An image capturing apparatus, comprising: a stage on which a sample is placed; a stage control unit for scanning the stage at a predetermined speed; a light source for radiating light to the sample; a light guiding optical system including a light dividing unit for dividing an optical image of the sample into a first optical path for capturing an image and a second optical path for focus control; a first imaging unit for capturing a first image by a first optical image divided into the first optical path; a second imaging unit for capturing a second image by a second optical image divided into the second optical path; a focus control unit for analysing the second image to control a focus position of the image pickup by the first imaging unit based on the analysis result; a region control unit for setting at an imaging area of the second imaging unit a first imaging region and a second imaging region for capturing a partial image of the second optical image; and an optical path difference producing member which is disposed on the second optical path to give an optical path difference to the second optical image along an in-plane direction of the imaging area, characterized in that the optical path difference producing member is disposed in such a manner that the optical path difference of the second optical image is symmetrical to the axis of the imaging area orthogonal to a direction at which the second optical image moves in association with scanning of the sample, and in that the first imaging region and the second imaging region are set in a direction perpendicular to a direction at which the second optical image moves in association with scanning of the sample; and in that the focus control unit is operatively arranged to analyse the second image including determining a difference between a contrast value of the image captured at the first imaging region and a contrast value captured at the second imaging region in the second imaging unit; and the region control unit is operatively arranged to reverse a position of the first imaging region and a position of the second imaging region on the imaging area with respect to the axis in association with reversal of the scanning direction of the sample. a stage on which a sample is placed; a stage control unit for scanning the stage at a predetermined speed; a light source for radiating light to the sample; a light guiding optical system including a light dividing unit for dividing an optical image of the sample into a first optical path for capturing an image and a second optical path for focus control; a first imaging unit for capturing a first image by a first optical image divided into the first optical path; a second imaging unit for capturing a second image by a second optical image divided into the second optical path; a focus control unit for analysing the second image to control a focus position of the image pickup by the first imaging unit based on the analysis result; a region control unit for setting at an imaging area of the second imaging unit a first imaging region and a second imaging region for capturing a partial image of the second optical image; and an optical path difference producing member which is disposed on the second optical path to give an optical path difference to the second optical image along an in-plane direction of the imaging area, characterized in that the optical path difference producing member is disposed in such a manner that the optical path difference of the second optical image is symmetrical to the axis of the imaging area orthogonal to a direction at which the second optical image moves in association with scanning of the sample, and in that the first imaging region and the second imaging region are set in a direction perpendicular to a direction at which the second optical image moves in association with scanning of the sample; and in that the focus control unit is operatively arranged to analyse the second image including determining a difference between a contrast value of the image captured at the first imaging region and a contrast value captured at the second imaging region in the second imaging unit; and the region control unit is operatively arranged to reverse a position of the first imaging region and a position of the second imaging region on the imaging area with respect to the axis in association with reversal of the scanning direction of the sample.