In this Tools of the Trade article, Anne C. Rios describes the development of multispectral large-scale single-cell resolution three-dimensional (mLSR-3D) imaging and the analytical pipeline, segmentation analysis by parallelization of 3D datasets (STAPL-3D), which enables the extraction of hundreds of molecular, spatial and volumetric features from millions of cells imaged in 3D, revealing details of the structural organization of human tumours.