We present a software package called Microscopy Image Segmentation Tool (MIST). MIST is designed for analysis of microscopy images which contain large collections of small regions of interest (ROIs). Originally developed for analysis of porous anodic alumina scanning electron images, MIST capabilities have been expanded to allow use in a large variety of problems including analysis of biological tissue, inorganic and organic film grain structure, as well as nano- and meso-scopic structures. MIST provides a robust segmentation algorithm for the ROIs, includes many useful analysis capabilities, and is highly flexible allowing incorporation of specialized user developed analysis. We describe the unique advantages MIST has over existing analysis software. In addition, we present a number of diverse applications to scanning electron microscopy, atomic force microscopy, magnetic force microscopy, scanning tunneling microscopy, and fluorescent confocal laser scanning microscopy.