, which enables separation (Fig. 3 b). Using Eq. 1 , we predicted the unit masses of potential parent ions for the [M-H] − detected at the multiple maxima of the V T spectra. These were then used to identify peaks in the experimental mass spectra and are listed in Table 1 with their putative assignments. Table 1 Predicted unit masses of potential “parent ions” for the [M-H] - detected at the multiple maxima of V T spectra of raffinose and melezitose and identified peaks in the experimental mass spectra. V T of maximum (V) Calculated parent unit mass m/z in spectrum Molecular formula Putative assignment deviation (ppm) -30 - 503.1617 C 18 H 31 O 16 [M-H] − -0.2 -207 547 547.1515 C 19 H 31 O 18 [M - H + CO 2 ] − -0.1 -262 561 561.1610 C 20 H 33 O 18 [M - H + C 2 H 2 O 2 ] − -0.4 -320 575 575.1828 C 21 H 35 O 18 [M - H + C 3 H 4 O 2 ] − -0.1 -375 589 589.1985 C 22 H 37 O 18 [M - H + C 4 H 6 O 2 ] − -0.1 -435 603 603.2141 C 23 H 39 O 18 [M - H + C 5 H 8 O 2 ] − -0.2 The potential parent ions in Table 1 have masses higher than the molecular ion of the sugars. This indicates that they are weakly bonded to impurities in the sample, or generated via recombination during the sputtering process, or characteristic of its crystal structure. This shows that a V T spectrum has the potential to be informative on structure at molecular level but also at a crystal level. The identified potential parent ions are all detected as secondary ions and their V T spectra show the simple behaviour with a single maximum at the standard – 30 V ( Supplementary Fig. 2.5 ). This indicates that they are directly formed in the secondary ion emission process and not from metastable decay of a larger entity such as a dimer. In addition to the V T spectra data, we also investigated the effect of the collision cell He pressure, P He , with V T set at the two values where maxima occur, -30 V and − 207 V. The intensity of the quasi-molecular ion [M-H] − has different optimal P He for transmission for each of the isomers (Fig. 3 d-e). This is because the collision cross-section depends on the shape of the molecule. We further expanded our ion optics simulation framework to assess transmission of secondary ions after elastic collisions with a He atom with a given collision cross section ( Supplementary Note 3 ). We benchmarked our OrbiSIMS results with ion mobility mass spectrometry (IMS) of solutions of each compound (Fig. 3 c). The IMS peak drift times of the structural isomers are inversely proportional to their respective optimal P He for OrbiSIMS transmission, the lower the pressure for optimal transmission in OrbiSIMS, the higher the drift time in IMS (Fig. 3 f). This is generally as expected since a lower P He is used for molecules with a larger collision cross-section and these are consequently slower travelling through the IMS drift region. The OrbiSIMS instrument also has a bent flatapole filled with N 2 gas that can vary with the He pressure. 20 Therefore, to confirm that this behaviour is ruled by He rather than N 2 , we have repeated experiments allowing only He to vary ( Supplementary Fig. 2.6 ). In all three methods (V T spectra, varying P He and IMS) a more distinct separation is found for maltotriose with raffinose and melezitose being more similar. This is in agreement with their V T spectra, molecular structure and collisional cross sections. 38 Spatially distinguishing structural isomers in mixtures, on surfaces To assess whether the signal from isomers could be resolved and be relatively quantified in a mixture, we prepared thin film samples of 1:1 and 2:1 mixtures of raffinose and maltotriose and performed V T and P He scans (at V T = -26V and − 220 V) using the same conditions as before (Fig. 4 a-b). At -220 V, the optimal P He for transmission of the [M-H] − ion varies approximately linearly with the % of raffinose in the mixture. Whereas at -26 V, the P He initially increases linearly but after 60% drops (Fig. 4 c). The full mass spectra for each V T and P He value were merged into a single data matrix and processed with unsupervised machine learning or multivariate statistical analysis methods, such as principal component analysis (PCA), to find trends that are characteristic of each isomer 39 – 41 (details in Supplementary Note 4 ). PCA reveals distinguishing behaviour in the V T spectra of specific secondary ions such as C 6 H 11 O 6 − (galactose/fructose) and C 14 H 23 O 2 − ( Supplementary Fig. 4.1 ). In the V T spectra of mixtures, the oscillating features of the [M-H] − ion that are characteristic of raffinose are present, as well as the distinguishing maxima of C 14 H 23 O 12 − and C 6 H 11 O 6 − at -340 V (Fig. 4 d-e). Those features can be used to check whether the signal from the mixtures shows a linear proportion to the amount of each compound. Figure 4 g shows the V T spectrum for C 6 H 11 O 6 − in the range of -250 V to -450 V for pure raffinose and maltotriose revealing two distinct peaks for raffinose and one for maltotriose. These peak shapes are then used to fit to the V T spectra for the two mixtures (Fig. 4 g). For each V T spectrum, a linear background was removed prior to fitting. Peak fitting to each mixture’s V T spectrum was conducted by fixing the V T peak centres and peak widths and allowing the peak areas to vary. Qualitatively, the mixtures present features from both isomers but the areas of the fitted peaks do not have a linear correspondence to the proportions in the mixture. This is likely because the two compounds phase separate within the thin film, evidenced by the optical images of the films acquired using a micro camera in the instrument that show a much more homogenous film thickness (based on reflected light) for the pure samples (Fig. 4 i-k). OrbiSIMS imaging of the [M-H] − (C 18 H 31 O 3 − ) and C 14 H 23 O 12 − show that the two monomers can be spatially resolved using OrbiSIMS (Fig. 4 i) even at V T = -30 V, whereas analysis using the ToF analyser (used in the same instrument) show no distinction between the domains for either of the secondary ions. We further confirmed this with imaging data of pure raffinose (Fig. 4 j) and maltotriose (Fig. 4 k) that show homogeneous signal distribution. Discussion Controlling V T and P He give OrbiSIMS capability that couples phenomena used in IMS (separation of molecules based on collisions with a gas) and PSD (post-source dissociation of molecules with patterns that relate to their structure) methods, with the added capability of imaging (spatially resolve phase separation, depth profiling, localisation in complex biological systems) and direct detection on surfaces. We developed a simple ion dissociation model to understand the effects and