Table 1. Status flags for path classification and lineage construction. begin end mitosis cell death lost begin lost end merged The path begins in the first frame of the image series. The path ends in the last frame of the image series. border begin Path without predecessor starting near the image border*. border end Path without successor ending near the image border*. The path ends with a mitosis. The path ends with an apoptosis/cell death. No predecessor but neither status "begin" nor "border begin". No successor but neither status "end" nor "border end". Two or more cells are not separable and detected as one. The first 6 flags (bold) determine terminal starting / end points of paths. The next 2 flags indicate starting / end points which are in need for completion through either connection, correction, or removal from the tracking evaluation. Although the last flag ("merged") is not used to classify the ends of a path, it implies both a set "lost start" and "lost end" flag. *) The vicinity of the border is defined such that the distance between the centroid of the cell and the image border is smaller than a minimum distance (typically about 25–40 pixel, corresponding roughly to 50 mm). doi:10.1371/journal.pone.0027315.t001 Results and Discussion I) Analysis of tracking errors Validation algorithms are basically error detection strategies. We will therefore first analyze the errors, which may emerge in cell tracking. It turned out that many errors do not occur in cell detection only, but also in reconstructing their temporal continuity. Therefore, we give a description of typical error scenarios that we have encountered while inspecting our tracking Automated Tracking Turns Cell Culture into Numbers results. In fact, the error scenarios given below constitute a complete classification of all possible tracking errors. This allows us to assemble them in an error classification tree which in turn has been translated into an algorithm to automatically validate tracking results. Scenario 1: Cells at the image border. Cells in close vicinity to the image border may either leave or enter the observed area (see fig. 4a). Likewise, when most of the cell body lies outside the image, the visible part could become too small to be noticed by the cell detector. Since the identity of these cells cannot be determined with sufficient reliability, they are excluded from the tracking evaluation. That is, all paths with either a set border- begin or border-end flag cannot be taken into account for further cell tracking. This error scenario only reduces the number of paths that can be evaluated. Its impact can be minimized by observing larger areas, because the number of cells scales linearly with the area, whereas the number of border cells scales with the square root of the observed area only. Scenario 2: Undetected cells. The tracking algorithm may overlook "invisible" cells, i.e. cells, whose features do not meet the requirements of the detector. In our case this would happen, if a cell appeared too dark or too small with respect to the image threshold values (see fig. 4b). This detection error can only be corrected if the "invisible" cell itself, or its ancestor/successor is detected at some point. If otherwise, a cell (and its whole progeny) would pass unnoticed in all images, its consequence is the removal of the overlooked path/tree from the overall tracking results. Scenario 3: Merged cells. Two or more cells, which are located in very close proximity to one another may optically merge and appear as only one cell (see fig. 4c). This is the most critical error in cell tracking, as it appears to occur rather often and cannot be resolved trough optimized detection. Not only do merged cells cause falsely terminated paths, but they may also Figure 4. Error scenarios. A: Cells at the image border. Two cells first leave the observed area (image border at the right) and subsequently re- enter. An unambiguous temporal assignment is not possible. B: Undetected cell A cell may pass unnoticed, when its properties do not meet the requirements of the cell detector. In the depicted case, cells become too dark (arrows). Such "vanishing" or re-appearing cells result in a "lost cellpath" flag. C: Merged Cells Two cells appear to merge into one. It is not possible to tell the merged cells apart by detection alone; the error can only be noticed by taking into account the temporal nexus. D: Debris A non-cell/dead-cell object moves fast through the observed area. doi:10.1371/journal.pone.0027315.g004 PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27315