Validation
A synthetic video of five cells with changing shape was used in the evaluation of tracking software. Each cell was tracked using respective tracking software, and their coordinates in pixels were obtained.
The following steps were taken to analyse tracking data:
 Convert synthetic trajectory coordinate points into pixels.
 Calculate the displacement in each frame for both synthetic and tracking data
 Calculate the distance travelled in each frame for both synthetic and tracking data
 Obtain the errors in coordinates, dislacement and distance travelled by the cell
The errors associated with manual tracking and Volocity software are shown below. All errors are given in terms of pixels.
Manual Tracking
Error in X Coordinate
 Error in Y Coordinate



Mean = 1.7354, Standard Deviation = 1.7187
 Mean = 1.2524, Standard Deviation = 1.0568

Error in X Displacement
 Error in Y Displacement
 Error in Distance




Mean = 0.0022, Standard Deviation = 1.0002
 Mean = 0.0018, Standard Deviation = 0.6795
 Mean = 0.1506, Standard Deviation = 0.9196

Volocity
Error in X Coordinate
 Error in Y Coordinate



Mean = 1.7149, Standard Deviation = 0.1376
 Mean = 0.4907, Standard Deviation = 0.1614

Error in X Displacement
 Error in Y Displacement
 Error in Distance




Mean = 8.2732e005, Standard Deviation = 0.1111
 Mean = 6.1416e004, Standard Deviation = 0.0712
 Mean = 1.4262e004, Standard Deviation = 0.0947

Evaluation
Volocity
Pros
 Lower error in terms of tracking centroid than other tracking software.
 Less time consuming.
Cons
 Failure to track cells continuously over a long number of frames > 70
 Baised to cells which run, rather than tumble
Manual Tracking
Pros
 Able to discern cells visually
 Able to track over larger number of frames
Cons
 Time consuming
 Higher error in terms of tracking centroid as compared to tracking software.
Conclusion
 Manual tracking will be used to track B.Subtilis.
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