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Isotopically Encoded Nanotags for Multiplexed Ion Beam Imaging

High‐dimensional profiling of markers and analytes using approaches, such as barcoded fluorescent imaging with repeated labeling and mass cytometry has allowed visualization of biological processes at the single‐cell level. To address limitations of sensitivity and mass‐channel capacity, a nanobarcoding platform is developed for multiplexed ion beam imaging (MIBI) using secondary ion beam spectrometry that utilizes fabricated isotopically encoded nanotags. Use of combinatorial isotope distributions in 100 nm sized nanotags expands the labeling palette to overcome the spectral bounds of mass channels. As a proof‐of‐principle, a four‐digit (i.e., 0001–1111) barcoding scheme is demonstrated to detect 16 variants of 2H, 19F, 79/81Br, and 127I elemental barcode sets that are encoded in silica nanoparticle matrices. A computational debarcoding method and an automated machine learning analysis approach are developed to extract barcodes for accurate quantification of spatial nanotag distributions in large ion beam imaging areas up to 0.6 mm2. Isotopically encoded nanotags should boost the performance of mass imaging platforms, such as MIBI and other elemental‐based bioimaging approaches.