A microscope device includes an objective (1); an actuator (6) for adjustment of a distance in a focusing direction z, an absolute z-position detector (7) for measuring a z-position. The device further includes an autofocus light source (11); a first optical arrangement (12, 13) for generating a focused spot (31) of autofocus light from the light source in the backfocal plane (14) of the objective (1) at a position offset from the optical axis of the objective by a lateral offset distance (d) The objective generates a collimated incoming beam (22) of autofocus light, which is directed at an oblique angle (β) relative to the optical axis (15) of the objective onto the substrate. The microscope may further include a second optical arrangement (13, 16) for generating collimated outgoing beams (34, 35) of autofocus light and directing the collimated outgoing beam onto a detector-array (17).