IGEM:IMPERIAL/2008/New/Motility/Validation: Difference between revisions
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=Validating Tracking Software= | = Validating Tracking Software = | ||
ImageJ was used primarily to analyse images. Various plugins were tested, such as SpotTracker and Particle Tracker, but both failed to producing any form of convincing result. Microscopy videos were captured using Volocity software, which also provided a cell tracking function. Thus analysis of synthetic videos was done '''manually''' and using 64-Bit '''Volocity''' analysis software. The synthetic video shown to the right was used as an input for the tracking of synthetic cells. | ImageJ was used primarily to analyse images. Various plugins were tested, such as SpotTracker and Particle Tracker, but both failed to producing any form of convincing result. Microscopy videos were captured using Volocity software, which also provided a cell tracking function. Thus analysis of synthetic videos was done '''manually''' and using 64-Bit '''Volocity''' analysis software. The synthetic video shown to the right was used as an input for the tracking of synthetic cells. | ||
==Volocity== | == Volocity == | ||
Volocity 64-Bit was used to analyse synthetic videos created. The following protocol was followed: | Volocity 64-Bit was used to analyse synthetic videos created. The following protocol was followed: | ||
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#Begin tracking using Ctrl-U | #Begin tracking using Ctrl-U | ||
==Manual Tracking== | == Manual Tracking == | ||
Synthetic cells were tracked using ImageJ via the Manual Tracking Plugin. The following protocol was followed: | Synthetic cells were tracked using ImageJ via the Manual Tracking Plugin. The following protocol was followed: | ||
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#Once tracking is complete, end track and save results with a .xls extension. | #Once tracking is complete, end track and save results with a .xls extension. | ||
=Validation Process= | = Validation Process = | ||
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. | 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. | ||
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#Calculate the displacement in each frame for both synthetic and tracking data | #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 | #Calculate the distance travelled in each frame for both synthetic and tracking data | ||
#Obtain the errors in coordinates, | #Obtain the errors in coordinates, displacement 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. | The errors associated with manual tracking and Volocity software are shown below. All errors are given in terms of pixels. | ||
==Manual Tracking== | == Manual Tracking == | ||
{| border="1" cellpadding="5" cellspacing="0" align="center" | {| border="1" cellpadding="5" cellspacing="0" align="center" | ||
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|} | |} | ||
==Volocity== | == Volocity == | ||
{| border="1" cellpadding="5" cellspacing="0" align="center" | {| border="1" cellpadding="5" cellspacing="0" align="center" | ||
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|} | |} | ||
=Evaluation= | = Evaluation = | ||
{| border="1" cellpadding="5" cellspacing=" | {| border="1" cellpadding="5" cellspacing="0" align="center" | ||
|- | |- | ||
| | | | ||
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|- | |- | ||
|Pros | |Pros | ||
| | |Lower error in terms of tracking centroid than other tracking software. <br> Less time consuming. | ||
| | |Able to discern cells visually. <br> Able track over larger number of frames. | ||
|- | |- | ||
|Cons | |Cons | ||
| | |Failure to track cells continuously over a long number of frames > 70. <br> Biased to cells which run, rather than tumble. <br> Failure to discern motile from immotile or dead cells. | ||
| | |Time consuming. <br> Higher error in terms of tracking centroid as compared to tracking software. | ||
|- | |- | ||
|} | |} | ||
==Conclusion== | == Conclusion == | ||
*Manual tracking will be used to track ''B. | *Manual tracking will be used to track ''B. subtilis''. | ||
{{Imperial/EndPage}} | {{Imperial/EndPage}} | ||
Latest revision as of 20:01, 16 September 2008
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Manual TrackingSynthetic cells were tracked using ImageJ via the Manual Tracking Plugin. The following protocol was followed:
Validation ProcessA 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:
The errors associated with manual tracking and Volocity software are shown below. All errors are given in terms of pixels. Manual Tracking
Volocity
Evaluation
Conclusion
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