Mean Intensity values Mismatch

[amoghj]

Hey!

I have been trying to measure the mean gray intensity value in defined ROIs
in stacks. I first tried to use the Mean Gray Value option in Analyze>Set
Measurements, and then Analyze>Analyze Particles. I used the Mean column in
the Summary Table and plotted values to get an intensity profile through my
stack.
Then I discovered the Plot Z Profile function under Images>Stacks which I
thought would make my life easier. I read on
http://rsbweb.nih.gov/ij/docs/guide/146-28.html#sub:Plot-Z-axis-Profile...
that this should plot the Mean Gray Intensities versus the frame.
The problem is that though the profile of the plot seems similar in both
the cases (i.e my manual plot from Summary and the plot from the Plot Z
Profile function), the actual values of the Mean Gray Intensities are very
different.
Can someone tell me how both these methods calculate the Mean values, and
which I values I can use with more confidence?

Thanks a lot!
Amogh

PS. The definition of Mean Gray Intensity=-sum of gray values in
selection/number of pixels definitely help if ImageJ throws up 2 values.

PSS. I can attach a plot showing what I mean if it is allowed. I'm new to
this forum, so please let me know if any other information is required from
my part!

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[amoghj]

Hi Wayne!

Thanks for your reply!
Here is a link to the dropbox folder:

https://www.dropbox.com/sh/cz8ve6ofkjry401/AAC0g01KpiDB7zWiB4Rq6NHUa?dl=0

It contains a sample stack .tif, the macro that I recorded, the output of Plot Z Profile, and the output results table of both the Analyze Particle command as well as the Plot Z Profile.

In summary:
I duplicated the stack, adjusted Threshold of the duplicate with the 'Mean' method of thresholding (coincidence!) and Set Measurements to redirect output to the original stack. I got the mean from the Analyze particles function.
Next I selected th duplicated stack and sent the overlays To ROI manager. Then I transferred the overlays to the original stack and used that to plot the Z Profile.

Is this the correct way to do things?

Any help appreciated!
Thanks a lot!