This article describes the configuration and application of an image processing software developed for extracting periodic atomic features from scanning tunneling microscopy (STM) images. This was done with a view to calibrating the lateral and/or vertical scale of images acquired by a STM with two tunneling units, where a crystal can be used as the scale reference with one of the tunneling units. The image processing software was applied to consecutively acquired images of graphite with a size of 150×5 nm. The number of lattices automatically counted in a line scan of 150 nm, scanned in 400 ms, showed a matching of 99.95% from one image to the other. On the other hand, the matching in the feed direction, where image width was only 5 nm, but took 240 s, was 95%. As an example of application of the developed software, nonlinear movement of piezoscanners were readily monitored in the 100 nm order.