Histology-Guided Serial-Section Framework for Quasi-3D ToF-SIMS Molecular Imaging of Rat Brain Lipid Ions

Abstract Time-of-flight secondary ion mass spectrometry (ToF-SIMS) provides label-free molecular images of biological tissues, but an isolated two-dimensional brain section cannot represent variation along the anterior–posterior axis. Here, we developed a histology-guided workflow for serial, sparsely sampled sections that organizes ToF-SIMS ion images into anatomically referenced quasi-three-dimensional (quasi-3D) representations. Adjacent hematoxylin and eosin (H&E)-stained sections guided cross-modal alignment, and intensity-filtered point clouds supported brain-wide and region-of-interest (ROI) visualization. Parameter-sensitivity tests showed that major high-contrast anatomical patterns remained stable across intermediate threshold and interpolation settings; lower-intensity signals were progressively lost as the threshold increased, whereas greater interpolation density primarily improved display smoothness. Application to four independently acquired single-brain datasets showed that the same workflow accommodated differences in tissue geometry and signal distribution. Section-resolved principal component analysis and total-ion-current-normalized abundance summaries were used as exploratory outputs rather than treatment-effect tests because one brain was imaged per condition. This framework provides a reproducible way to organize serial, sparsely sampled ToF-SIMS data for quasi-3D visualization and anatomically referenced exploratory profiling when dense volumetric acquisition is not feasible within the available acquisition time and data volume.