established an ion optics simulation framework that can aid the planning of future experiments. More work needs to be done to fully understand why and how molecules fragment and the dependence on V T . We show that the transmission characteristics in OrbiSIMS can be leveraged and used analytically to resolve structurally isomeric trisaccharides raffinose, maltotriose and melozitose that cannot be separated by mass alone. To further validate the method, we benchmarked our results with ion mobility spectrometry (IMS) demonstrating a good correlation between the He collision cell pressure, P He , and the ion mobility drift time. We demonstrate the OrbiSIMS capability to spatially separate isomers within a mixture of raffinose and maltotriose in a thin film, whereas with ToF-SIMS the distribution appears homogenous. This shows promise for high spatial resolution imaging of polysaccharides. The ability to spatially resolve polysaccharides in mixtures and on surfaces and nanoscale interfaces can be transformative as these types of molecules play important biological roles; acting as building blocks of macromolecules, composing cell walls and carrying information for proteins. Furthermore, we expect the method can be applied for direct imaging of biomolecules that may have isomeric ambiguity such as lipids and nucleotides. Since OrbiSIMS allows direct analysis of molecules at nanoscale interfaces, for example in organic electronics 40 and biofilms 42 , this additional separation dimension provides an exciting new capability for a wide range of science and technology applications. Declarations Acknowledgements This work was funded by the UK National Measurement System (NMS). NPL is operated by NPL Management Ltd, a wholly-owned company of the UK Department of Science, Innovation and Technology (DSIT). The authors would like to thank Dr Alexander Shard (NPL) for insightful discussion about ion dissociation. Authors contributions All authors contributed to the manuscript and approved the final manuscript. The concept was devised by GFT, AP and ISG. GFT and RP acquired OrbiSIMS experimental data with advice from AP and ISG. RP and GFT developed the ion optics simulation framework. GFT, AP and ISG processed and interpreted OrbiSIMS data. BY proposed the trisaccharide isomer samples, acquired and interpreted IMS data and prepared the cytochrome C sample for OrbiSIMS. JLV and GFT prepared spin-coated samples. GFT oversaw the study. Data availability All OrbiSIMS data reported in this manuscript have been deposited as imzML files to figshare ( DOI to be provided ). The IMS data reported in this manuscript have been deposited as an xlsx file to figshare ( DOI to be provided ). imzML files can be processed in MATLAB using the imzML parser of SpectralAnalysis (https://github.com/AlanRace/SpectralAnalysis). Code availability This manuscript did not require the development of code that is central to the main work described. Conflicts of interests The authors declare the following competing financial interest: AP is an employee of IONTOF GmbH, the manufacturer of the OrbiSIMS instrument used in this research. References Hale OJ, Cooper HJ (2021) Native Mass Spectrometry Imaging of Proteins and Protein Complexes by Nano-DESI. Anal Chem 93:4619–4627 Yang M et al (2023) Nano-DESI Mass Spectrometry Imaging of Proteoforms in Biological Tissues with High Spatial Resolution. Anal Chem 95:5214–5222 Holčapek M, Jirásko R, Lísa M (2012) Recent developments in liquid chromatography–mass spectrometry and related techniques. 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Anal Chem 90 Trindade GF et al (2023) Direct identification of interfacial degradation in blue OLEDs using nanoscale chemical depth profiling. Nat Commun 14:1–9 Keenan MR, Trindade GF, Pirkl A, Newell CL (2024) Orbitrap noise structure and method for noise-unbiased multivariate analysis. Pre-print Kotowska AM et al (2023) Toward Comprehensive Analysis of the 3D Chemistry of Pseudomonas aeruginosa Biofilms. Anal Chem 95 Additional Declarations Yes there is potential Competing Interest. The authors declare the following competing financial interest: Alexander Pirkl is an employee of IONTOF GmbH, the manufacturer of the OrbiSIMS instrument used in this research. Supplementary Files SupportingInformationOrbiSIMSisomersv01.pdf Supplementary Information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6558145","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":455240147,"identity":"bf13dc6c-d0d3-4593-9314-a57fc5f2f140","order_by":0,"name":"Gustavo Trindade","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYHADxgYGHgZmBn4g6wBYgAefUrA8VIskkEusFjDBzGBwgIAW+Yj05w9+ttkw2EsfbnvwpsZazvh274EDDDU2DAZnDmDVYngjx7Cxty2NgYcvsd1wzrF0Y7M75xIOMBxLYzA424Bdy4wcxgaeM4cZeHgY26R52A4nbruRY3CAseEwg8F57A4znJH+sPEPXMu/w/WbZxDQIi+RYNjMUwHVwtt2OMFAAqYFh8MMeN4YzpapSOPhOcPYJjm3L91wxo28hAMJx9J4JHF4X749/cHHNwY2cuw97M8k3nyzluefkXvwwYcaGzm+MwnYbYGahBwHQHYCnoiUx+Je3LE+CkbBKBgFIxMAALAYXkBDTK2jAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-6998-814X","institution":"National Physical Laboratory","correspondingAuthor":true,"prefix":"","firstName":"Gustavo","middleName":"","lastName":"Trindade","suffix":""},{"id":455240148,"identity":"6b841ad6-5943-4a2b-ae93-54747e3b6b0d","order_by":1,"name":"Richard Paterson","email":"","orcid":"","institution":"National Physical Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Paterson","suffix":""},{"id":455240149,"identity":"94527c9c-75ea-478c-9a6c-08b64cb87676","order_by":2,"name":"Bin Yan","email":"","orcid":"https://orcid.org/0000-0002-7562-9562","institution":"National Physical Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Yan","suffix":""},{"id":455240150,"identity":"89f76f69-8aea-489e-a805-4cefa9dacfee","order_by":3,"name":"Jean-Luc Vorng","email":"","orcid":"","institution":"National Physical Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Jean-Luc","middleName":"","lastName":"Vorng","suffix":""},{"id":455240151,"identity":"e70f929b-c6ac-4905-a14e-57441f08ac70","order_by":4,"name":"Alexander Pirkl","email":"","orcid":"","institution":"ION-TOF","correspondingAuthor":false,"prefix":"","firstName":"Alexander","middleName":"","lastName":"Pirkl","suffix":""},{"id":455240152,"identity":"cc5137ed-7f7f-4d47-a055-1269bc8e543d","order_by":5,"name":"Ian Gilmore","email":"","orcid":"https://orcid.org/0000-0002-0981-2318","institution":"National Physical Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Ian","middleName":"","lastName":"Gilmore","suffix":""}],"badges":[],"createdAt":"2025-04-29 16:00:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6558145/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6558145/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82672121,"identity":"b963b833-f05e-49a3-b620-28d4bc2987b5","added_by":"auto","created_at":"2025-05-14 