I am not completely new to ImageJ, but to the board. I hope you can help me since I assume it is more a technical problem. My aim is to analyze the bright structures (focal adhesion) on the first image. I am interested how much of those cover the cell. I am going to describe how I do it manually, since this works fine. However, I have a problem that is does not work as batch nor as normal macro.
Unfortunately, I am restricted to upload only 1 image and 5 links so I tried to work around a bit.
Figure 1: This is the original image
Figure 2: Image is converted into 16 bit
Figure 3: The background is subtracted (sliding paraboloid, rolling ball radius = 50 pixel)
Figure 4: The CLAHE (Contrast Limited Adaptive Histogram Equalization) plugin is used to enhance the constrast (blocksize: 15, histogram bins: 256, maximum slope = 6.00)
Figure 5: The LoG3D plugin is used (Sigma X = 4, Sigma Y = 4)
Figure 6: Threshold is used using the Otsu method
Figure 7: Particles are analyzed according to their size
I used the macro recorder and got following code:
run("Subtract Background...", "rolling=50 sliding");
run("CLAHE ", "blocksize=15 histogram=256 maximum=6");
run("LoG 3D", "sigmax=4 sigmay=4");
run("Analyze Particles...", "size=1-Infinity circularity=0.05-1.00 display clear add");
I had to add the sigma values to the LoG3D plugin since the recorder did not write it. The macro runs fine until the threshold method. I figured that out by running the macro with the first command line, then the second, etc.
As it is displayed above I get this image in the end with other measurements than the ones I get running it manually.
Figure 8: Outcome after running the macro
I hope somebody can help me since I have over 300 images.
Thanks in advance!