Real-time focusing in a slide-scanning system. In an embodiment, focus points are added to an initialized focus map while acquiring a plurality of image stripes of a sample on a glass slide. For each image stripe, a plurality of frames, collectively representing the image stripe, may be acquired using both an imaging line-scan camera and a tilted focusing line-scan camera. Focus points, representing positions of best focus for trusted frames, are added to the focus map. Outlying focus points are removed from the focus map. In some cases, one or more image stripes may be reacquired. Finally, the image stripes are assembled into a composite image of the sample.