03:10:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":527414,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVarying target potential in OrbiSIMS selects dissociated ions. a \u003c/strong\u003eSchematic of instrument showing key parameters V\u003csub\u003eT\u003c/sub\u003e and P\u003csub\u003eHe. \u003c/sub\u003e\u003cstrong\u003eb \u003c/strong\u003eMass spectra of reference silver sample at 4 different values of V\u003csub\u003eT\u003c/sub\u003e. \u003cstrong\u003ec \u003c/strong\u003eV\u003csub\u003eT\u003c/sub\u003e spectra of reference silver showing signal for clusters sized 1 to 11. \u003cstrong\u003ed\u003c/strong\u003e V\u003csub\u003eT\u003c/sub\u003e values for maximum transmission in function of \u003cem\u003em/z \u003c/em\u003efor clusters sized up to 33. Dashed lines represent estimated values based on model of dissociation in a field-free region. \u003cstrong\u003ee \u003c/strong\u003eIon optics simulation model with example dissociation of Ag\u003csub\u003e13\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e ions before they enter the 90 degrees deflector. \u003cstrong\u003ef \u003c/strong\u003eOverlay of a measured (blue), calculated (dashed green) and simulated V\u003csub\u003eT\u003c/sub\u003e spectra for Ag\u003csub\u003e11\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e. \u003cstrong\u003eg \u003c/strong\u003eIon optics simulation model with example dissociation of Ag\u003csub\u003e13\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e ions for V\u003csub\u003eT\u003c/sub\u003e values of 57.5 V, 230 V and 440 V.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6558145/v1/66731e9571636ddf8818ef99.jpg"},{"id":82671372,"identity":"369da739-2994-4b58-a617-dcc7728bbe8d","added_by":"auto","created_at":"2025-05-14 03:02:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":464786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eV\u003c/strong\u003e\u003c/em\u003e\u003csub\u003e\u003cem\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/em\u003e\u003c/sub\u003e\u003cstrong\u003e spectra show dissociation of secondary ions of heme C molecule in cytochrome C. a \u003c/strong\u003eStructure of cytochrome C and heme C\u003cstrong\u003e \u003c/strong\u003e(Wikipedia.org).\u003cstrong\u003e b \u003c/strong\u003eOrbiSIMS mass spectrum showing peptide fragments and heme C fragments (intensity scale between \u003cem\u003em/z\u003c/em\u003e 100 and 300 is multiplied by 25 for better visualisation.\u003cstrong\u003e c \u003c/strong\u003ePrediction of dissociation of heme C secondary ions.\u003cstrong\u003e d \u003c/strong\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e\u003csub\u003e \u003c/sub\u003espectra of 12 heme C secondary ions.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6558145/v1/ccd96af2418a71f1333d4896.jpg"},{"id":82671075,"identity":"eaae9a3b-a76d-4480-9543-8b532e7ea20f","added_by":"auto","created_at":"2025-05-14 02:54:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":341707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUnambiguous direct detection of structural isomeric trisaccharides. a OrbiSIMS \u003c/strong\u003emass spectra of raffinose, maltotriose and melezitose with inset showing de-protonated quasi-molecular ion [M-H]\u003csup\u003e-\u003c/sup\u003e for each compound. \u003cstrong\u003eb \u003c/strong\u003eV\u003csub\u003eT \u003c/sub\u003espectra for all three compounds with inset showing a zoomed in view of intensities between V\u003csub\u003eT\u003c/sub\u003e of -180 V and -400 V. \u003cstrong\u003ec \u003c/strong\u003eIMS\u003cstrong\u003e \u003c/strong\u003eresults for each compound. P\u003csub\u003eHe\u003c/sub\u003e scans at V\u003csub\u003eT\u003c/sub\u003e values of \u003cstrong\u003ed\u003c/strong\u003e -30 V and \u003cstrong\u003ee \u003c/strong\u003e-207 V. \u003cstrong\u003ef \u003c/strong\u003eComparison between mean drift time in the IMS results and optimal P\u003csub\u003eHe\u003c/sub\u003e for transmission of the molecular ion for each compound. A P\u003csub\u003eHe\u003c/sub\u003e scan was not acquired for melezitose at \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e = -30 V.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6558145/v1/4be53bfd5d9961fc019d90b9.jpg"},{"id":82671085,"identity":"f1cef7f6-387b-4ef1-9173-5d9c97c5f9dd","added_by":"auto","created_at":"2025-05-14 02:54:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":727253,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatially distinguishing structural isomeric trisaccharides in mixtures. \u003c/strong\u003eP\u003csub\u003eHe \u003c/sub\u003escans for pure raffinose, pure maltotriose and 1:1 and 2:1 mixtures at \u003cstrong\u003ea\u003c/strong\u003e V\u003csub\u003eT\u003c/sub\u003e= -30 V and \u003cstrong\u003eb \u003c/strong\u003eV\u003csub\u003eT\u003c/sub\u003e= -207 V. \u003cstrong\u003ec \u003c/strong\u003eP\u003csub\u003eHe \u003c/sub\u003efor maximum transmission in function of % of raffinose in the mixture. V\u003csub\u003eT\u003c/sub\u003e spectra at P\u003csub\u003eHe\u003c/sub\u003e = 0.04 mbar for pure samples and mixtures showing intensities of \u003cstrong\u003ed\u003c/strong\u003e C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e31\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, \u003cstrong\u003ee\u003c/strong\u003e C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003csup\u003e- \u003c/sup\u003eand \u003cstrong\u003ef\u003c/strong\u003e C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e. \u003cstrong\u003eg \u003c/strong\u003eRelative quantification of V\u003csub\u003eT\u003c/sub\u003e spectrum signal in mixtures based on signal for pure samples. \u003cstrong\u003ei \u003c/strong\u003eOptical image (left) and OrbiSIMS (right, at different values of V\u003csub\u003eT\u003c/sub\u003e) and ToF-SIMS images of relative signal intensity of C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e31\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e\u003csup\u003e- \u003c/sup\u003eand C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e for the 1:1 mixture sample. Optical (left) and OrbiSIMS (right) images of relative signal intensity of C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e31\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e\u003csup\u003e- \u003c/sup\u003eand C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e for the pure \u003cstrong\u003ej \u003c/strong\u003eraffinose and \u003cstrong\u003ek \u003c/strong\u003emaltotriose samples. For each ion, the OrbiSIMS images were fixed to the same intensity scale to enable comparison. In \u003cstrong\u003ej-k\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003c/sub\u003enotice\u003cstrong\u003e \u003c/strong\u003esignal was detected for all ions but at different proportions. The white scale bars represent 100 µm. The dashed squares on the optical images of \u003cstrong\u003ei-k \u003c/strong\u003erepresent the area from which OrbiSIMS images were acquired.