3 NA oil immersion objective (equipped with a DIC prism). Reflection and fluorescence channels were included as described above. We evaluated the results from TIAM against manually established ground truth by visual inspection as well as by the use of quantitative metrics.

We have also compared the performance of TIAM with other tools. We chose two benchmark datasets on fluorescent-labeled T cells subjected to antigen-induced and chemokine-induced motility that provided different experimental and acquisition settings as well as different motility characteristics (Table 1). We collected both DIC and fluorescence images in parallel, in order to perform tracking using both image series and compare the results. Tracking of cells in Pifithrin-�� mouse transmitted light image series in TIAM is performed by a two-tiered approach that involves linkage of neighboring cells in consecutive frames followed by joining of short segments by a global optimization routine (Fig. S3). To validate the segment joining algorithm in a principled manner, we computed the ATA before and after running the algorithm on a set of ground truth Ibrutinib tracks that had been synthetically broken. The accuracy improved drastically after joining the broken

segments, which implies correct pairs of segments were joined by the algorithm (Fig. S6). Including the segment-joining algorithm in TIAM improved the ATA values for both the benchmark experiments (Fig. S7). The improvement in ATA, expectedly, was more when less than optimal r-value was used for the nearest neighbor association. Tracks of cells obtained from TIAM showed good overlap with those from manually established ‘ground truth’ (Fig. 3a, Videos S1 and S2). This suggests that detection and tracking results from TIAM are reliable. Visual inspection of videos revealed that the fastest moving cells escaped being tracked. In some other cases cells were not tracked continuously, leading

to shorter tracks and/or multiple shorter segments (sub-tracks) corresponding to the same cell. This is most likely due to the failure of the nearest neighbor linkage during the periods of fast motility, especially in crowded areas. This observation provides an explanation for obtaining more tracks than in the ground truth and for under-estimation of mean track-length (Table 1, see below). Guanylate cyclase 2C While the modified nearest neighbor algorithm attempts to minimize the wrong track assignment by not doing any track assignment in case of ambiguity, tracking errors can nonetheless occur. In order to further characterize tracking errors, we manually recorded different types of errors in the track assignment by visual inspection using the stand-alone track visualization module of TIAM. Overall, the error rate in track assignment was estimated to be around 1% (Fig. S8). Thus, TIAM provides reliable detection and tracking of cells in transmitted light image series.