Check out this discussion at RG on intensity measurements:

https://www.researchgate.net/post/How_can_I_automatically_measure_intensity_of_a_nuclear_staining_using_ImageJ

You can find some relevant methods which works for you.

@Sathya

Hi Akshay Venkatakrish, You can try ImageJ which has the options to analyze intensity and average it.

https://imagej.nih.gov/ij/

Hi there,

First of all try to acquire images with a higher magnification objective, it will be easier...

Basic workflow:

1) Image > Color > Split channels (You have attached an RGB image)

2) select the blue (DAPI) channel

3) Process > Substract Background (Rolling ball radius=20; the diameter of your nuclei are around this value in pixels)

4) Image > Adjust > Threshold (Press on Auto and Apply)

5) Process > Binary > Watershed*

6) Analyze > Analyze particles (Leave the default values and check the add to manager option)

7) Open the original image (In 8 or 16 bits NOT in RGB)

9) Save the ROI manager

10) in the ROI manager > Multi measure

11) Copy the results

To set the parameters you want to measure go to:

Analyze > Set measurements

*The watershed doesn't work really well in your original image, try to get higher mag pictures in order to perform better this operation

In macro would be something like this (just up to getting the ROI manager, please note that split channels is not necessary if you work with 8 bits images):

macro "name"

{

img_name = getTitle();

run("Split Channels");

selectWindow(img_name+" (green)");

close();

selectWindow(img_name+" (red)");

close();

selectWindow("Dapi1.jpg (blue)");

run("Subtract Background...", "rolling=20");

setAutoThreshold("Default dark no-reset");

//run("Threshold...");

setAutoThreshold("Default dark no-reset");

setOption("BlackBackground", true);

run("Convert to Mask");

run("Watershed");

run("Analyze Particles...", "clear add");

}

Hope it helps,

Cheers