Automated Tracking Turns Cell Culture into Numbers Figure 9. Proliferation curve of stem cells from the exocrine pancreas of rattus norwegicus. The cell count increases roughly tenfold during the time-lapse observation. Reference data are shown as a solid line; automatically detected cells are displayed as a dotted line. The automatically calculated curve displays a very low error (mostly ,5%). The cell number correlates well with the number of mitotic events (histogram bars). doi:10.1371/journal.pone.0027315.g009 the growth area, because the threshold based detector finds only the cell body while chopping off all fine details of the cells (e.g. podia). When compared with the reference data, our fully automated cell detection delivers very accurate figures (error ,2%). Life/Cycle time distribution. One particularly telling parameter to characterize stem cell populations is the distribution of life times, i.e. the time span between two mitoses (length of complete paths). In the ideal case that all cells behaved the same, their life time would be fairly identical. Thus, the shape and width of life time-distributions can be used as a parameter to assess the heterogeneity of a cell population. This is of specific importance for adult stem cell cultures: It is a commonly held believe, that stem cells proliferate rather slow, but give rise to faster expanding progenitor cells; these progenitor cells would then - after some generations - produce terminally differentiated cells, which are believed to be not proliferative at all (e.g. Watt et al. [34] or Dingli et al [35]). Such cell fractions - short living progenitors, longer living stem cells and "everliving" terminally differentiated cells - should appear in the life time distribution figures. Depending on their difference in life time and frequency, they would either give rise to isolated peaks in the distribution function or at least broaden it. Figure 11a displays the life time distribution of reference data set A, calculated from 1635 complete paths. Although the distribution function features only one distinctive peak, one might reason from its asymmetric shape, i.e. the pronounced tail towards longer life times, that the pancreatic stem cell population is not very homogeneous and contains various subpopulations. This conclusion, however, would erroneously neglect the effect of contact inhibition. Therefore, figure 11b gives a more detailed account of life time distributions; here, each life time (path length) is plotted at the time-point, when the path ends. This figure reveals that after about 36 hours, the average life time starts to increase from about 12 hours (subconfluency) to roughly 20 hours (con- fluency). Still, life time distributions seem to be rather broad, even within the two distinct growth regimes (see fitted distributions in fig 11a), indicating diverse cell types or different stages of differentiation. Cellular genealogies. True single cell information about in vitro culture can only be gained if the whole connectivity matrix, i.e. the genealogical nexus of each cell, is taken into account. Several advanced measures have been proposed to compare and identify different cell fates based on the topological features of PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27315