Posted by
sdegan on
Nov 11, 2020; 12:16am
URL: http://imagej.273.s1.nabble.com/Problem-with-thresholding-tp5024162.html
Hi All,
I am having a problem quantifying an antibody staining where the value of
the total area comes up smaller than the value of the cells stained by the
antibody (it should be the opposite).
In the past with the color threshold plugin I was able to sample the
staining and have the values for both total area and the staining. Now it
this not working so I tried some workaround. Here is the macro for the
reactive area to the antibody where I run the color threshold and then I
inverted the image and re-run the threshold (this was one of the many
attempts).
I am attaching the original image and the image generated after running the
macro for the threshold antibody calculation in the output folder (black
background). You can also check a third image (white background) that was
generated during the second threshold calculation in the macro.
Does anybody have any idea what I am missing since it was working in the
past?
Thank you
// Color Thresholder 1.52k
// Autogenerated macro, single images only!
min=newArray(3);
max=newArray(3);
filter=newArray(3);
a=getTitle();
run("HSB Stack");
run("Convert Stack to Images");
selectWindow("Hue");
rename("0");
selectWindow("Saturation");
rename("1");
selectWindow("Brightness");
rename("2");
min[0]=117;
max[0]=229;
filter[0]="stop";
min[1]=170;
max[1]=255;
filter[1]="pass";
min[2]=102;
max[2]=255;
filter[2]="pass";
for (i=0;i<3;i++){
selectWindow(""+i);
setThreshold(min[i], max[i]);
run("Convert to Mask");
if (filter[i]=="stop") run("Invert");
}
imageCalculator("AND create", "0","1");
imageCalculator("AND create", "Result of 0","2");
for (i=0;i<3;i++){
selectWindow(""+i);
close();
}
selectWindow("Result of 0");
close();
selectWindow("Result of Result of 0");
rename(a);
// Colour Thresholding-------------
run("Invert");
run("Duplicate...", " ");
setAutoThreshold("Intermodes");
//setThreshold(0, 125);
run("Convert to Mask");
run("Set Scale...", "distance=340 known=100 pixel=1 unit=um");
run("Measure");
<
http://imagej.1557.x6.nabble.com/file/t187616/photomicrograph.png>
<
http://imagej.1557.x6.nabble.com/file/t187616/44773_Tra%2BHCQ_F40_dapi_Merge_001.jpg>
<
http://imagej.1557.x6.nabble.com/file/t187616/Screen_Shot_2020-11-10_at_5.png>
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