Motivation:Cell division inEscherichia coliis morphologically symmetric. However, as unwanted protein aggregates are segregated to the cell poles and, after divisions, accumulate at older poles, generate asymmetries in sister cells’ vitality. Novel single-molecule detection techniques allow observing aging-related processesin vivo, over multiple generations, informing on the underlying mechanisms. Results:CellAging is a tool to automatically extract information on polar segregation and partitioning in division of aggregates inE.coli, and on cellular vitality. From time-lapse, parallel brightfield and fluorescence microscopy images, it performs cell segmentation, alignment of brightfield and fluorescence images, lineage construction and pole age determination, and it computes aging-related features. We exemplify its use by analyzing spatial distributions of fluorescent protein aggregates from images of cells across generations. Availability:CellAging, instructions and an example are available athttp://www.cs.tut.fi/%7esanchesr/cellaging/.