The invention provides a method of producing an in-focus image of an area on a sample plate for an ion source, e.g., a matrix-based ion source or any other type of ion source that employs a sample plate onto which samples are deposited. The method generally includes: a) positioning an area of the sample plate in the field of view of an imaging device; b) producing a plurality of images of the area having different in-focus regions; and c) generating an in-focus image of the area using the plurality of images. The in-focus image may be two-dimensional or three-dimensional. Systems and programming for performing the methods are also provided.