A sample observation device, comprising: an emission optical system configured to emit planar light to a sample along an optical axis of the emission optical system, defining a Z' axis direction; a scanning unit configured to scan the sample in a Y axis direction within a scanning surface so as to pass through an emission surface of the planar light, a width direction of the sample is an X axis direction and the depth direction of the sample is a Z axis direction such that each of the X, Y, and Z axis directions are perpendicular to one another, the X axis is perpendicular to the Y axis within a plane perpendicular to the optical axis of the emission optical system, and the emission surface is a surface within a XZ' plane; an imaging optical system configured to form an image of observation light generated in the sample by emission of the planar light; an image acquisition unit that has a plurality of pixels arranged in a two-dimensional manner and which is configured to acquire a plurality of pieces of image data corresponding to an optical image of the observation light formed by the imaging optical system; and an image generation unit configured to generate observation image data of the sample based on the plurality of pieces of image data acquired by the image acquisition unit, wherein, when an angle formed by an optical axis (Pl) of the emission optical system and a normal of the scanning surface along the Z axis direction is Θ1 and an angle formed by an optical axis of the imaging optical system and the normal of the scanning surface is θ2, both θ1 and θ2 are 80° or less and a sum of θ1 and θ2 is 100° or more, and in the image acquisition unit, an image acquisition region of an (n+1)-th pixel is shifted from an image acquisition region of an n-th pixel in a scanning direction of the sample according to a scanning amount of the sample in an exposure time of one frame, wherein the image acquisition region of a pixel is the region in the XZ' plane which is imaged by the imaging optical system onto a pixel of the image acquisition unit and the respective image acquisition regions of the (n+1)-th pixel and the n-th pixel are aligned along the Z' direction. an emission optical system configured to emit planar light to a sample along an optical axis of the emission optical system, defining a Z' axis direction; a scanning unit configured to scan the sample in a Y axis direction within a scanning surface so as to pass through an emission surface of the planar light, a width direction of the sample is an X axis direction and the depth direction of the sample is a Z axis direction such that each of the X, Y, and Z axis directions are perpendicular to one another, the X axis is perpendicular to the Y axis within a plane perpendicular to the optical axis of the emission optical system, and the emission surface is a surface within a XZ' plane; an imaging optical system configured to form an image of observation light generated in the sample by emission of the planar light; an image acquisition unit that has a plurality of pixels arranged in a two-dimensional manner and which is configured to acquire a plurality of pieces of image data corresponding to an optical image of the observation light formed by the imaging optical system; and an image generation unit configured to generate observation image data of the sample based on the plurality of pieces of image data acquired by the image acquisition unit, wherein, when an angle formed by an optical axis (Pl) of the emission optical system and a normal of the scanning surface along the Z axis direction is Θ1 and an angle formed by an optical axis of the imaging optical system and the normal of the scanning surface is θ2, both θ1 and θ2 are 80° or less and a sum of θ1 and θ2 is 100° or more, and in the image acquisition unit, an image acquisition region of an (n+1)-th pixel is shifted from an image acquisition region of an n-th pixel in a scanning direction of the sample according to a scanning amount of the sample in an exposure time of one frame, wherein the image acquisition region of a pixel is the region in the XZ' plane which is imaged by the imaging optical system onto a pixel of the image acquisition unit and the respective image acquisition regions of the (n+1)-th pixel and the n-th pixel are aligned along the Z' direction.