An image acquisition device comprising: a stage for a sample to be mounted thereon; stage control means for scanning the stage at a predetermined speed; a light source for applying light to the sample; a lightguide optical system including light splitting means for splitting a optical image of the sample into a first optical path for image acquisition and a second optical path for focus control; first imaging means for acquiring a first image formed by a first optical image split into the first optical path; second imaging means for acquiring a second image formed by a second optical image split into the second optical path; focus control means for performing an analysis of the second image and controlling a focal position in imaging by the first imaging means, based on the result of the analysis; and an optical path difference generating member arranged in the second optical path and adapted to generate an optical path difference in the second optical image along an in-plane direction of an imaging surface of the second imaging means, wherein the optical path difference generating member has a first face and a second face tilted relative to a plane orthogonal to an optical axis of the second optical path, wherein the first face and the second face are each configured to refract the light from the second optical path inward so as to impinge upon the imaging surface of the second imaging means, and characterized in that a light reduction portion for reducing light reaching the imaging surface is provided between the first face and the second face, so as to narrow a light superimposed region on the imaging surface of the second imaging means where beams refracted at the first face and at the second face of the optical path difference generating member are superimposed, the image acquisition device further comprising: region control means for setting a first imaging region and a second imaging region on the imaging surface of the second imaging means so as to avoid the light superimposed region, the focus control means being configured to calculate a difference between a contrast value of the first imaging region and a contrast value of the second imaging region. a stage for a sample to be mounted thereon; stage control means for scanning the stage at a predetermined speed; a light source for applying light to the sample; a lightguide optical system including light splitting means for splitting a optical image of the sample into a first optical path for image acquisition and a second optical path for focus control; first imaging means for acquiring a first image formed by a first optical image split into the first optical path; second imaging means for acquiring a second image formed by a second optical image split into the second optical path; focus control means for performing an analysis of the second image and controlling a focal position in imaging by the first imaging means, based on the result of the analysis; and an optical path difference generating member arranged in the second optical path and adapted to generate an optical path difference in the second optical image along an in-plane direction of an imaging surface of the second imaging means, wherein the optical path difference generating member has a first face and a second face tilted relative to a plane orthogonal to an optical axis of the second optical path, wherein the first face and the second face are each configured to refract the light from the second optical path inward so as to impinge upon the imaging surface of the second imaging means, and characterized in that a light reduction portion for reducing light reaching the imaging surface is provided between the first face and the second face, so as to narrow a light superimposed region on the imaging surface of the second imaging means where beams refracted at the first face and at the second face of the optical path difference generating member are superimposed, the image acquisition device further comprising: region control means for setting a first imaging region and a second imaging region on the imaging surface of the second imaging means so as to avoid the light superimposed region, the focus control means being configured to calculate a difference between a contrast value of the first imaging region and a contrast value of the second imaging region. region control means for setting a first imaging region and a second imaging region on the imaging surface of the second imaging means so as to avoid the light superimposed region, the focus control means being configured to calculate a difference between a contrast value of the first imaging region and a contrast value of the second imaging region.