Team:SUSTech Shenzhen/Cell Tracking Algorithm
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Cell Tracking Analysis
We have developed a control program to dynamic control cell secretion. Next we want to analyze the migration behavior of mammalian cells. To this end, we chose chemotaxis as our testing model which is described as the directed migration of cells towards a chemoattractant. We used IL8 as the chemoattractant which can promote the activation and chemotaxis of neutrophils.
Overview
Even have we taken use of microfluidic chips to show the migration of neutrophils, it is still not clear enough to display the detailed migration. Therefore, we needed more statistical data to support our results. Based on CSR-DCF and OpenCV, we wrote a tracking program that can track cells on bright-field for a long time. The program could output a visible plot and all position information for those eligible cells. With that information, we could do some statistic analysis to verify the effectiveness of chemotaxis. We chose three basic relevant chemotaxis parameters, forward migration index, the center of mass and directness.
Forward Migration Index (FMI)
Forward migration index(FMI) is a statistic index that indicates how efficiently the forward migration of cells is. Generally speaking, it needs a forward migration index for both orthometric axes in a plane. By parity of reasoning, three forward migration indexes are needed for a spatial pattern. The formula of forwarding migration index shows in formula (1),(2),
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In our project, we defined the x-axis as parallel to the gradient while the y-axis perpendicular to the gradient. Besides, n is the number of cells that we tracked, xi,end & yi, end are coordinates of the endpoints of cells, and di, accum is the whole path length of every cell. Considering the practical meaning of FMI, the larger the absolute value of FMI, the stronger the chemotactic effect is on the axes.
The Center of Mass (Mend)
The center of mass (Mend) is the coordination of the average of all single-cell endpoints. Usually, we use this index to observe the average distribution of endpoints. The formula of the center of mass is shown as formula (3),
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in which n is the number of cells that we tracked, and xi, end & yi, end are coordinates of the endpoints of cells. The position of the center of mass can display how the group of cells primarily traveled.
Directness(D)
Directness(D) is a measure of how straightness the cell trajectories are. For every single cell, the method of calculation is indicated as formula (4),
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in which Di is the directness of every single cell, di, euclid is the euclidean distance of cell, in other words, the straight-line distance between initial point and endpoint. Besides, di, accum is still the whole path length of every cell. Since there is no coordinate in the formula, directness is always a positive value between 0 and 1. And the larger the value, the straighter the route. To display the global condition, the average of the directness of all cells is calculated by the formula (5),
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where n is the number of cells. Synthesize all the indexes above, we can speculate whether the chemotaxis works.
Analysis
Making use of the cell tracking program and statistical analysis methods, we can output a visible plot and three indexes to analyze.
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From Fig 1, a rough impression came to us and told us how the cells mainly migrate while data in Fig 2 showed more specific details. Since IL8 was placed on the negative axis of our microfluidic chips in this experiment, the coordinates of endpoints showed a tendency. Specifically, these points tend to be negative on the x-axis while more randomly distributed on the y-axis. Although there was a difference in tendency, it was not very clear and accurate. Still, the absolute value of FMI in parallel direction was far larger than in the perpendicular direction, which indicated a stronger migration in the parallel direction. And the minus indicated that they migrated towards IL8, which provided evidence that our IL8 was still active biologically. Besides, the center of mass showed the primary migration of the whole cell group. The coordinates of the center of mass in this experiment were both negative and significantly biased towards the negative x-axis. And directness here is about 0.148, which could show some influence of IL8 on the cells. This result also reminded us that cell is a complex system after all. What we have done was attempting to explore a feasible method to control it slightly.
In conclusion, we utilized machine vision and statistical tools to analyze the migration experiment result. It was satisfactory that we found those neutrophils tend to approach IL8 which were secreted by our modified cells. These results provided a wonderful guide to our experiment.