A system 100 and method are provided for imaging a sample in a sample holder. For providing autofocus, a 2D pattern is projected onto the sample holder 050 via an astigmatic optical element 120. Image data 172 of the sample is acquired by an image sensor 140 via magnification optics 150. A difference in sharpness of the two-dimensional pattern in the image data is measured along a first axis and a second axis. Based on the difference, a magnitude and direction of defocus of the camera subsystem is determined with respect to the sample holder. This enables the sample holder, and thereby the sample, to be brought into focus in a fast and reliable manner.