A spot scanning imaging system with run-time alignment includes a beam scanning device configured to linearly scan a focused beam of illumination across a sample, one or more detectors positioned to receive light from the sample, and a controller communicatively coupled to the beam scanning apparatus, the sample stage, and the one or more detectors. The controller is configured to store a first image, transmit a set of drive signals to at least one of the beam scanning device, the sample stage, or the one or more detectors, compare at least a portion of the second sampling grid to at least a portion of the first sampling grid to determine one or more offset errors, and adjust at least one drive signal in the set of drive signals based on the one or more offset errors such that the second sample grid overlaps the first sample grid.