A scanning electron microscopy system includes an electron beam source, a sample stage that includes a first alignment feature, an electron-optical column that includes electron-optical elements that include a lens having a second alignment feature, and an alignment plate having a third alignment feature. The system additionally includes a reference target, and a detector assembly. The electron-optical elements configurable to simultaneously focus on a substrate and the reference target. The system also includes a controller communicatively coupled to at least one or more portions of the electron-optical column and the sample stage, to make adjustments in order to align the electron beam to at least one of the first set of alignment features, the second set of alignment features, the third set of alignment features, the reference target or the substrate. The controller also makes adjustments to simultaneously focus the electron beam at a first and second high resolution plane.