with a decrease in A, the program first determines whether the trace contains any A values less than 230 pixels; if not, phase for the entire trace is set to interphase. In HeLa cells expressing H2B- GFP, a cutoff of 230 included 97.2% of true divisions (of 109 divisions analyzed), with a false positive rate of 4.5% (later steps in the algorithm eliminate most of these false divisions). If the trace contains any A value less than 230, all local minima ,230 are then found using a 47 frame search window, identifying one frame containing the local minimum (Fmin) within the period window. The search window width was chosen because cells do not divide more often than once every 10 hours in 12 min interval imaging. Once Fmin is identified in each search window, the frame containing the maximum A value in the preceding 50 frames is identified (Fmax). Therefore, each Fmin value is associated with an Fmax, which gives an approximate indication of duration of mitosis. This approach results in a maximum potential mitotic duration of 50 frames (10 hours if imaged every 12 minutes), which may lead to underestimation of mitotic duration under conditions where cells arrest in mitosis for very long periods. In summary, the first part of the algorithm identifies a series of Fmin/ Fmax pairs, with each pair representing a potential division. The algorithm next determines the IPT using information based on either I or A, depending on the characteristics of the trace. The algorithm first calculates the 1st derivative of the average intensity (dI/dt; abbreviated dI) for all frames between Fmax and Fmin. We found that there are often rapid increases (dI.30 units/frame) in dI at the IPT (Figure S4b). Biologically, this correlates with condensation of chromatin, producing a nucleus with greater average fluorescence intensity. If there is at least one frame between Fmax and Fmin with a dI.30, the algorithm chooses IPT as the frame closest to Fmin whose dI value exceeds 30. However, many traces do not contain rapid increases in I as nuclei enter mitosis (Figure S4c), and thus an intensity-based method cannot be used to identify IPT for all traces. In these cases, the algorithm identifies the IPT based on A. The algorithm begins at Fmax and determines the number of frames to Fmin. If Fmax and Fmin are sequential frames, all frames are set to interphase, as this is unlikely to be a true division, because manual analysis of HeLa cell divisions indicated that no divisions were shorter than 30 minutes (2 frames) in untreated cells. The algorithm then searches forward in time from Fmax for the first frame that shows dA,250, indicating a significant decrease in A. Biologically, this represents the decrease in nuclear area associated with chromo- some condensation and the formation of the metaphase plate. Setting a threshold at this value enabled sensitive identification of mitotic divisions, but use of this criterion by itself led to a high false positive rate. We identified two reasons for identification of false positives. First, single-frame spikes in A, which were generally due to segmentation errors, could produce a dA,250 in the subsequent frame. To address this problem, the algorithm requires that the A in the current frame be less than 120% of the value of the previous frame. If this criterion is not met, it suggests that an artifactual spike in A has occurred, and the program continues to search for another frame with a dA,250. The second source of false-positive identifications is temporary decreases in A that are not true divisions. To eliminate these false-positives, the algorithm requires that A of the selected frame be substantially greater than each of the subsequent A values to Fmin. The algorithm uses different thresholds (either .120% or .111% of the A value in each subsequent frame) depending on the number of frames between Fmax and Fmin, because the rate in change of A is dependent on the duration of mitosis. If the current frame does not meet these criteria, the algorithm continues to search. If all of these criteria are met, however, the current frame is selected as the IPT. Automated Cell Cycle Analysis The final stage of the algorithm identifies the metaphase- anaphase transition (MAT) based on analysis of A. In this case, MAT is defined as the first frame of anaphase. The result from the IPT algorithm is passed along to the MAT algorithm, which searches from the IPT for the A minimum in the following 50 frames. In most cases, this value represents the first frame of anaphase, because all subsequent frames show increases in area, reflecting decondensation of chromatin in early telophase (see Figure 1b for example). However, in some cases, we found that the local minimum corresponded to the second rather than the first frame of anaphase. These cases could be identified by comparing the value of the local minimum to the value in the previous frame. DCELLIQ detects only about half of all mitotic events before trace exclusion. Some factors that are involved in reducing the number of events obtained by automated analysis are: (1) all incomplete traces due to cells entering or exiting the frame during the movie are removed, (2) at the step of segmentation, any cell that is touching the border of the image (even by a pixel) is eliminated and is not registered as an object. For example, a cell that stays in the frame during the whole movie but touches the border just once will not result in a trace. The likelihood that a trace will be excluded from a movie for this reason increases with the length of the movie, because there is a higher likelihood that a cell will enter or exit the frame at least once if the imaging time is longer. Effect of Removing Incorrect Traces Accurate measurement of interphase and mitotic duration is highly dependent on accurate tracking of a nucleus from frame to frame. The accuracy of tracking tends to decline if cell density is high, if the experiment is long, if the cells are highly motile, or if the frequency of imaging is low. One reason for this is that these conditions make it more likely that two nuclei will cross over one another during the experiment, making accurate tracking difficult. The chances of such an event occurring increase the longer the experiment is performed. Determination of interphase duration therefore places especially high demands on accurate tracking because the cells need to be followed for a long period of time. We therefore determined the accuracy of the segmentation and tracking of DCELLIQ using two movies that lasted 48 hours. We found that 27% of the traces contained at least one tracking error (Figure S5a). Of these errors, 36% were a consequence of segmentation errors, 51% were due to incorrect selection of neighboring cells (due to overlapping nuclei or nuclei that approached closely to one another during the movie), and 12% were due to abnormal division of the cells (formation of more than two daughter nuclei during division). Traces containing segmen- tation and tracking errors show sudden increases in A that are not observed in correct traces. There is no biological reason for a rapid increase in A; however, incorrect segmentation or two nuclei crossing frequently produced a rapid increase in A within one or two frames. We found that incorrect traces frequently contained a large dA (1st derivative of the A trace, dA/dt; abbreviated dA) value (.130); in contrast, correct traces rarely had a dA value exceeding 130 (Figure S5b). At this threshold, we could remove 79% of incorrect traces while excluding only 6% of correct traces. We analyzed the effect of implementing this approach in the analysis of an independent experiment (48 hrs movies of DMSO- treated cells analyzed in Figure 3d). Of 106 traces identified by DCELLIQ, the exclusion criterion led to elimination of 66 traces. Of these 66 traces, 19 (28%) were a consequence of segmentation errors, 11 (16%) were due to abnormal cell division, and 36 (54%) were due to nuclei that overlapped during the course of the experiment. None of the remaining 40 traces contained a tracking PLoS ONE | www.plosone.org 12 September 2011 | Volume 6 | Issue 9 | e25511