An image acquisition device (M) comprising: a stage for mounting a sample (S); a light source for emitting light to the sample (S); a lightguide optical system including an objective lens arranged so as to oppose the sample (S) on the stage and light splitting means for splitting an optical image of the sample into a first optical path (L1) for image acquisition and a second optical path (L2) for focus control; field drive means adapted to move a field position of the objective lens with respect to the sample (S); first imaging means adapted to acquire a first image formed by a first optical image split into the first optical path (L1); second imaging means adapted to acquire a second image formed by a second optical image split into the second optical path (L2); focus calculation means adapted to analyse the second image and adapted to calculate focus information of the sample (S) 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 (S) 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 (S). a stage for mounting a sample (S); a light source for emitting light to the sample (S); a lightguide optical system including an objective lens arranged so as to oppose the sample (S) on the stage and light splitting means for splitting an optical image of the sample into a first optical path (L1) for image acquisition and a second optical path (L2) for focus control; field drive means adapted to move a field position of the objective lens with respect to the sample (S); first imaging means adapted to acquire a first image formed by a first optical image split into the first optical path (L1); second imaging means adapted to acquire a second image formed by a second optical image split into the second optical path (L2); focus calculation means adapted to analyse the second image and adapted to calculate focus information of the sample (S) 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 (S) 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 (S). <EOS>