Automated Tracking Turns Cell Culture into Numbers Figure 7. Reliability of the threshold based cell detection. False Rejection Rate (FRR), False Acceptance Rate (FAR, left axis) and distribution of mitotic events (right axis). Inset: Counting false rejection (FR) and false acceptance (FA) errors; detected cells are displayed in red, reference (true) cells in blue. It is apparent from the schematic scenarios, that FA and FR errors often occur interrelated. doi:10.1371/journal.pone.0027315.g007 Evaluation of cell tracking (II): Complete path detection rate. To evaluate the tracking method, i.e. the algorithm which connects the detected cells through time, we found the number of correct complete paths to be an instructive measure (cf. table 2). However, it would be misleading to equate a high number of correct complete paths with a high quality of the tracking algorithm. In fact, algorithms, which return high numbers of correct complete paths are often afflicted with returning equally high numbers of wrong complete paths; valuable tracking results, on the other hand, require the correct paths to be of high "purity". Therefore, to evaluate a tracking algorithm, we relate the number of correct complete paths to the total number of complete paths; the quotient #correct complete paths #complete paths trustworthiness". Table 2 compares different path classification counts. Our first path construction algorithm (cf. section IV: First validation/path fragments) returns a four times higher number of short path fragments than the true number of paths is. This number can be reduced by a factor of two using an unvalidated, naive tracking approach to connect/elongate path fragments (3 rd column); the trustworthiness of these paths, however, is extremely low (56.4%). In contrast, our conservative validation algorithm leaves many path fragments unconnected, but the returned complete paths are can be called "tracking Table 2. Path classification count for different tracking approaches. Number of: paths detected mitoses correctly detected mitoses mitosis detection trustworthiness complete paths (mitosis-mitosis) correctly detected complete paths tracking trustworthiness 4684 2019 2019 100% 1635 1635 100% 17932 1012 865 85.5% 268 253 94.9% The first column shows the reference data, i.e. the manually determined number of paths and mitoses. Next, the number of path fragments is given (cf. section IV: first validation/path fragments). Connecting path fragments with naive tracking results in the numbers, displayed in the 3 rd column (cf. section IV: naive tracking). Validating the path continuations leads to significantly less complete paths, the reliability of which is, however, much higher (rightmost column). doi:10.1371/journal.pone.0027315.t002 PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27315 reference initial path fragments + initial adjacency naive tracking without validation with validation 8704 3299 1644 49.8% 1400 789 56.4% 11455 1283 1114 86.8% 377 360 95.5%