Live cell assay that maximizes signal-to-noise ratio while minimizing phototoxicity. To define a standardized protocol for our assay, we established a range of imaging conditions that maintained cell viability, i.e., 80% of cells progressed through mitosis with normal timing. The kinetochore detection results under these imaging conditions were compared with the “ground truth,” determined by detecting kinetochores in very high signal-to-noise ratio (SNR) images (in which detection sensitivity was maximized at the expense of cell viability). With this, imaging conditions that allowed the automatic detection of >90% of kinetochores while maintaining cell viability were established. Although not depicted, we also optimized temporal sampling of the time-lapse imaging protocol (see Materials and methods). We found that recording image stacks every 7.5 s was sufficient to capture the dynamics of sister oscillation and breathing while maintaining satisfactory cell viability (Fig. 2, j and k). NEBD, nuclear envelope breakdown. Bar, 5 µm.