When the microscope zoom-in value was set to 3.2, the picture of a ruler with a minimum scale of 0.5mm was as shown in the figure below. The real distance between point A and point B is 0.5mm. After Python work of this image, the coordinates of the two points were determined: A (1670,400), B (2690,400). Therefore, the pixel difference between points A and B was 1020 pixels, which meant that when the zoom-in value was 3.2, the real size corresponding to one pixel is 0.5mm/1020≈0.5μm/pixel.