A method for enhancing contrast in fluorescence imaging is provided. The method comprises providing a patterned illumination source for illuminating one or more regions corresponding to a scan step, scanning at least a portion of a surface of a subject using a plurality of scan steps, acquiring image frames corresponding to two or more scan steps, deducting a background fluorescence from the image frames corresponding to the two or more scan steps to form one or more processed image frames, and reconstructing an image using one or more of the processed image frames.