The approach begins with the evaluation of the area, A, as a function of time . Because mitotic division is associated 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 H2BGFP, a cutoff of 230 included 97.2% of true divisions , with a false positive rate of 4.5% . 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 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 . 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 , 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 purchase Bortezomib identifies a series of Fmin/ Fmax pairs, with each pair representing a potential division. The algorithm next determines the IPT using information based on PCI-32765 cost either I or A, depending on the characteristics of the trace. The algorithm first calculates the 1st derivative of the average intensity for all frames between Fmax and Fmin. We found that there are often rapid increases in dI at the IPT . 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 , 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 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.