Here’s a simple method to deal with varying intensities in a large run of images, assuming you have a known number of cells in a field. If I assume I should see, say, more than X cells per area, I can adjust my threshold to slowly increase it’s aggressiveness until that minimum counted value is met. The code can be downloaded as an imageJ macro here. but here’s a walk through of this simple script. My comments are preceded by a *.:
rnum = 0; *this creates a new variable, or container, for our # of regions
tval = 2000; *this is the starting object threshold value.
while (rnum<500) *this is a "while" loop. The loop runs until the argument is satisfied.
{
setAutoThreshold("Default");
setThreshold(tval, 4095); *note that tval = the minimum threshold value
run("Analyze Particles...", "size=0-Infinity circularity=0.00-1.00 show=Nothing display clear add");
rnum = roiManager("count");
tval = tval - 100; *tval continues to become more aggressive in counting objects until the argument is met.
}
Simple routines like these can reduce your headache factor by a large margin if you learn how to harness the power of macros!
-Austin