An image acquisition device comprising: a stage for mounting a sample; a light source for emitting light to the sample; a lightguide optical system including an objective lens arranged so as to oppose the sample on the stage and light splitting means for splitting an optical image of the sample into a first optical path for image acquisition and a second optical path for focus control; field drive means adapted to move a field position of the objective lens with respect to the sample; first imaging means adapted to acquire a first image formed by a first optical image split into the first optical path; second imaging means adapted to acquire a second image formed by a second optical image split into the second optical path; focus calculation means adapted to analyse the second image and adapted to calculate focus information of the sample according to a result of the analysis; operation control means adapted to control operations of the field drive means and second imaging means; and an optical path difference generating member (21, 41A to 41E, 51) 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 second imaging means has a two-dimensional image pickup element, including a plurality of pixel columns, characterized in that the second imaging means is adapted to perform rolling readout and in that the operation control means synchronizes movement of a predetermined part (Sa) of the sample within the field of the objective lens caused by the field drive means with rolling readout of the two-dimensional image pickup element such that each pixel column of the two-dimensional image pickup element is exposed to the optical image of the predetermined part (Sa) in the sample. a stage for mounting a sample; a light source for emitting light to the sample; a lightguide optical system including an objective lens arranged so as to oppose the sample on the stage and light splitting means for splitting an optical image of the sample into a first optical path for image acquisition and a second optical path for focus control; field drive means adapted to move a field position of the objective lens with respect to the sample; first imaging means adapted to acquire a first image formed by a first optical image split into the first optical path; second imaging means adapted to acquire a second image formed by a second optical image split into the second optical path; focus calculation means adapted to analyse the second image and adapted to calculate focus information of the sample according to a result of the analysis; operation control means adapted to control operations of the field drive means and second imaging means; and an optical path difference generating member (21, 41A to 41E, 51) 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 second imaging means has a two-dimensional image pickup element, including a plurality of pixel columns, characterized in that the second imaging means is adapted to perform rolling readout and in that the operation control means synchronizes movement of a predetermined part (Sa) of the sample within the field of the objective lens caused by the field drive means with rolling readout of the two-dimensional image pickup element such that each pixel column of the two-dimensional image pickup element is exposed to the optical image of the predetermined part (Sa) in the sample.