16. Open the Marker positioning menu by clicking “n” and tick
the “Use automatic maximum finding” and “Subpixel position
by Centre of Mass” boxes again.
17. Return to the last marker available in the track by pressing “9”
and “0.”
18. Press “W” (Shift+w) again to restart the tracking.
19. Once track 1 is correct and complete, select a different nucleus
at the beginning of its migration and press “k.” This will create
marker number 2. Repeat steps 11–13 for each nucleus you
need to track.
20. To save all tracks, hover the cursor over command window
(bottom right) and press “m.” Importantly, do this periodically
as the program does not save automatically and you may lose all
your work if the program or the computer freezes. Pressing
“m” shows the raw data on the command window and creates a
text file in the selected “MarkerFileOut” folder with your
tracking data.
In case of cell division during a track, refer to steps 21 or
22 to either follow only one daughter cell as a continuation of
the on-going track (step 21) or to follow both daughter cells
after division (step 22).
21. To follow only one daughter cell as part of the same track
(a single track showing cell1 plus one of its daughters, but
ignoring the second daughter), place the marker on that
daughter cell once the division is completed and press “W”
(Shift+w) to continue the track (if the program has not done so
automatically).
22. To record the division in the data file and follow both daughter
cells as related tracks, go to the first time point after the division
and press “\” (backslash). This will create two new related
tracks labeled “1a” and “1b.” Move the “1a” marker to one
of the daughter cells; move to the next time point (second time
point after division) and delete all tracking after this point by
pressing “Q”; press “W” (Shift+w) and follow steps 8–11 to
re-track the first daughter path. Do the same for the second
daughter.
3.5 Data Analysis:
View5D Output
The output from View5D will be saved in the selected folder as a
text file. By default, it will be named “markers,” or have any name
assigned in step 6. This can be opened in Microsoft Excel to easily
access the data. This file will include 24 columns with all the
tracking data. The ones that are relevant for this work are:
3.5.1 Position in Pixels
The position of each nucleus in every axis at any given time point
measured in pixels are in columns C, D, and E called “PosX
[pixels],” “PosY [pixels],” and “PosZ [pixels],” respectively. This
Tools to Study Neural Crest Migration in Zebrafish
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the “Use automatic maximum finding” and “Subpixel position
by Centre of Mass” boxes again.
17. Return to the last marker available in the track by pressing “9”
and “0.”
18. Press “W” (Shift+w) again to restart the tracking.
19. Once track 1 is correct and complete, select a different nucleus
at the beginning of its migration and press “k.” This will create
marker number 2. Repeat steps 11–13 for each nucleus you
need to track.
20. To save all tracks, hover the cursor over command window
(bottom right) and press “m.” Importantly, do this periodically
as the program does not save automatically and you may lose all
your work if the program or the computer freezes. Pressing
“m” shows the raw data on the command window and creates a
text file in the selected “MarkerFileOut” folder with your
tracking data.
In case of cell division during a track, refer to steps 21 or
22 to either follow only one daughter cell as a continuation of
the on-going track (step 21) or to follow both daughter cells
after division (step 22).
21. To follow only one daughter cell as part of the same track
(a single track showing cell1 plus one of its daughters, but
ignoring the second daughter), place the marker on that
daughter cell once the division is completed and press “W”
(Shift+w) to continue the track (if the program has not done so
automatically).
22. To record the division in the data file and follow both daughter
cells as related tracks, go to the first time point after the division
and press “\” (backslash). This will create two new related
tracks labeled “1a” and “1b.” Move the “1a” marker to one
of the daughter cells; move to the next time point (second time
point after division) and delete all tracking after this point by
pressing “Q”; press “W” (Shift+w) and follow steps 8–11 to
re-track the first daughter path. Do the same for the second
daughter.
3.5 Data Analysis:
View5D Output
The output from View5D will be saved in the selected folder as a
text file. By default, it will be named “markers,” or have any name
assigned in step 6. This can be opened in Microsoft Excel to easily
access the data. This file will include 24 columns with all the
tracking data. The ones that are relevant for this work are:
3.5.1 Position in Pixels
The position of each nucleus in every axis at any given time point
measured in pixels are in columns C, D, and E called “PosX
[pixels],” “PosY [pixels],” and “PosZ [pixels],” respectively. This
Tools to Study Neural Crest Migration in Zebrafish
97