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Instrument development in the SIMS field has yielded devices that enable 3D visualization of small molecules with subcellular resolution, suggesting the potential for multiplexed analysis of dozens of biomolecules and small molecules at the nanoscale 17, 18, 19, 20, 21, 22, 23, 24. Although MIBI can define subcellular distribution of dozens of biomolecules, resolution limits (determined by the ion gun, vibration effects, electromagnetic lenses, and the working distance from the gun to the sample) are currently the “filters” that define the limits for 3D cellular imaging at the nanoscale.
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A similar approach, imaging mass cytometry (IMC), allows protein and RNA to be visualized in tissues using laser ablation at 1000 nm resolution 15, 16. In MIBI, proteins of interest are stained with isotope-tagged antibodies, and the sample is rasterized with an oxygen-based primary ion beam to generate a two-dimensional composite image of up to 50 proteins, down to 260 nm resolution 14. We previously demonstrated the ability of secondary ion beam imaging (SIMS), a form of MSI, to image multiple targeted-proteins in tissue samples using multiplexed ion beam imaging (MIBI) 13. Images are obtained via the rastering of a biological sample with a laser 11 or an ion beam 12 to liberate molecules to be analyzed by a mass spectrometer. This will complement the tissue mapping efforts now ongoing worldwide by bringing subcellular maps of the 3D nucleome and proteome to more public use and application.Īn alternative modality of biomolecular imaging is mass spectrometry imaging (MSI). The ability to directly image atomic components as labels would, with sufficiently high resolution, allow for a three-dimensional (3D) reconstruction of the distribution of multiple biomolecules in whole cells. Often, imaging of small molecules, such as drugs, metabolites, or lipids, require the conjugation of chemical tags, and can vastly affect biological activities 10. Acquisition time for imaging and sample positional shifts between cycles, which become more pronounced at super-resolution scales 9, places practical limits on the utility of these approaches. These techniques have allowed a range of studies from single cells at super-resolution 5 to whole tissues 6 and have revealed novel insights on the spatial distribution of DNA and RNA in their biological context 5, 7, 8. Recent advancements using cyclic-hybridization protocols enable multiparameter visualization of proteins, RNA, and DNA 1, 2, 3, 4, in part overcoming the spectral issue. Development of multiparameter high-resolution approaches that visualize relevant drugs and multiple cellular components at the molecular scale will be crucial to overcome limitations in our current understandings.įluorescence microscopy is currently the de-facto choice for biomolecular imaging, although multiplexing with fluorophores can be challenging due to the spectral overlap of fluorophores. When current understandings of cell function falter short of clinical utility, this drives a need for technologies that enable more precise analysis of cellular biomolecular components within cells in more native contexts. Interactions of molecular entities drive function in biological systems. Multiplexed high-resolution imaging techniques, such as HD-MIBI, will enable studies of biomolecules and drug distributions in biologically relevant subcellular microenvironments by visualizing the processes themselves in concert, rather than inferring mechanism through surrogate analyses. Unexpectedly, cells surviving multi-drug treatment with cisplatin and the BET inhibitor JQ1 demonstrated near total cisplatin exclusion from the nucleus, suggesting that selective subcellular drug relocalization may modulate resistance to this important chemotherapeutic treatment.
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Cisplatin was preferentially enriched in nuclear speckles and excluded from closed-chromatin regions, indicative of a role for cisplatin in active regions of chromatin. We correlated the subcellular localization of the chemotherapy drug cisplatin simultaneously with five subnuclear structures. With this technology, the atomic constituents of the biomolecules themselves can be used in our system as the “tag” and we demonstrate measurements down to ~30 nm lateral resolution. We present here high-definition multiplex ion beam imaging (HD-MIBI), a secondary ion mass spectrometry approach capable of high-parameter imaging in 3D of targeted biological entities and exogenously added structurally-unmodified small molecules. Simultaneous visualization of the relationship between multiple biomolecules and their ligands or small molecules at the nanometer scale in cells will enable greater understanding of how biological processes operate.