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6558145/v1/903e141ae2eb37258e9e1ef3.jpg"},{"id":82672559,"identity":"7cee6cf3-c33b-48eb-8d27-e8c84db93fb5","added_by":"auto","created_at":"2025-05-14 03:18:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3006374,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6558145/v1/42f12084-ccfc-464e-91f8-c367d1ea9eb3.pdf"},{"id":82671088,"identity":"467bd397-0702-40fa-aa6c-bc6a3314097e","added_by":"auto","created_at":"2025-05-14 02:54:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2735590,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupportingInformationOrbiSIMSisomersv01.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6558145/v1/0f08acd68daeb8224070ccec.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nThe authors declare the following competing financial interest: Alexander Pirkl is an employee of IONTOF GmbH, the manufacturer of the OrbiSIMS instrument used in this research.","formattedTitle":"OrbiSIMS spatially resolves isomeric molecules on surfaces","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMass spectrometry imaging (MSI) has been a transformative suite of methods over the past 50 years with current technology being able to detect and spatially map various kinds of molecules in parallel and without the use of labels. However, MSI is only able to detect mass-to-charge ratios (\u003cem\u003em/z\u003c/em\u003e), which carries no direct information of a molecules higher order structure. Indirect information of structure in MSI can be inferred from charge state detected in desorption electrospray ionisation (DESI).\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e When no accurate spatial information is needed and samples can be prepared in solution, molecular structure can be inferred by coupling MS with liquid chromatography for physical separation\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e or by adding additional reaction steps for fragmenting precursor molecules (tandem MS also known as MS/MS\u003csup\u003e4,5\u003c/sup\u003e) and infer structural information from fragmentation patterns of molecules in collision-induced, surface-induced dissociation or hydrogen-deuterium exchange.\u003csup\u003e\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Ion mobility spectrometry (IMS) is another established method to study molecular structure, where ions of the same \u003cem\u003em/z\u003c/em\u003e but different shapes are separated depending on how they transit a gas-filled collision cell.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e IMS is suitable to be coupled with the imaging capability of MSI as previously shown with matrix-assisted laser desorption/ionization (MALDI), DESI and liquid extraction surface analysis (LESA) sources, allowing parallel imaging and structural information.\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e MALDI and on-tissue chemical derivatisation have also been used to separate isomers using MSI.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSecondary ion mass spectrometry (SIMS) is an MSI method based on the detection of ionised species resulting from the impact of a primary ion beam on a sample surface, with the ability to map in three dimensions.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Over the last decade, SIMS has had significant impact for biological imaging, with constant developments towards improved ion beams and analysers for biomolecules,\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e higher spatial resolution, better accuracy in molecular identification and preserving the native state with cryogenic analysis. Orbitrap SIMS (OrbiSIMS) is a recent technique that is growing in popularity in a wide range of applications in life sciences and materials science.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e An OrbiSIMS (also known as Hybrid SIMS) instrument features a dual analyser configuration with a time-of-flight (ToF) analyser and an Orbitrap\u0026trade; analyser (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). As a consequence this instrument has the ability to combine the mass spectrometry performance usually found in state-of-the-art proteomics and metabolomics with fast 3D imaging at the microscale and from nanolayers of less than 1 nm of material, which enables molecules to be studied directly from surfaces and tissues, unlocking new capability in important applications such as proteomics\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, lipidomics\u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, metabolomics,\u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e and drug delivery.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Secondary ion emission is a heavily fragmenting process, therefore fragmentation patterns can be used to differentiate signals of structural isomers but may carry a lot of ambiguities in complex samples. Recently, fragmentation patterns in SIMS have been combined with the high accuracy of the Orbitrap analyser to infer structure of proteins,\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e biomarkers\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and nucleotides.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e However, there are no established methods using a SIMS platform for the direct detection, separation and mapping of structural isomers on solid surfaces and interfaces (via 3D analysis).\u003c/p\u003e \u003cp\u003eIn OrbiSIMS, for secondary ions to be efficiently transferred to the Orbitrap analyser, their potential energy is matched to the Orbitrap acceptance window by biasing the sample with a target potential \u003cem\u003e(V\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e) and their kinetic energy distribution from the SIMS collision process is reduced through elastic collisions with a helium gas at a pressure \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eHe\u003c/em\u003e\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We previously conducted a systematic assessment of \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eHe\u003c/em\u003e\u003c/sub\u003e on the transmitted secondary ion intensities of inorganic and organic molecules and revealed interesting patterns, indicating the possibility for additional information from secondary ion molecules.