A method for autofocusing a microscope at a correct autofocus position in a sample includes the steps: generating a reference pattern by an autofocus light device, projecting the reference pattern towards a sample, whereby the reference pattern is backscattered by at least two interfaces being located at or close to the sample, projecting the backscattered reference pattern towards a detector which provides spatial resolution, obtaining a superposition of a number of detection patterns, each detection pattern related to one of the interfaces, on the detector, analyzing the superposition of detection patterns to identify at least one autofocus detection pattern related to at least one of the interfaces, and analyzing the at least one autofocus detection pattern to determine the direction and/or magnitude of deviation of the microscope's current focus position from the correct focus position.