Fig. 2: Metabolic tagging and endogenous elemental contrasts enable ion beam nanoscopic tomography.

A single Nalm6, B cell lymphoblast, imaged for Secondary electron (SE), ¹²C (red), ¹⁴N (measured as ¹²C¹⁴N, cyan), ³¹P for DNA (blue), ⁸¹Br for newly synthesized transcripts (labeled with ⁸¹Br-rU, magenta), ¹²⁷I for replication loci (labeled with ¹²⁷I-dU, green), and ³⁴S (yellow) for detection of total proteins. ① A volumetric scan across 1000 slices was performed by ion beam imaging from the top to the bottom of a cell for seven mass channels. Potentially 100 distinct mass channels can be analyzed. Raw ion images showed highly pixelated and noisy ion beam data. Scale bar 2 µm. ② Standard image sum analysis of 20 ion stacks for four mass channels. Global spatial patterns in subcellular volumes were visible albeit being blurry. Scale bar 2 µm. ③ Deconvolution-IBT analysis of four channels resolved finer structures in total DNA (blue), RNA (magenta), replicated DNA (green), and proteins (yellow). Chromatin showed folding fiber structures and other channels exhibited subcellular distributions around chromatin. Scale bar 2 µm. ④ Highly precise IBT using SILM analysis provided even sharper ion reconstructions that generated spatially localized data that is more precise than the practical resolution limit of ion-beam-width. Localized ion signals from each channel were digitally combined across twenty ion slices to create a tomographic image. Scale bar 2 µm. ⑤ Ion-beam tomograms are shown in the deconvolution-IBT image format (upper images, n = 600 stacks) and SILM-IBT image arrays (lower images, n = 600 stacks) with the three channels (selected out of seven acquired channels) for combining as a 3D cell, in the form of chromatin, transcription, and replication tomographic representations. 3D Visualization tool of Volocity (Perkin Elmer) was used to render IBT data in 3D.