How to measure the AREA and Mean Intensity of Stack Images using ImageJ

Link to convert source code to macro video tutorial:
• How to install PLUGINS...
Macro source code:
Please replace the (angle bracket) with a "less than symbol angle bracket" at line 11 of the code. Angle brackets are not allowed in the RUclips description.
// Initialize Area and Mean Intensity measurement variables
{
//Manually run("8-bit")
//Manually setThreshold
// Save current settings
saveSettings;
// Enable "Stack position" option
setOption("Stack position", true);
Slice = nSlices;
// Loop through all slices and measure
for (n = 1; n(angle bracket)=nSlices; n++) {
setSlice(n);
//Measure the Area and Mean Intensity
run("Measure");
}
// Restore previous settings
restoreSettings;

[@Dana-py3hx]

Hi, when you set the threshold, you did not press apply. Is this correct for us to do?
When I press apply, it wants to “convert stack to binary”.

[@nrttaye4033]

This methods is applicable for analysis that involves "limit to threshold" option selection in the "set measurement window".

[@merveemen3838]

Hi, thank you for this informative video. I would like to ask when I try to set a threshold, I see the background as red, not the sample I stained. What could be the reason of this?

[@nrttaye4033]

Hello, welcome. Please try adjusting the bottom slider in the threshold window too and adjust the upper slider accordingly. Auto threshold is also another option you could try out. If it still does not solve the issue , please send me a sample image for troubleshooting

[@merveemen3838]

​@@nrttaye4033 Thank you so much for the response. I will try it. My other question is how could I perform the analysis? I mean how can I compare this kind of data with other group of z stack images? Thank you.

[@nrttaye4033]

This way of analysis is same as comparing the MFI of different single images. Using the same number of slices, thresholding data, brightness/contrast and maintaing all other parameters equal for all the z stack images for comparison should be good enough to get the data.

[@merveemen3838]

@@nrttaye4033 Thank you so much for the response. For instance, I have 15 slices for one sample and 18 slides for the other one. Then, to compare the number of slices must be equal? Then, should the step size be equal for each sample?

[@nrttaye4033]

hello, welcome. with regards to your sample size, it alright to compare between two groups of unequal size. The statistics is needed to be considered here. Since, a stundent's t test cannot be performed in this scenerio, you may have to consider a Welch's t test. Have a look at this discussion (www.researchgate.net/post/How-to-compare-two-samples-with-different-sample-size). In addition, you might have to be cautious while doing the brightness and contrast settings as this changes the pixel values. If not required, avoid the brightness and contrast adjustments. In case you want to change it, there are other methods to increase the brightness without changing the pixel values. all the best with your quantification.