20. In ImageJ, select the plugin “Transform Virtual Stack Slices”
by clicking “Plugins,” “Transform,” “Transform Virtual Stack
Slices.”
21. In the source directory, select the folder containing the images
for one of the channels.
22. In the output directory, create a new folder where the corrected images will be saved.
23. In the transform directory, select the folder containing the
correct number of matrices created in step 6.g.
24. Keep the “interpolate” option selected and press “OK.”
25. Repeat step 6-b–f for every channel in the movie.
Adjust all movies to the same frame size, and equivalent
orientation by following steps 26–28.
26. Orientation: All samples must be oriented in the same way to
homogenize the angle of migration. For in vivo imaging, the
position of the anterior-posterior (AP) and the dorsal-ventral
(DV) axes must remain constant in all movies (e.g., X axis:
anterior to posterior; Y axis: dorsal to ventral; Z axis: left to
right). To do this, open the file in Fiji select “Image > Transform > Flip Horizontal or Flip Vertical or Rotate” until all axis
are positioned as required. It is advisable to take note of
sequence applied (flips and/or rotation angles) and include
these in the database. When rotating, click the option “Enlarge
Image To Fit Result” in order to not lose data. Apply the same
transformations to all channels of the movie.
27. Framing: Ensure that the samples are centred within the frame
of the movie. If the sample is off-centre, select the “Rectangle”
tool in Fiji (the left-most button on the Fiji menu) and draw a
rectangle around the sample so that it is located in the middle.
Then select “Image > Crop” to make this the new frame.
28. Size: Once all the corrections have been done, the file can be
returned to its original size by selecting “Image > Adjust >
Canvas Size.” Maintain this to a standard size for all stacks.
3.4 Image
Processing: 3D
Tracking Using
View5D
With your corrected image you can proceed to semi-automatically
track cells/nuclei/objects in 3D. After opening the image and
making sure its size is correct you will indicate the objects to follow
in the first frame they appear. Thereafter the program will try to
automatically follow them over time. You can define the parameters
to find the objects (e.g., intensity levels, maximal radius in which to
look in the next time point, etc.). Once the automatic tracking is
done, you can manually correct it.
1. Open the corrected stack in Fiji and ensure the properties of
the image correspond to real units. Go to “Image > Properties” and correct number of slices and time frames, as well as the
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by clicking “Plugins,” “Transform,” “Transform Virtual Stack
Slices.”
21. In the source directory, select the folder containing the images
for one of the channels.
22. In the output directory, create a new folder where the corrected images will be saved.
23. In the transform directory, select the folder containing the
correct number of matrices created in step 6.g.
24. Keep the “interpolate” option selected and press “OK.”
25. Repeat step 6-b–f for every channel in the movie.
Adjust all movies to the same frame size, and equivalent
orientation by following steps 26–28.
26. Orientation: All samples must be oriented in the same way to
homogenize the angle of migration. For in vivo imaging, the
position of the anterior-posterior (AP) and the dorsal-ventral
(DV) axes must remain constant in all movies (e.g., X axis:
anterior to posterior; Y axis: dorsal to ventral; Z axis: left to
right). To do this, open the file in Fiji select “Image > Transform > Flip Horizontal or Flip Vertical or Rotate” until all axis
are positioned as required. It is advisable to take note of
sequence applied (flips and/or rotation angles) and include
these in the database. When rotating, click the option “Enlarge
Image To Fit Result” in order to not lose data. Apply the same
transformations to all channels of the movie.
27. Framing: Ensure that the samples are centred within the frame
of the movie. If the sample is off-centre, select the “Rectangle”
tool in Fiji (the left-most button on the Fiji menu) and draw a
rectangle around the sample so that it is located in the middle.
Then select “Image > Crop” to make this the new frame.
28. Size: Once all the corrections have been done, the file can be
returned to its original size by selecting “Image > Adjust >
Canvas Size.” Maintain this to a standard size for all stacks.
3.4 Image
Processing: 3D
Tracking Using
View5D
With your corrected image you can proceed to semi-automatically
track cells/nuclei/objects in 3D. After opening the image and
making sure its size is correct you will indicate the objects to follow
in the first frame they appear. Thereafter the program will try to
automatically follow them over time. You can define the parameters
to find the objects (e.g., intensity levels, maximal radius in which to
look in the next time point, etc.). Once the automatic tracking is
done, you can manually correct it.
1. Open the corrected stack in Fiji and ensure the properties of
the image correspond to real units. Go to “Image > Properties” and correct number of slices and time frames, as well as the
94
Zain Alhashem et al.