colour. Number assignation will allow to easily distinguish
tracks in this situation.
9. Set up parameters for automatic maximum finding and subpixel position by center of mass. These parameters determine
how the program localizes the nucleus after it has moved from
the previous time point. A very low radius will minimize jumping to a different nucleus but will lose the tracked nucleus in big
movements. A high radius will allow for following big leaps in
migration, but it will make it easier for the track to jump to a
different nucleus. What constitutes a “low” or “high” value will
depend on the relative size of the nuclei and the distance
between them and will need to be decided by trial and error.
In the case of TNC, appropriate values for X, Y, and Z range
between 3 and 8. These can be adjusted throughout the tracking to accommodate for more or less clustered migration.
10. Press “OK” to start tracking.
11. Set the cursor on top of the nucleus to track in the first time
point it appears and press “m.” This nucleus will now have
track number 1. At this point take note of which track correspond to which cell, so later on you can compile data from
different cell populations (e.g., cell1 at the front of the migratory group, Leader; cell 2 in the middle of the group, Follower
2; or cell 1a Leader’s front daughter; etc.)
12. Press “W” (Shift+w) for automatic tracking of the nucleus. A
track line will appear. The colour of the track line can be
changed by pressing “w” while working on that track.
13. Use the keys “9” and “0” to circulate through the track and
ensure that it follows the selected nucleus in all planes XY, XZ,
and YZ (look at the different windows, all should display the
corresponding track).
To correct an erroneous location along the track, drag the
corresponding mark to the appropriate location follow steps
14–18.
14. If the track returns to the wrong nucleus after dragging it to
the right one, open the Marker positioning menu and remove
the ticks from “Use automatic maximum finding” and “Subpixel position by of Mass.” This will make the tracking manual
until the boxes are ticked again, allowing for the correct positioning of the marker.
15. Once the marker is positioned correctly, advance to the following time point by pressing “0.” The track will still follow the
trajectory traced automatically by the computer beforehand.
You can manually correct it as before or delete the rest of the
track and re-track the path for the remaining time points. To
delete the track in this and all following time points press “Q”
(Shift+q).
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tracks in this situation.
9. Set up parameters for automatic maximum finding and subpixel position by center of mass. These parameters determine
how the program localizes the nucleus after it has moved from
the previous time point. A very low radius will minimize jumping to a different nucleus but will lose the tracked nucleus in big
movements. A high radius will allow for following big leaps in
migration, but it will make it easier for the track to jump to a
different nucleus. What constitutes a “low” or “high” value will
depend on the relative size of the nuclei and the distance
between them and will need to be decided by trial and error.
In the case of TNC, appropriate values for X, Y, and Z range
between 3 and 8. These can be adjusted throughout the tracking to accommodate for more or less clustered migration.
10. Press “OK” to start tracking.
11. Set the cursor on top of the nucleus to track in the first time
point it appears and press “m.” This nucleus will now have
track number 1. At this point take note of which track correspond to which cell, so later on you can compile data from
different cell populations (e.g., cell1 at the front of the migratory group, Leader; cell 2 in the middle of the group, Follower
2; or cell 1a Leader’s front daughter; etc.)
12. Press “W” (Shift+w) for automatic tracking of the nucleus. A
track line will appear. The colour of the track line can be
changed by pressing “w” while working on that track.
13. Use the keys “9” and “0” to circulate through the track and
ensure that it follows the selected nucleus in all planes XY, XZ,
and YZ (look at the different windows, all should display the
corresponding track).
To correct an erroneous location along the track, drag the
corresponding mark to the appropriate location follow steps
14–18.
14. If the track returns to the wrong nucleus after dragging it to
the right one, open the Marker positioning menu and remove
the ticks from “Use automatic maximum finding” and “Subpixel position by of Mass.” This will make the tracking manual
until the boxes are ticked again, allowing for the correct positioning of the marker.
15. Once the marker is positioned correctly, advance to the following time point by pressing “0.” The track will still follow the
trajectory traced automatically by the computer beforehand.
You can manually correct it as before or delete the rest of the
track and re-track the path for the remaining time points. To
delete the track in this and all following time points press “Q”
(Shift+q).
96
Zain Alhashem et al.