★Track: Inferred counting and tracking of replicating DNA loci

Fluorescent microscopy is the primary method to study DNA organization within cells. However the variability and low signal-to-noise commonly associated with live-cell time lapse imaging challenges quantitative measurements. In particular, obtaining quantitative or mechanistic insight often depends on the accurate tracking of fluorescent particles. Here, we present ★Track, an inference method that determines the most likely temporal tracking of replicating intracellular particles such DNA loci while accounting for missing, merged and spurious detections. It allows the accurate prediction of particle copy numbers as well as the timing of replication events. We demonstrate ★Track’s abilities and gain new insight into plasmid copy number control and the volume dependence of bacterial chromosome replication initiation. By enabling the accurate tracking of DNA loci, ★Track can help to uncover the mechanistic principles of chromosome organisation and dynamics across a range of systems. Significance Microscopy is one of the main tools for studying the intracellular organisation of cells. In particular, fluorescent proteins allow us to study the dynamics of many cellular processes. However, this requires the accurate tracking of fluorescent foci. Here, we present ★Track a tool tailored to the tracking of replicating persistent subcellular particles such as DNA loci. ★Track provides accurate predictions of particle copy number and replication timing even in the presence of substantial noise. The knowledge of these quantities are critical for uncovering the mechanisms behind many cell-cycle dependent processes, such the control of chromosome and plasmid replication initiation.