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Here, we explore OrbiSIMS \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e spectra and use them analytically to detect and spatially resolve the signal of mixtures of structural isomeric trisacharides. Identifying and separating molecules by their higher order structure, or shape, combined with their spatial localisation can bring new biological insight, for example understanding the function of glycans in cell walls,\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e unambiguous determination of lipids distribution within a tissue\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e and better understanding of protein and nucleotide function.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eVarying target potential selects dissociated ions\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe acquired OrbiSIMS mass spectra of a reference silver sample with a scanned V\u003csub\u003eT\u003c/sub\u003e potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) to produce V\u003csub\u003eT\u003c/sub\u003e spectra of various clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Experimental details are in \u003cb\u003eSupplementary Note 1\u003c/b\u003e. The standard target potential for OrbiSIMS analysis of conducting materials is V\u003csub\u003eT1\u003c/sub\u003e = 57.5 V which is suitable to detect all stable Ag\u003csub\u003en\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e clusters from a reference silver sample.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e However, if V\u003csub\u003eT\u003c/sub\u003e values are increased up to 400 V, a more complex distribution pattern is revealed in the silver cluster intensities, with changing maxima in transmission which vary with cluster size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb-c \u003cb\u003eand Supplementary Figs.\u0026nbsp;2.1-2\u003c/b\u003e). The V\u003csub\u003eT\u003c/sub\u003e values for the multiple maxima are inversely proportional to \u003cem\u003em/z\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed) in agreement with our model (\u003cb\u003eSupplementary Note 2)\u003c/b\u003e that suggests Ag\u003csub\u003en\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e clusters are dissociating into smaller clusters at some point before they are decelerated to enter the collision cell and the ones that dissociate in an electric field-free region are successfully transmitted to the Orbitrap. For singly charged molecules (\u003cem\u003ez\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1), Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e predicts the position of all maxima, \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT2\u003c/sub\u003e, for daughter ions with mass \u003cem\u003em\u003c/em\u003e\u003csub\u003e2\u003c/sub\u003e where the maximum for the parent ion with mass \u003cem\u003em\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u003cem\u003em\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;\u003cem\u003e+\u0026thinsp;Δ\u003c/em\u003e is given by \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT1\u003c/sub\u003e:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:{V}_{T2}^{max}=\\left({V}_{T1}+\\:{V}_{E}\\right)\\:\\frac{{m}_{2}}{{(m}_{2}+\\varDelta\\:)}-{V}_{E}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eWhere \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eE\u003c/em\u003e\u003c/sub\u003e is the extraction potential. For silver clusters, \u003cem\u003eΔ\u003c/em\u003e can only be multiples of the masses of the two Ag isotopes (\u003csup\u003e107\u003c/sup\u003eAg and \u003csup\u003e109\u003c/sup\u003eAg) and V\u003csub\u003eT1\u003c/sub\u003e is the optimised potential of 57.5 V. This is sufficient to estimate all possible dissociations and the experimental data has great agreement with the model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed and \u003cb\u003eSupplementary Figs.\u0026nbsp;2.1-2\u003c/b\u003e). To aid the interpretation of the results on secondary ion dissociation, we established an ion optics simulation model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-g, details in \u003cb\u003eSupplementary Note 3\u003c/b\u003e). The number of simulated Ag\u003csub\u003e11\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e ions transmitted to the Orbitrap as a function of V\u003csub\u003eT\u003c/sub\u003e shows good agreement with the experimental data for the same secondary ions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). The small differences are likely due to second-order effects such as kinetic energy of formation, decays inside electric field gradients (which may also lead to background signal in \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e spectra) and sub-optimal transmission and restrictions on the simulations.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e This shall be investigated in future work.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess whether the model can predict dissociation of more complex organic molecules, we have acquired \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e spectra of a cytochrome C sample (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea experimental details in \u003cb\u003eSupplementary Note 1\u003c/b\u003e). The OrbiSIMS mass spectrum of cytochrome C in the \u003cem\u003em/z\u003c/em\u003e range between 100 and 700 has several peptide fragments as well as multiple peaks of higher intensity relating to the heme C molecule (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Many of these ions will have more than one peak in their \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e spectra and by identifying the voltage for those maxima, the \u003cem\u003em/z\u003c/em\u003e of parent ions and neutral losses can be estimated using Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and \u003cb\u003eSupplementary Fig.\u0026nbsp;2.4\u003c/b\u003e). For the heme C molecule, the predicted losses are multiples of HS, CH, CH\u003csub\u003e2\u003c/sub\u003e, CH\u003csub\u003e3\u003c/sub\u003e and COOH, which based on their putative assignments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), all relate to dissociation of the functional side chains on the four pyrrole rings. Molecules with different structures may have different side chain stability and present different \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e spectra. Therefore, the fact that we can separate dissociated fragments in \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e spectra, together with our previous observations on the dependency transmission of secondary ions on \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e and the collision cell pressure,\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e reveal that there is potential for the study of further molecular properties using OrbiSIMS that extend beyond the \u003cem\u003em/z\u003c/em\u003e scale to separate molecules based on their structure.