Disclosed herein is a computer-implemented method for generating calibration data usable for analysis of a sample. The method includes: (i) identifying targets in an image frame pertaining to a scanned area of a sample; (ii) computing displacements of the targets relative to positions thereof as given by, or derived from, reference data of the scanned area; (iii) based at least on the computed target displacements, determining values of coordinate transformation parameters (CTPs) relating coordinates of the image frame to coordinates of the scanned area as given by, or derived from, the reference data; and (iv) using at least the CTPs to obtain displacements of multiple segments in the image frame, thereby generating a displacement mapping of the image frame or at least a part thereof.