An important consideration in such Gefitinib experiments is the frequency of imaging. More frequent imaging provides better temporal resolution, but fewer positions can be analyzed at once, and thus fewer conditions can be tested in a given experiment. We therefore assessed how changing the frequency of image acquisition influences the ability of the time-series method to accurately measure mitotic duration as compared to manual analysis. We Semaxanib imaged HeLa H2B-GFP cells with an imaging interval of 4 minutes for 24 hours. This short imaging interval enables many images to be captured during mitosis . Under these conditions, manual and automated analysis measured identical median mitotic durations of 52 minutes, with the automated method measuring a slightly shorter mean mitotic duration . This difference was not statistically significant , but we explore the basis for this trend below. To model what would happen when the imaging frequency is decreased, we repeated manual and automated analysis on the same movies, but used every other frame or every third frame to reflect imaging intervals of 8 minutes or 12 minutes, respectively. In this case, the mean and median mitotic duration increased as imaging frequency decreased. Because manual and automated analysis provided very similar results, the change in imaging frequency, rather than the analysis method, must be responsible for the difference. We determined that the increase in measured duration results from the fact that the interphase-prophase transition point is chosen as the last frame of interphase when early prophase is not imaged. As the imaging interval increases, the likelihood that early prophase will be missed increases, leading to a slight overmeasurement of sample median and mean . Thus there is an inherent trade-off between imaging frequency and accuracy of measurement of mitotic duration. However, this trade-off is inherent to time-lapse imaging approaches, and is not a consequence of implementation of the automated analysis method per se. Based on this analysis we used frequent imaging for perturbations that were expected to shorten mitotic duration, or less frequent imaging for cases that were predicted to extend mitotic duration.