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStructural isomers on surfaces\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the potential for OrbiSIMS to separate molecules by shape using \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e spectra, we studied three trisaccharide structural isomers, raffinose, maltotriose and melezitose. These have the same molecular formula C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e and are detected as the [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e ion but have different structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Thin films of each sugar were produced on top of a silicon wafer using a spin coater to avoid electric charge build up during the analysis and to allow for the preparation of mixtures (further details in \u003cb\u003eSupplementary Note 1\u003c/b\u003e). The mass spectrum intensity of the [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e is not useful to distinguish between these structural isomers. V\u003csub\u003eT\u003c/sub\u003e spectra of the [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e ion at standard He cell pressure (0.04 mbar) for organic materials for each sample show that optimal transmission occurs at approximately \u0026minus;\u0026thinsp;30 V, however, both raffinose and melezitose show multiple maxima in their V\u003csub\u003eT\u003c/sub\u003e spectra with an oscillating pattern with clear peaks at -207 V, -262 V, -320 V, -375 V and \u0026minus;\u0026thinsp;435 V, which have a mean difference of 59 V. Those features are absent for maltotriose, which enables separation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eUsing Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, we predicted the unit masses of potential parent ions for the [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e detected at the multiple maxima of the V\u003csub\u003eT\u003c/sub\u003e spectra. These were then used to identify peaks in the experimental mass spectra and are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e with their putative assignments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePredicted unit masses of potential \u0026ldquo;parent ions\u0026rdquo; for the [M-H]\u003csup\u003e-\u003c/sup\u003e detected at the multiple maxima of V\u003csub\u003eT\u003c/sub\u003e spectra of raffinose and melezitose and identified peaks in the experimental mass spectra.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eV\u003csub\u003eT\u003c/sub\u003e of maximum (V)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCalculated parent unit mass\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003em/z\u003c/em\u003e in spectrum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMolecular formula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePutative assignment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003edeviation (ppm)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e503.1617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e31\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e547.1515\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e31\u003c/sub\u003eO\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[M - H\u0026thinsp;+\u0026thinsp;CO\u003csub\u003e2\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e561\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e561.1610\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e33\u003c/sub\u003eO\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[M - H\u0026thinsp;+\u0026thinsp;C\u003csub\u003e2\u003c/sub\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e575\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e575.1828\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e35\u003c/sub\u003eO\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[M - H\u0026thinsp;+\u0026thinsp;C\u003csub\u003e3\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e589\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e589.1985\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e37\u003c/sub\u003eO\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[M - H\u0026thinsp;+\u0026thinsp;C\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e-435\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e603\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e603.2141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eC\u003csub\u003e23\u003c/sub\u003eH\u003csub\u003e39\u003c/sub\u003eO\u003csub\u003e18\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e[M - H\u0026thinsp;+\u0026thinsp;C\u003csub\u003e5\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e]\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe potential parent ions in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e have masses higher than the molecular ion of the sugars. This indicates that they are weakly bonded to impurities in the sample, or generated via recombination during the sputtering process, or characteristic of its crystal structure. This shows that a V\u003csub\u003eT\u003c/sub\u003e spectrum has the potential to be informative on structure at molecular level but also at a crystal level. The identified potential parent ions are all detected as secondary ions and their V\u003csub\u003eT\u003c/sub\u003e spectra show the simple behaviour with a single maximum at the standard \u0026ndash; 30 V (\u003cb\u003eSupplementary Fig.\u0026nbsp;2.5\u003c/b\u003e). This indicates that they are directly formed in the secondary ion emission process and not from metastable decay of a larger entity such as a dimer.