Automated Cell Cycle Analysis Figure 1. ‘Area’ and ‘Average Intensity’ features are sufficient to identify the boundaries of mitosis. (a) A portion of an image of HeLa cells expressing the fluorescent marker H2B-GFP is shown. The red asterisk marks an example of a nucleus automatically tracked throughout a division. Images were acquired every 12 minutes. At the time of division (between frames 4 and 5) each daughter nucleus is tracked independently to produce a ‘‘trace’’ for the division. The images of the trace are then used for the time-series analysis as shown in (b). (b) An example of a trace, showing the values of average intensity (I) and area (A) graphed over time with montage of nucleus images. The interphase-prophase transition (IPT) and metaphase-anaphase transition (MAT) points are identified. doi:10.1371/journal.pone.0025511.g001 late relative to manual analysis. The MAT was identified accurately 98% of the time. For the remaining 2% of cells, the TSA identified the transition point one frame late. This problem occurred when sister chromatids had not separated far apart enough to independently segment the two daughter nuclei. Overall, the TSA method was very accurate, as the TSA yielded a mean mitotic duration of 66.4 minutes relative to the manual measurement of 65.9 minutes, (p = 0.986; Figure 3a). We conclude that the time series method is very accurate in measuring mitotic duration for nuclei that are successfully tracked. A potential limitation of the DCELLIQ method is that it does not track all cells that divide during the course of the movie. Traces are removed from analysis if nuclei go out of the frame or touch the border of the frame, or if nuclei cross over one another during interphase or mitosis (see Trace Removal in Methods). During long movies necessary for determination of interphase duration, a significant proportion of cells are eliminated from analysis for these reasons. The longer the movie, or the greater the density of cells imaged, the smaller the proportion of the total number of mitotic divisions analyzed. In the current experiment lasting 48 hours, DCELLIQ analyzes one-third of all mitotic events. The analysis of a subpopulation of cells by DCELLIQ has the potential to introduce a selection bias into the results. In order to reveal a possible selection bias, we manually determined the mitotic duration of cells that were successfully tracked by DCELLIQ and compared the measurement to that of cells that were excluded from analysis (Figure 3a). The median and mean mitotic durations were longer for cells that were not tracked successfully (72 and 77.2 minutes, respectively) compared to those that were tracked (60 and 65.9 minutes, respectively), equal to a difference of one imaging PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e25511