Abstract: A method of precision calibration of a microscope magnification with calculating a magnification scale as a quotient obtained when an image size of a test object viewed or collected with the microscope is divided by a true test object size, the methods comprising the steps of obtaining a magnification reference by taking a diffraction grating with a tested pitch value as the test object; distributing a brightness level between 30-70% amplitude in one of an image of the diffraction grating and a video signal obtained in the microscope; calculating a position of the video signal “center of mass” for each of formed “islands” of the brightness distribution; considering an average distance between neighboring “center of mass” as being a grating pitch in a microscope image of the object; and recognizing that a magnification scale of the microscope is a result of a division of an average pitch dimension by true grating pitch.