Unit of Length (e.g., from “Pixels” to “μm”), Width, Height,
and Depth that correspond to the size of voxel. If your instrument is properly calibrated this information should be found,
and correctly read by Fiji, from the metadata (see Note 8).
2. Start View5D through “Image > Stack > View5D > start
viewer.”
3. In the window that opens, make sure that the dimension Order
is the one your data has. Usually CZT (channel, z, time). Make
sure that SliceCount, Size Z; TimeCount, Number of Time
point and Colours are correct. Tick the option “Aspects with
data scaling” and press “OK.” This will open the 5D viewer,
which divides the screen in four: left-top is the XY plane; righttop is the XZ plane; left-bottom is the YZ plane; and right
bottom is the line of commands. The actions taken will only
apply to the part of the screen the cursor is on, which is highlighted by a colour frame on the image.
4. Adjust the size and colours for optimal visualization. To adjust
the size, hover over the XY top-left screen and press Shift+A
(“A”) to zoom in or “a” to zoom out. To adjust the colours,
hover over the different coloured squares representing each
channel on the bottom-right corner and press “c” until the
desired colour is reached (list of View5D commands Supplementary Table 1).
5. Allow for the full movie to load onto the viewer by scrolling
through it once. You can scroll backwards or forward in time by
pressing “,” and “.” or using the slider at the righthand side of
the image.
6. Define the parameters to display, the directory where to save
the tracking and the parameter tracking by opening the
“Marker positioning” window. Position the cursor in any of
the three image windows (XY, XZ, or YZ) and press “n.”
7. Choose the directory where to save the markers (tracking) by
pressing “MarkerFileOut.”
8. Choose the display mode of the tracking in the image by
ticking the boxes you find convenient in the list on the
top-left corner. Here, “line” applies to the line connecting
each consecutive marker in the track; “list” is the number
associated with each track in the movie (1, 2, 3, etc.); “trees”
are the individual tracks, referred to as trees for the branches
representing daughter cells (see step 21 for an explanation on
tracking with cell divisions). All the “Display” or “Show”
options on this list do not affect the tracking, only how the
tracks are seen on the screen; tick or untick them as convenient
throughout the process. Make sure the option “Annotate with
List Nr.” is selected. 5DView will randomly assign colours to
the tracks; hence it is possible that two tracks have the same
Tools to Study Neural Crest Migration in Zebrafish
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and Depth that correspond to the size of voxel. If your instrument is properly calibrated this information should be found,
and correctly read by Fiji, from the metadata (see Note 8).
2. Start View5D through “Image > Stack > View5D > start
viewer.”
3. In the window that opens, make sure that the dimension Order
is the one your data has. Usually CZT (channel, z, time). Make
sure that SliceCount, Size Z; TimeCount, Number of Time
point and Colours are correct. Tick the option “Aspects with
data scaling” and press “OK.” This will open the 5D viewer,
which divides the screen in four: left-top is the XY plane; righttop is the XZ plane; left-bottom is the YZ plane; and right
bottom is the line of commands. The actions taken will only
apply to the part of the screen the cursor is on, which is highlighted by a colour frame on the image.
4. Adjust the size and colours for optimal visualization. To adjust
the size, hover over the XY top-left screen and press Shift+A
(“A”) to zoom in or “a” to zoom out. To adjust the colours,
hover over the different coloured squares representing each
channel on the bottom-right corner and press “c” until the
desired colour is reached (list of View5D commands Supplementary Table 1).
5. Allow for the full movie to load onto the viewer by scrolling
through it once. You can scroll backwards or forward in time by
pressing “,” and “.” or using the slider at the righthand side of
the image.
6. Define the parameters to display, the directory where to save
the tracking and the parameter tracking by opening the
“Marker positioning” window. Position the cursor in any of
the three image windows (XY, XZ, or YZ) and press “n.”
7. Choose the directory where to save the markers (tracking) by
pressing “MarkerFileOut.”
8. Choose the display mode of the tracking in the image by
ticking the boxes you find convenient in the list on the
top-left corner. Here, “line” applies to the line connecting
each consecutive marker in the track; “list” is the number
associated with each track in the movie (1, 2, 3, etc.); “trees”
are the individual tracks, referred to as trees for the branches
representing daughter cells (see step 21 for an explanation on
tracking with cell divisions). All the “Display” or “Show”
options on this list do not affect the tracking, only how the
tracks are seen on the screen; tick or untick them as convenient
throughout the process. Make sure the option “Annotate with
List Nr.” is selected. 5DView will randomly assign colours to
the tracks; hence it is possible that two tracks have the same
Tools to Study Neural Crest Migration in Zebrafish
95