Provided herein are methods, systems, devices, and computer-readable storage media for measuring flatness (e.g., along a Z-axis) of a detection stage. In some aspects, measuring flatness of the detection stage includes obtaining two or more images representing different Z coordinates of a first XY coordinate on a substantially flat substrate positioned on the detection stage; determining a sharpest Z coordinate at the first XY coordinate based on sharpness of the two or more images; obtaining two or more images representing different Z coordinates of a second XY coordinate spaced apart from the first XY coordinate on the substantially flat substrate; determining a sharpest Z coordinate at the second XY coordinate based on sharpness of the two or more images; and calculating a difference between the sharpest Z coordinates at the first and the second XY coordinates to measure the flatness of the detection stage.