Method for determining z-stack boundary of image of object

The invention relates to a method for automatically determining a boundary of a z-stack of an image of an object, an optical instrument for imaging an object, and a computer program. A z-stack of images is to be acquired by imaging an object at different focal positions by an optical instrument comprising instrument optics and a focus adjustment unit for imaging the object at different focal positions by the instrument optics, the method comprising the steps of: generating a set of images of the object, each image is captured at a different focus position; applying an ambiguity-W metric function to each of the images of the set of images, the ambiguity-W metric function calculating a metric value for each of the images, different metric values associated with different image sharpness or ambiguity of the corresponding image, if an ambiguity-W metric function with focus positions as variables shows a primary extreme value and two secondary extreme values adjoining the primary extreme value, the z-stack boundar