I've got a couple options that work well. Both of these methods basically
determine the number of pixels in the cell-free area. For images with
varying background levels or colors, the first method is much better because
it has automated algorithms for determining appropriate threshold values.
1) Tscratch which is a free, published, Matlab-based program for analysis.
The necessary Matlab components can be installed for free.
http://www.cse-lab.ethz.ch/index.php?&option=com_content&view=article&id=363&catid=49
If that link doesn't work go to
http://www.cse-lab.ethz.ch/software.html and
click on TScratch
2) In ImageJ, I've made a macro to analyze batches of images. The analysis
procedure is a modified version of the procedure I found at
http://www.le.ac.uk/biochem/microscopy/wound-healing-assay.htmlI generally use this macro on stacks of time-lapse images taken of cells on
glass cover slips that have been sealed with a small volume of medium
inside. Under these conditions the color of the medium doesn't change
resulting in uniform background levels/color for all the images.
Here is the code for the macro. You'll probably need to edit it a little to
make it suit your purpose.
dir1 = getDirectory("Choose Source Directory ");
dir2 = getDirectory("Choose Destination Directory");
list = getFileList(dir1);
setBatchMode(true); //use this to save time by not displaying images
for (i=0; i<list.length; i++){
showProgress(i+1, list.length);
open(dir1+list[i]);
run("8-bit"); //my experience is that I get better results in 8-bit.
run("Duplicate...", "title=copy duplicate");
selectWindow(list[i]);
run("Sharpen", "stack"); //this step really helps a lot for thin cells with
thin lamellopodia
run("Find Edges", "stack");
setThreshold(0,20); //very important to get an appropriate threshold
run("Convert to Mask", " ");
run("Analyze Particles...", "size=12000-Infinity circularity=0.00-1.00
show=Outlines summarize stack");
selectWindow("Summary of "+list[i]);
saveAs("Text", dir2+list[i]);
selectWindow("Drawing of "+list[i]);
run("Red");
run("Invert LUT");
run("RGB Color");
selectWindow("copy");
run("RGB Color");
imageCalculator("Add stack", "copy", "Drawing of "+list[i]);
run("Size...", "width=600 constrain interpolate");
saveAs("Tiff", dir2+"Drawing "+list[i]);
close();
close();
close();
}
On 9/21/09 10:15 AM, "Pascal Lorentz" <
[hidden email]> wrote:
> Dear all,
>
> I was wondering, what is the best way the measure scratches in a wound
> assay.
> So far I just used the polygon selection to line out the cap between the
> cells and took the area as a measurement.
> Is there a better way to do it? Maybe a plugin or any other automated
> procedure?
> Any help is appreciated.
>
> Best regards
>
> Pascal
>
> Pascal Lorentz
> Department of Biomedicine
> University of Basel
> Mattenstrasse 28
> 4058 Basel
> Switzerland
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