Microscope observation method, and image processing program

A When the three-dimensionally to obtain separately the information of the sample into a plurality of layers, aligning the information in multiple layers. A microscope includes an illumination optical system for projecting illumination light to a sample containing a fluorescent substance, an observation optical system including an objective lens, an imaging unit that captures an image of the observation optical system is formed, the results of imaging includes an image processing unit for performing image processing, and a control unit for moving at least one of the stage and the objective lens in the optical axis direction of the objective lens, the use, the image processing unit, the focal position is a first position of the objective lens the position information of the fluorescent substance in the first area obtained by the imaging results when in the, the fluorescent material in the second region obtained by the image pickup result obtained when the focal position of the objective lens is in the second position of the positional information, using the position information of the fluorescent substance in at least part of the overlap region between the first region and the second region, it corrects the position information of the fluorescent material in the second region.