This section describes the tracking process. The purpose of this step is to track cells from one image to the next and to build a lineage tree that contains every cell in every analyzed image and their relations to each other.
The Tracking step of the workflow is controlled by 2 panels: The Tracking panel contains elements to choose the tracking method and to execute the tracking process. The Edit lineage tree panel serves to manually edit the resulting lineage tree.
In the tracking step, cells can be selected by clicking on them either with the left or the right mouse button on both screens. It is possible to select two cells at once. The cell selected by a left mouse click is marked red, the cell selected by a right mouse click is marked cyan (alternatively, two cells/nodes can be selected by holding down the Strg/Ctrl key). When selecting a cell in the lineage tree it is shown on the actual image too an vice versa. Aditionally it is possible to select connections between cells in the lineage tree by a left click outside of the rectangles marking the cells. Cells and connections between cells can be deselected by clicking on a free area far away of any objects in the lineage tree. By marking cells it is possible to remove them or to edit the connections between them manually using the tools in the 'Edit lineage tree'-Panel.
The Edit button opens the Edit tracking panel that allows the selection of the tracking method and parameter settings.
The Execute button conducts the tracking process using the selected tracking method. For all tracking methods it is necessary that the displacement of the cells between subsequent images is moderate. Preferentially, according cells should completely overlap.
Edit lineage tree Panel
With the Add button a new connection is introduced between the two currently selected cells. This connection can be seen as a line in the lineage tree. The same function can be accessed by pressing the 'Insert' button on your keyboard. If nothing or only one cell is selected nothing will happen.
The Remove button is used to remove cells or connections between cells. If one cell or one connection between two cells is selected it will be removed. If two cells are selected, the connection between them will be removed. If there is no connection between these two cells nothing will happen. The same function can also be accessed by pressing the Remove button on the keyboard.
When removing a cell, all the data assigned to this cell, comprising the cell properties determined within the segmentation procedure, is deleted. The removal of cells is irreversible unless the segmentation is reexecuted. In this case, the complete lineage tree is restored. The removal of connections between cells is reversible by adding them with the Add button.
With the Remove group button a group of cells can be removed from the lineage tree. The Remove group panel allows to select which cells shall be removed.
The following cell groups can be selected from the dropdown menu:
- Single nodes: Cells that are not connected to any other cells. Usually they are aligned at the bottom left corner of the lineage tree.
- Children nodes: Cells descendant from the currently selected cell. With this option it is possible to remove whole branches of the tree. If no cell or two cells are selected nothing will happen.
- Not growing nodes: Cells that do not divide over the entire observation time. Often these objects aren't even cells but background particles. In the lineage tree they emerge as long straight branches without any bifurcations.
Select the desired group and click Ok to remove the according cells from the tree.
With the Layout button the layout of the lineage tree is recalculated, which can be useful after editing operations within the tree (addition or removal of cells or connections between cells).
With the Next button in the Workflow panel or selecting the appropriate entry in the workflow menu you enter the next step in the workflow, the Export.
Usually, the preselected tracking method 'overlapping regions' with no prediction of movement works good. If the cells displace alot between two subsequent images another tracking method shall be used. The first step is to modify the tracking method by pressing the 'Edit'-button. After that the tracking can be executed by pressing the 'Execute'-button. As a result a lineage tree is shown on screen 2.
Now it is necessary to inspect the lineage tree for logical correctness. This can be done by regarding the tree itself (it can be zoomed using the tools in the toolbar) or by moving through the sequence and regarding the association of cells from one image to the next on screen 1. The blue rectangles here mark the cell in the current frame, the green rectangles mark the cells in the last frame and the yellow lines mark the assignment of cells between the two frames.
If there are wrong assignments or background objects which were recognized as cells, the tree can be edited using the tools in the 'Edit lineage tree'-panel. Connections can be added or removed wheresas cells can only be removed. The cells and connections to modify can be selected by left- and right click on screen 1 and 2. After that, recalculate the layout using the 'Layout'-button.
Logical errors, like a cell which has been recognized as two cells in the last and the next frame, but as one cell in this frame, have to be corrected by going one step back to the segmentation (press the 'Back'-button). Here the image can be modified on screen 1 using the paint tool and the other tools from the 'Manual correction' panel or the toolbar. If you use one of the predefined segmentation processed these modification are done best at the end of the first process pipeline, so press the 'Edit'-button and in the appearing window select the first pipeline and the last operation in it. Then divide the cell manually using the paint tool by clicking the right mouse-button on screen 1 (can be zoomed using the tools in the toolbar). After that reexecute the segmentation for this image by pressing the 'Execute'-button in the 'Manual correction' panel. Now you can go back to the tracking by pressing the 'Continue'-button and reexecute the tracking process by pressing the 'Execute'-button.
When the lineage tree is corrected completely continue to the next step using the 'Continue'-button.