colocalization greater than 100%

Posted by Gretchen Unger, Ph.D. on Dec 19, 2007; 4:18am
URL: http://imagej.273.s1.nabble.com/colocalization-greater-than-100-tp3697720.html

Hi all,

I used the division feature of Image J, v. 1.38 to estimate maximum
colocalization between two optical sections, one was green (reporter
gene) and the other red (cells). It looked like it worked but in one
section I calculated a value over 100%. Can anyone explain how that
might happen?

I transformed final images processed against background into
grayscale using Photoshop for the Image J calculations. In Image J, I
calculated a joint (green+red) area by pixel-by-pixel division of
green/red. This would calculate a maximum colocalization as a pixel
with any signal above background in both images would divide to a
"1". Because of drift or jumping or whatever, I had to manually alter
alignment in some of the joint picture calculations.

Next, I calculated "area fractions" in both the joint and red image
using the "threshold" feature, applying threshold , and then
"measuring" following selection of "area fraction" as an attribute.
Because my final calculation was Colocalization =
(green/red)*100/red, I selected the thresholding level from my
denominator using the auto function. I then manually set the
threshold to the same value (whatever it was) for the area fraction
measurement of the joint image (the numerator). I got a number around
170% (17.25 divided by 10.37),  I have used this method in the past
and got numbers ranging from 50% to 98%, which seemed reasonable.  I
don't know why this set of aligned panels exceeds 100% and can't
explain to anyone how that happened or what it can mean. Should I
auto-threshold both images ? did I alter a calculation basis somehow
when I manually aligned the image pair?

Any thoughts or comments would be appreciated.

Gretchen Unger