\u003c/p\u003e \u003cp\u003eIn addition to the \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e spectra data, we also investigated the effect of the collision cell He pressure, \u003cem\u003eP\u003c/em\u003e\u003csub\u003eHe\u003c/sub\u003e, with \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e set at the two values where maxima occur, -30 V and \u0026minus;\u0026thinsp;207 V. The intensity of the quasi-molecular ion [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e has different optimal \u003cem\u003eP\u003c/em\u003e\u003csub\u003eHe\u003c/sub\u003e for transmission for each of the isomers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed-e). This is because the collision cross-section depends on the shape of the molecule. We further expanded our ion optics simulation framework to assess transmission of secondary ions after elastic collisions with a He atom with a given collision cross section (\u003cb\u003eSupplementary Note 3\u003c/b\u003e). We benchmarked our OrbiSIMS results with ion mobility mass spectrometry (IMS) of solutions of each compound (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The IMS peak drift times of the structural isomers are inversely proportional to their respective optimal P\u003csub\u003eHe\u003c/sub\u003e for OrbiSIMS transmission, the lower the pressure for optimal transmission in OrbiSIMS, the higher the drift time in IMS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). This is generally as expected since a lower P\u003csub\u003eHe\u003c/sub\u003e is used for molecules with a larger collision cross-section and these are consequently slower travelling through the IMS drift region. The OrbiSIMS instrument also has a bent flatapole filled with N\u003csub\u003e2\u003c/sub\u003e gas that can vary with the He pressure.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Therefore, to confirm that this behaviour is ruled by He rather than N\u003csub\u003e2\u003c/sub\u003e, we have repeated experiments allowing only He to vary (\u003cb\u003eSupplementary Fig.\u0026nbsp;2.6\u003c/b\u003e). In all three methods (V\u003csub\u003eT\u003c/sub\u003e spectra, varying P\u003csub\u003eHe\u003c/sub\u003e and IMS) a more distinct separation is found for maltotriose with raffinose and melezitose being more similar. This is in agreement with their \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e spectra, molecular structure and collisional cross sections.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003ch3\u003eSpatially distinguishing structural isomers in mixtures, on surfaces\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess whether the signal from isomers could be resolved and be relatively quantified in a mixture, we prepared thin film samples of 1:1 and 2:1 mixtures of raffinose and maltotriose and performed \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eP\u003c/em\u003e\u003csub\u003eHe\u003c/sub\u003e scans (at V\u003csub\u003eT\u003c/sub\u003e = -26V and \u0026minus;\u0026thinsp;220 V) using the same conditions as before (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-b). At -220 V, the optimal \u003cem\u003eP\u003c/em\u003e\u003csub\u003eHe\u003c/sub\u003e for transmission of the [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e ion varies approximately linearly with the % of raffinose in the mixture. Whereas at -26 V, the \u003cem\u003eP\u003c/em\u003e\u003csub\u003eHe\u003c/sub\u003e initially increases linearly but after 60% drops (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eThe full mass spectra for each \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eHe\u003c/em\u003e\u003c/sub\u003e value were merged into a single data matrix and processed with unsupervised machine learning or multivariate statistical analysis methods, such as principal component analysis (PCA), to find trends that are characteristic of each isomer\u003csup\u003e\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e (details in \u003cb\u003eSupplementary Note 4\u003c/b\u003e). PCA reveals distinguishing behaviour in the \u003cem\u003eV\u003c/em\u003e\u003csub\u003e\u003cem\u003eT\u003c/em\u003e\u003c/sub\u003e spectra of specific secondary ions such as C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (galactose/fructose) and C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e (\u003cb\u003eSupplementary Fig.\u0026nbsp;4.1\u003c/b\u003e). In the \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e spectra of mixtures, the oscillating features of the [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e ion that are characteristic of raffinose are present, as well as the distinguishing maxima of C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e and C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e at -340 V (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-e). Those features can be used to check whether the signal from the mixtures shows a linear proportion to the amount of each compound. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg shows the \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e spectrum for C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e in the range of -250 V to -450 V for pure raffinose and maltotriose revealing two distinct peaks for raffinose and one for maltotriose. These peak shapes are then used to fit to the \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e spectra for the two mixtures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). For each \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e spectrum, a linear background was removed prior to fitting. Peak fitting to each mixture\u0026rsquo;s V\u003csub\u003eT\u003c/sub\u003e spectrum was conducted by fixing the V\u003csub\u003eT\u003c/sub\u003e peak centres and peak widths and allowing the peak areas to vary. Qualitatively, the mixtures present features from both isomers but the areas of the fitted peaks do not have a linear correspondence to the proportions in the mixture. This is likely because the two compounds phase separate within the thin film, evidenced by the optical images of the films acquired using a micro camera in the instrument that show a much more homogenous film thickness (based on reflected light) for the pure samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei-k). OrbiSIMS imaging of the [M-H]\u003csup\u003e\u0026minus;\u003c/sup\u003e (C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e31\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) and C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e show that the two monomers can be spatially resolved using OrbiSIMS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei) even at \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e = -30 V, whereas analysis using the ToF analyser (used in the same instrument) show no distinction between the domains for either of the secondary ions. We further confirmed this with imaging data of pure raffinose (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej) and maltotriose (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek) that show homogeneous signal distribution.