Abstract
Time-lapse microscopy offers a powerful approach for analyzing cellular activity. In particular, this technique is valuable for assessing the behavior of bacterial populations, which can exhibit growth and intercellular interactions in a monolayer. Such time-lapse imaging typically generates large quantities of data, limiting the options for manual investigation. Several image-processing software packages have been developed to facilitate analysis. It can thus be a challenge to identify the software package best suited to a particular research goal. Here, we compare four software packages that support the analysis of 2D time-lapse images of cellular populations: CellProfiler, SuperSegger-Omnipose, DeLTA, and FAST. We compare their performance against benchmarked results on time-lapse observations of Escherichia coli populations. Performance varies across the packages, with each of the four outperforming the others in at least one aspect of the analysis. Not surprisingly, the packages that have been in development for longer showed the strongest performance. We found that deep learning-based approaches to object segmentation outperformed traditional approaches, but the opposite was true for frame-to-frame object tracking. We offer these comparisons, together with insight into usability, computational efficiency, and feature availability, as a guide to researchers seeking image-processing solutions. IMPORTANCE: Time-lapse microscopy provides a detailed window into the world of bacterial behavior. However, the vast amount of data produced by these techniques is difficult to analyze manually. We have analyzed four software tools designed to process such data and compared their performance, using populations of commonly studied bacterial species as our test subjects. Our findings offer a roadmap to scientists, helping them choose the right tool for their research. This comparison bridges a gap between microbiology and computational analysis, streamlining research efforts.