Mean gray intensity, Integrated density and Raw integrated density

intensity
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#1

Hi everyone, I am a new user of ImageJ and was suggested by my colleague to use Mean gray intensity, Integrated density and Raw integrated density to measure the brightness of my image. I used these measurements to quantify a brown stain called Perls (that stain for iron present in tissues) and now I have to present my results to lay audience. What would be the best way to explain about these three measurements to people lay audience?
Thanks very much


#2

Dear,

as a novice it’s always a good idea to read the manual and docs—no?

Here are the relevant sections:
https://imagej.nih.gov/ij/docs/guide/146-30.html#toc-Subsection-30.1
https://imagej.nih.gov/ij/docs/guide/146-30.html#sub:Set-Measurements…

What distinguishes a novice from lay people?

Regards

Herbie


#3

Hi Herbie,

I’ve read the manual on the links supplied and I cannot find where difference the between IntDen and RawIntDen is defined:

Integrated density The sum of the values of the pixels in the image or selection. This is equivalent to the product of Area and Mean Gray Value. With IJ 1.44c and later, Raw integrated density (sum of pixel values) is displayed under the heading RawIntDen when Integrated density is enabled. The Dot Blot Analysis tutorial demonstrates how to use this option to analyze a dot blot assay.

The DotBlot analysis doesn’t even mention Raw intensity.

Playing around with some images it appears that:
• RawIntDen = sum of pixel values in selection
• Mean = RawIntDen / (Area in pixels )
• IntDen = RawInden * (Area in scaled units) / (Area in pixels)

Cheers,

Chris


#4

Good day Christian!

Without playing around I found in the ImageJ user guide:

30.7 Set Measurements…

Mean gray value Average gray value within the selection. This is the sum of the gray values of all the pixels in the selection divided by the number of pixels. Reported in calibrated units (e.g., optical density) if Analyze▷Calibrate…↓ was used to calibrate the image.

Integrated density The sum of the values of the pixels in the image or selection. This is equivalent to the product of Area and Mean Gray Value. With IJ 1.44c and later, Raw integrated density (sum of pixel values) is displayed under the heading RawIntDen when Integrated density is enabled.

The last sentence refers to the whole paragraph not specifically to “RawIntDen”.

I think both paragraphs tell all you’ve found out by playing around.

Regards

Herbie


#5

Hi Herbie,

OK, I can see how the documentation says that now, but it is difficult (for me at least) to parse that. The relationship between IntDen and RawIntDen require going through the mean grey value definition and is not very enlightening.

So while the manual is technically correct, I suggest that the manual should say something like:

  • RawIntDen = (sum of pixel values in selection)
  • IntDen = (sum of pixel values in selection) * (area of one pixel)

NB: if the image is unscaled then IntDen = RawIntDen

This makes the relationship between IntDen and RawIntDen much clearer - it’s about a conversion to scaled coordinates.

Cheers,

Chris


#6

Good day,

i’m by no means resposible for the user guide, as there is nobody here on the Forum responsible for anything. That’s how volteers and open source work …

Regards

Herbie


#8

Hi evenhuis,

I also have read the manuals several times but still could not figure out the difference between IntDen and RawIntDen. I know several students in my department who have the same confusion.

Thank you for explaining the difference. I find your answer more helpful than Herbie’s.

Best,

Stone


#9

Stone,

please read carefully:

Integrated density
[…]
This is equivalent to the product of Area and Mean Gray Value.

Mean gray value
[…]
Reported in calibrated units (e.g., optical density) if Analyze▷Calibrate…↓ was used to calibrate the image.

Consequently, not only the “Mean gray value” depends on Calibration but “Integrated density” as well, because, as explained above, the latter refers to the former.
Then of course “Raw integrated density” means “Integrated density” without Calibration.
(If no Calibration is present it holds: “Raw integrated density” = “Integrated density”)

Where is the problem?

Regards

Herbie


#10

Hi Herbie,

Your updated explanation makes a lot sense. Thank you!

Best,
Lei


#11

Hi,
The difference between Integrated density and Raw Integrated density is in image scaling and not in Calibration. In imagej “calibration” is the assignment of a physical meaning to the grey values of the pixels (e.g. Optical Density units, photon number, electron number etc). For the calibration of the interpixel distance is used the term “image scale”.
If the image brightness is calibrated both the mean grey value and the Integrated density are reported in calibrated units.
If the image interpixel distance is calibrated,i.e. a scale is set (a physical distance unit is assigned to the interpixel distance ) the Integrated density is different from the Raw Integrated density in the already explained dependancy:
Raw Integrated density is the sum of all pixel values (or ODU, etc) in an area, while the
Integrated density equals the Mean Grey value (or mean OD, or mean electron number etc) times the area in scaled units . Thus Integrated density=Raw Integrated density * Area of a region with size one pixel
E.g. if interpixel distance pixel in the image is scaled to 0.5 micrometers then IntDen=RawIntDen*0.25 sq um but it uses the same pixel brightness units (either calibrated or not).