Most common: cut the tissue into continuous slices of a fixed thickness (e.g. 10–20 μm), image each slice independently to obtain a 2D molecular map, then align each layer using microscopic coordinates or histological features and stack them into a volume.
This method applies to planar imaging sources such as MALDI, DESI and LDPI; the key is not losing sections and keeping coordinates traceable (see section thickness).
Some sources can repeatedly sample at different depths on the same surface or have z-direction resolution, directly obtaining 3D information and avoiding physical section stacking. But depth resolution is limited by desorption/sputtering longitudinal diffusion and is mostly used for the top few micrometers.
The sputtering of SIMS itself has depth-profiling capability and can accumulate 3D voxels while profiling, making it one of the representative in-situ 3D routes.
Section deformation and tissue loss caused by staining/imaging order can both cause inter-layer misalignment. Non-rigid registration using continuous-section morphological features or fiducial markers is needed.
Registration quality directly determines 3D credibility; it is recommended to report registration error and the validation method in the methodology.
3D MSI is used for intra-organ drug penetration volume, 3D tumor heterogeneity and stereoscopic plant-tissue metabolism, among others. The output is 'one volume map per molecule', which can connect to spatial omics analysis.
The data volume is large and requires supporting analysis software and standard-format management.
To obtain detailed specifications, compatible models, or a quotation for the MSI LDPI / DPI full series imaging ion sources, visit the Neo-Source official website, or contact the official team for compatibility advice tailored to your mass spectrometer (Agilent / SCIEX / Thermo and other mainstream MS).