2. To de-noise the Tiff files, download the nd-safir de-noising
code. The code is available at http://serpico.rennes.inria.fr/
doku.php?id¼software:nd-safir:index.
3. Open a command prompt and type: cd C:\ndsafir-3.0-win64.
4. Type: ndsafir—i ‘input\input.tif’—o ‘output\output.tif’.
Replace input with your input image and replace output with
your output image.
5. Repeat for all files and once denoised the images can be analyzed (see Notes 18 and 19).
3.3 Tracking
and Image Analysis
1. First create a time-lapse stack using a single slice in either
MatLab or ImageJ.
2. Invert the stack in ImageJ, go to “edit/invert.”
3. Bandpass filter the stack to remove high and low frequency
noise, go to “Process/FFT/Bandpass filter.”
4. Subtract background using a rolling ball radius of 20 pixels, go
to “Process/Subtract background.”
5. Save the processed stack, go to “file/Save as.”
6. Track the processed stack using ImageJ trackmate plugin, go to
“plugins/Tracking/Trackmate.”
7. Determine the parameters for cell detection in trackmate and
apply to all further data sets.
8. Run the analysis and save the results file.
9. To determine the directional concerted movement, isolate the
angle results from the results file. Plot the sum of all the values
and S.D.
10. For angle plots take the angle data, import into MatLab, and
apply angle plot in MatLab.
11. Plot track speed using analysis packages such as excel, MatLab
or (for this work) we used Origin.
3.4 Somite Size
Quantification
1. Open file in ImageJ.
2. Find the center of the somite and use the threshold tool
(“image\adjust\threshold”) to determine the area of the somite
center slice.
3. Set measurements to record perimeter (“Analyse/Set measurements,” then select perimeter). Measure perimeter, go to
“Analyse/measure.”
4. To model the volume of the somite, we assume the perimeter
to be equal to the circumference. From the circumference, we
calculate the radius using C ¼ 2πr.
5. Next we use the radius to model the volume of a sphere using
V ¼ 4/3πr
3 .
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code. The code is available at http://serpico.rennes.inria.fr/
doku.php?id¼software:nd-safir:index.
3. Open a command prompt and type: cd C:\ndsafir-3.0-win64.
4. Type: ndsafir—i ‘input\input.tif’—o ‘output\output.tif’.
Replace input with your input image and replace output with
your output image.
5. Repeat for all files and once denoised the images can be analyzed (see Notes 18 and 19).
3.3 Tracking
and Image Analysis
1. First create a time-lapse stack using a single slice in either
MatLab or ImageJ.
2. Invert the stack in ImageJ, go to “edit/invert.”
3. Bandpass filter the stack to remove high and low frequency
noise, go to “Process/FFT/Bandpass filter.”
4. Subtract background using a rolling ball radius of 20 pixels, go
to “Process/Subtract background.”
5. Save the processed stack, go to “file/Save as.”
6. Track the processed stack using ImageJ trackmate plugin, go to
“plugins/Tracking/Trackmate.”
7. Determine the parameters for cell detection in trackmate and
apply to all further data sets.
8. Run the analysis and save the results file.
9. To determine the directional concerted movement, isolate the
angle results from the results file. Plot the sum of all the values
and S.D.
10. For angle plots take the angle data, import into MatLab, and
apply angle plot in MatLab.
11. Plot track speed using analysis packages such as excel, MatLab
or (for this work) we used Origin.
3.4 Somite Size
Quantification
1. Open file in ImageJ.
2. Find the center of the somite and use the threshold tool
(“image\adjust\threshold”) to determine the area of the somite
center slice.
3. Set measurements to record perimeter (“Analyse/Set measurements,” then select perimeter). Measure perimeter, go to
“Analyse/measure.”
4. To model the volume of the somite, we assume the perimeter
to be equal to the circumference. From the circumference, we
calculate the radius using C ¼ 2πr.
5. Next we use the radius to model the volume of a sphere using
V ¼ 4/3πr
3 .
178
Gi Fay Mok et al.