IGEM:IMPERIAL/2008/Prototype/Drylab/Validation: Difference between revisions
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==Microscope Experimental Setup== | |||
==Microscope Video Characteristics== | ==Microscope Video Characteristics== | ||
Revision as of 03:33, 27 August 2008
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Validating the Tracking Software
From the video of the cells obtained via the wiedfield microscope, we determined using the measurement tool of ImageJ the average intensity and standard deviation of a few cells over a different frames. Identical measurements were made for the background. We identified a few static noisy patterns in the background as well. They might be due to optical effects.
We generated a synthetic video consisting of a low intensity blob of changing shape, moving about a darker background. The SpotTracker failed to track the moving blob. Although it should have spotted the brightest spot first ( which is somewhere on the blob), it wrongly identifies point of coordinates (2,2) in pixels as being the brightest spot. We hypothesised that perhaps the blobs should not be of uniform intensity.
Using the measure command of ImageJ we have acquired the following properties for the background: mean intensity, standard deviation of intensity, skewness and kurtosis.Similarly the same properties were extracted for a set of cells taken randomly over different frames. Additionally, from the contour of the cells, we obtained the dimensions of the ellipses that would best fit each cell's shape. This data can be found HERE.
The second synthetic video had a noisy Gaussian distributed background. We centred the synthetic background intensity to the mean intensity of the background of the microscope video. The standard deviation of the synthetic background's intensity was made to be the same as that of the background from the microscope video. We attempted to accurately model one cell as seen from the microscope. We represented it by 2 concentric ellipses: the inner one of brighter intensity, the outer one of darker intensity. Furthermore the ellipses were made to change shape, so as to mimic the fact that the cells in the microscope video swim away from the focal plane.
In theory when the Spot Tracker algorithm is launched it should spot the brightest spot in the image sequence. In practice using our experimental data (microscope video) we notice that it does actually the brightest spot. The software will then track the spot along all the frames. However, using our synthetic data the software spots the ellipse (representing a cell) but fails to spot the brightest part of the ellipse. Instead it is spotting a random point of the ellipse.
Approach
The following figure describes our approach in validating the tracking software.
Microscope Experimental Setup
Microscope Video Characteristics
From our experience on the microscope, we observe the following:
- Bacteria are rod shaped. As the bacteria transverse across the plate, it may change shape and orientation.
- Due to tumbling in the z-direction, bacteria may go in and out of focus. This leads to change in light intensity
- Background noise is also present
Bacteria Motility
Bacteria motility comprises of periods of "runs", referring to movement in a single direction at relatively constant velocity, and periods of "tumbles", where the bacteria changes its orientation and thus direction. During runs, the flagellum of the bacteria are bundled together and turn in an anti-clockwise direction, propelling the bacteria forward. Tumbling is caused when one or more flagellar flare up and break away from the bundle, turning in a clockwise direction. Runs usually last for ~1 seconds, while tumbles last for ~0.1 seconds.
The assumptions are used in generating synthetic motility data:
- Run velocity is Maxwell distributed.
- Tumbling Angle is Von Mises distributed.
- Run Duration and Tumbling Duration are Exponentially Distributed with parameter lamda of 1.0 and 10.0 respectively.
Synthetic Video
A synthetic video will be generated with MATLAB. A function is written to create the video. This function will call upon two other functions which return motility data and bacteria characteristic data which are used as inputs for the bacteria to be drawn on a plot.
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