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eControlling \u003cem\u003eV\u003c/em\u003e\u003csub\u003eT\u003c/sub\u003e and \u003cem\u003eP\u003c/em\u003e\u003csub\u003eHe\u003c/sub\u003e give OrbiSIMS capability that couples phenomena used in IMS (separation of molecules based on collisions with a gas) and PSD (post-source dissociation of molecules with patterns that relate to their structure) methods, with the added capability of imaging (spatially resolve phase separation, depth profiling, localisation in complex biological systems) and direct detection on surfaces. We developed a simple ion dissociation model to understand the effects and established an ion optics simulation framework that can aid the planning of future experiments. More work needs to be done to fully understand why and how molecules fragment and the dependence on V\u003csub\u003eT\u003c/sub\u003e. We show that the transmission characteristics in OrbiSIMS can be leveraged and used analytically to resolve structurally isomeric trisaccharides raffinose, maltotriose and melozitose that cannot be separated by mass alone. To further validate the method, we benchmarked our results with ion mobility spectrometry (IMS) demonstrating a good correlation between the He collision cell pressure, \u003cem\u003eP\u003c/em\u003e\u003csub\u003eHe\u003c/sub\u003e, and the ion mobility drift time. We demonstrate the OrbiSIMS capability to spatially separate isomers within a mixture of raffinose and maltotriose in a thin film, whereas with ToF-SIMS the distribution appears homogenous. This shows promise for high spatial resolution imaging of polysaccharides. The ability to spatially resolve polysaccharides in mixtures and on surfaces and nanoscale interfaces can be transformative as these types of molecules play important biological roles; acting as building blocks of macromolecules, composing cell walls and carrying information for proteins. Furthermore, we expect the method can be applied for direct imaging of biomolecules that may have isomeric ambiguity such as lipids and nucleotides. Since OrbiSIMS allows direct analysis of molecules at nanoscale interfaces, for example in organic electronics\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and biofilms\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, this additional separation dimension provides an exciting new capability for a wide range of science and technology applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThis work was funded by the UK National Measurement System (NMS). NPL is operated by NPL Management Ltd, a wholly-owned company of the UK Department of Science, Innovation and Technology (DSIT). The authors would like to thank Dr Alexander Shard (NPL) for insightful discussion about ion dissociation.\u003c/p\u003e\n\u003ch2\u003eAuthors contributions\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the manuscript and approved the final manuscript. The concept was devised by GFT, AP and ISG. GFT and RP acquired OrbiSIMS experimental data with advice from AP and ISG. RP and GFT developed the ion optics simulation framework. GFT, AP and ISG processed and interpreted OrbiSIMS data. BY proposed the trisaccharide isomer samples, acquired and interpreted IMS data and prepared the cytochrome C sample for OrbiSIMS. JLV and GFT prepared spin-coated samples. GFT oversaw the study.\u003c/p\u003e\n\u003ch2\u003eData availability\u003c/h2\u003e\n\u003cp\u003eAll OrbiSIMS data reported in this manuscript have been deposited as imzML files to figshare (\u003cem\u003eDOI to be provided\u003c/em\u003e). The IMS data reported in this manuscript have been deposited as an xlsx file to figshare (\u003cem\u003eDOI to be provided\u003c/em\u003e). imzML files can be processed in MATLAB using the imzML parser of SpectralAnalysis (https://github.com/AlanRace/SpectralAnalysis).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eCode availability\u003c/h2\u003e\n\u003cp\u003eThis manuscript did not require the development of code that is central to the main work described.\u003c/p\u003e\n\u003ch2\u003eConflicts of interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare the following competing financial interest: AP is an employee of IONTOF GmbH, the manufacturer of the OrbiSIMS instrument used in this research.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHale OJ, Cooper HJ (2021) Native Mass Spectrometry Imaging of Proteins and Protein Complexes by Nano-DESI. 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Anal Chem 95\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6558145/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6558145/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOrbitrap secondary ion mass spectrometry (OrbiSIMS) is growing in popularity in a wide range of applications in life sciences and materials science, with the ability to provide high confidence in molecular identification simultaneously with high confidence in localisation (spatially at the microscale and at the nanoscale within a layer). In OrbiSIMS, transmission of secondary ions depends on two key parameters, the target voltage and helium gas pressure in a cooling cell.\u003csub\u003e \u003c/sub\u003eHere, we show that controlling these parameters gives OrbiSIMS the capability to couple phenomena that enable the separation of molecules based on their dissociation patterns and transmission after gas-phase collisions, which are related to molecular structure. We show that these phenomena can be leveraged and used analytically to take OrbiSIMS beyond the limitations of the mass scale and spatially resolve the signal of mixtures of structural isomeric trisaccharides (raffinose, maltotriose and melezitose). We benchmarked our results with ion mobility spectrometry and showed a correspondence between the methods to distinguish structural isomers. The ability to spatially resolve isomeric molecules in mixtures and on surfaces using OrbiSIMS can be transformative as these types of molecules play important biological roles.\u003c/p\u003e","manuscriptTitle":"OrbiSIMS spatially resolves isomeric molecules on surfaces","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-14 02:54:24","doi":"10.21203/rs.3.rs-6558145/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3bf4e734-3348-4d75-bab9-ae5da9b48863","owner":[],"postedDate":"May 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":48390398,"name":"Physical sciences/Chemistry/Analytical chemistry/Mass spectrometry"},{"id":48390399,"name":"Biological sciences/Chemical biology/Small molecules"},{"id":48390400,"name":"Physical sciences/Nanoscience and technology/Techniques and instrumentation/Mass spectrometry"},{"id":48390401,"name":"Physical sciences/Physics/Techniques and instrumentation/Characterization and analytical techniques"}],"tags":[],"updatedAt":"2025-06-21T09:35:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-14 02:54:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6558145","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6558145","identity":"rs-6558145","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}