To avoid axial resolution loss, we binned 10 slices and incremented by one slice at a time over the entire section. a mathematical and technical framework for three-dimensional (3D) subcellular MIBI is usually offered. Ion-beam tomography (IBT) compiles ion beam images that are acquired iteratively across successive, multiple scans, and later put together into a 3D format without loss of depth resolution. Algorithmic deconvolution, tailored for ion beams, is usually then applied to the transformed ion image series, yielding 4-fold enhanced ion beam data cubes. To further generate 3D sub-ion-beam-width precision visuals, isolated ion molecules are localized in the natural ion beam images, creating an approach coined as SILM, secondary ion beam localization microscopy, providing sub-25?nm accuracy in initial ion images. Using deep learning, a parameter-free reconstruction method for ion beam tomograms with high accuracy is developed for low-density targets. In cultured malignancy cells and tissues, IBT enables R935788 (Fostamatinib disodium, R788) accessible visualization of 3D volumetric R935788 (Fostamatinib disodium, R788) distributions of genomic regions, RNA transcripts, and protein factors with 5?nm axial resolution using isotope-enrichments and label-free elemental analyses. Multiparameter imaging of subcellular features at near macromolecular resolution is implemented by the IBT tools as a general biocomputation pipeline for imaging mass spectrometry. that was varied between 3 and 20 slices (Supplementary Fig.?2, step 3 3). This process was then incremented by one layer at a time. For instance, ion signals from 1 to slices were combined to yield the first transformed image, ion signals from slices 2 to axis of IBT images. Here, the resolution was defined based on the error function (erf) fit21 of TEF2 the transmission drop at the edge, corresponding to the distance between 88 and 12% of the maximum ion beam transmission at the plateau region. With these settings, a presumptive 53?nm resolution image was obtained for ion beam images of a Jurkat cell (Supplementary Fig.?5). Using the oxygen source, only a 498?nm maximum resolution was obtained from three edge scans at the junction of aluminium and silicon (Supplementary Fig.?6). Thus, the resolution depends on ion current levels and aperture size. As expected, a cesium beam allowed for higher resolution imaging than an oxygen beam. Therefore, with a scalable resolution from 55?nm to 500?nm, ion beam imaging bridges a space between super-resolution optical imaging and wide-field microscopy (Supplementary Fig.?7). The axial depth resolution of the ion beam imaging was evaluated by two methods. In the first, a single replication site that was marked by 30?min incorporation of 127I-dU was evaluated: A 175?nm wide pattern crossed 40 slices, indicative of 5?nm axial resolution (Supplementary Figs.?8 and 9). In the R935788 (Fostamatinib disodium, R788) second approach, the cell height (~10?m) was divided by the total quantity of scans (1000), again indicative of 5C10?nm axial resolution (Supplementary Fig.?10). To produce high-precision IBT maps with sub-ion-beam-width accuracy, we were inspired by the stochastic optical reconstruction microscopy (STORM) method22,23 from fluorescence imaging. STORM utilizes blinking single molecules to localize individual fluorophores and combines the localized positions to synthesize super-resolved data. In ion beam imaging, we adopt each scan from ultra-thin layers (1C10?nm) as a single time-stamped frame with varying ion transmission levels and combine localized ion images from a series of sequential scans to generate a high-precision IBT reconstruction (Fig.?1b, SILM-IBT). We term this approach Secondary Ion-beam Localization Microscopy (SILM) that provides nanoscopic ion beam data cubes from a series of NanoSIMS scans (Supplementary Fig.?11). The highly precise ion-beam analysis benefits from the stochastic nature of a subset of ion signal levels appearing at each SIMS image, allowing us to localize only a subset of ion molecules. Unlike the STORM method, only mathematical localizations (similar to the FIONA23 approach) of individual molecules were used as a general spatially resolving theory, as also previously performed in photoacoustic imaging24 and label-free interferometric imaging25. Using a sliding windows of 20C100 frames, the depth resolution is usually retained back to accomplish highly precise tomograms of single cells. The high-precision tomograms, however, can be applied to mostly relatively dense subcellular markers and structures. For instance, a concentrated DNA image in the 127I-dU and 81Br-dU channels were analyzed by the SILM-IBT pipeline. Insets show the collection scans with precisely localized features of chromatin in the major peaks identified in the previous sum and deconvolution IBT images denoted by dashed squares (Fig.?1c). To quantify and validate the overall performance of SILM-IBT, we designed and fabricated multi-color nanotags with a 109.8??17.6?nm average diameter (Supplementary Fig.?12), akin to TetraSpeck microspheres26 used in fluorescence microscopy. These nanotags are composed of homogeneously distributed isotopes (19F) within a silica matrix (28Si) across the entire 109?nm spherical volume, making them an.