The multiplexed ion beam imaging (MIBI) platform was designed to bridge the gap between imaging mass spectrometry and the clinical lab, delivering high throughput, subcellular spatial resolution imaging for 40+ protein markers per sample. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) can be a powerful tool for tissue imaging. However, its applications in tissue imaging are often limited by its usability, acquisition time, and complex mass spectra, the latter making data analysis and interpretation difficult. MIBI has overcome these limitations with a high resolution, high throughput ToF-SIMS system to quickly analyze proteins of interest which are labeled using conjugated antibodies. Specifically, heavy metal atoms, which become the reporter ions measured, are conjugated to antibodies that target the proteins of interest and stain the tissue using a protocol much like that for other multiplexed imaging techniques, such as immunohistochemistry and multiplexed immunofluorescence. Antibody clones, which are known to be successful in other pathology research and clinical settings, can often be used to further facilitate the adoption of the MIBI platform in these settings.

The MIBIscope utilizes a high density Xe plasma ion source to enable rapid imaging of tissue with ToF-SIMS, acquiring an 800 µm x 800 µm ROI in as little as 35 minutes. By targeting protein species with labeled antibodies, the resulting mass spectra are less complicated and since the target analyte is not being fragmented, and the image data has higher signal to noise. An increase in throughput, simpler data analysis, and a sample prep procedure consistent with other common techniques have all allowed the MIBI platform to enter applications spaces that traditionally have been unobtainable for ToF-SIMS.

Metabolomics provides the chemical readout that is closest of all the omics to the phenotype of cells. We believe that this level of insight is necessary to interpret the effect of the environmental cues provided to cells by manmade biomaterials.¹

ToF SIMS struggles with its poor mass resolving power in complex biological systems when faced with myriad possible peak assignments for each secondary ion peak.² The 3D OrbiSIMS approach addresses that by combining an OrbiTrap with a time-of-flight SIMS instrument to undertake direct analysis of solid samples.³

Application examples from the field of novel biomaterials development will be provided that take advantage of the unique capability of this instrument, focussing on its ability to detect and identify small molecules with a high degree of certainty. Markers of immune cell polarisation for next generation implant materials have been found by assessing single macrophage cells rather than the 6 million cells required previously by LC-MS.⁴ Small molecules in complex bacterial biofilms are of interest in understanding the response to novel materials that resist bacterial colonisation and infection.⁵The utility of recently development software to allow chemical filtering to predict molecular formula from SIMS using existing databases⁶ is illustrated by reanalysis of the data from Zhang et al, to exemplify the massive increase in the proportion of the spectrum assigned using this automation of data interpretation for OrbiSIMS.⁷

These recent developments enable metabolomic analysis by OrbiSIMS to achieve a label-free, unbiased insight into cellular phenotype at the resolution of single mammalian cells in culture, but ultimately on explanted devices to interpret their responses to different biomaterials.

1. Single-cell metabolomics hits its stride Nature Methods Caroline Seydel
2. Mass Spectrometry and Informatics: Distribution of Molecules in the PubChem Database and General Requirements for Mass Accuracy in Surface Analysis Anal Chem 2011, Green et al
3. The 3D OrbiSIMS - Label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power. Nature Methods2017, Passarelli et al.
4. Single cell metabolomics of macrophages using 3D OrbiSIMS: correlations with phenotype Suvannapruk et al. under review 2022
5. Cryo-OrbiSIMS for 3D Molecular Imaging of a Bacterial Biofilm in Its Native State. Anal Chem2020, Zhang et al.
6. Molecular formula prediction for chemical filtering of 3D OrbiSIMS Datasets Anal Chem 2022 Edney et al.
7. Towards comprehensive analysis of the 3D chemical makeup of Pseudomonas aeruginosa biofilmsKotowska et al. under review 2022.

In MeV TOF-SIMS, heavy primary ions with higher energy produce higher secondary ion yield of heavy molecules, an important parameter for molecular imaging. These primary ions (such as 14 MeV copper ions) cannot be focused with magnetic lenses available at the RBI accelerator facility. For this reason, a new setup is developed that uses a simple round aperture with a 5 – 10 um opening to collimate the primary beam independently on primary ion mass. As the beam current is significantly reduced after collimation with the aperture, a common beam pulsing method for triggering the TOF measurement could not be utilized. Instead, two different options for the start signal for a continuous primary beam are available: for thin samples, a particle detector placed behind the target that detects primary ions that pass through the target, and for any target thickness an electron multiplier that detects secondary electrons created in the interaction of the primary ions with a 5 nm thick carbon foil placed over the aperture. The samples interesting for forensic (ink deposited on a paper and fingermark) and biological (section of brain tissue) applications of MeV TOF-SIMS were analyzed to show the imaging capabilities of the presented setup.