Exploring the impact of variability in cell segmentation and tracking approaches

Segmentation and tracking are essential preliminary steps in the analysis of almost all live cell imaging applications. Although the number of open-source software systems that facilitate automated segmentation and tracking continue to evolve, many researchers continue to opt for manual alternatives for samples that are not easily auto-segmented, tracing cell boundaries by hand and re-identifying cells on consecutive frames by eye. Such methods are subject to inter-user variability, introducing idiosyncrasies into the results of downstream analysis that are a result of subjectivity and individual expertise. Such methods are also susceptible to intra-user variability, meaning findings are challenging to reproduce. Here we demonstrate and quantify the degree of intra- and inter-user variability in manual cell segmentation and tracking by comparing the phenotypic metrics extracted from cells segmented and tracked by different members of our research team. Furthermore, we compare the segmentation results for a ptychographic cell image obtained using different automated software and demonstrate the high dependence of performance on their imaging modality optimisation. Our results show that choice of segmentation and tracking methods should be considered carefully in order to enhance the quality and reproducibility of results. ### Competing Interest Statement The authors have declared no competing interest.