Spatial intensity distribution

Hello all!
I am looking for a imagej plugin or any other software for analyzing fluorescence intensity distribution inside vesicles. Would you help me find a suitable one, thanks!

John G

Hi,

Fiji has inbuilt functions to measure fluorescent intensity:
https://imagej.nih.gov/ij/docs/menus/analyze.html#set

In image analysis the challenge is rather to select the specific parts of the image you want to measure in. If you provide us with an example image we could suggest ways to tackle your specific issue.

Hi,
Thanks for your quick response. That is very helpful to become aware of Imagej applications.
I have attached here an image showing vesicles with variations in fluorescence intensity distribution across cross sectional area.
My task is to choose a particular area within vesicles and measure pixel intensity variation within the selected area and plot a graph of pixel intensity vs pixel count. The same applies to all other vesicles. finally I can analyse how the pixel intensity varies within vesicles and among the vesicles.
Please let me know if you have any questions.

best,
John G
30mg__test.tif (2.1 MB)

Do you need 3D information or can you reduce that stack? Most of it is anyways just empty and the vesicles are cut off. That the vesicles are cut off can be an issue for your analysis. You need to make sure that you analyse comparable sections or - if 3D - volumes of your vesicles.

What do you mean by that: “choose a particular area within vesicles”?
Do you need to choose a specific area were you have certain criteria?
Or is the choice arbitrary or do you need to get the entire crosssection?

So you want something like a histogram within a specific ROI?
Should be easy to get even manually. Just draw a ROI. Then press:
Analyze > Histogram

Gives you the counts per gray value. Which you can extract via pressing List in the Histogram.

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great! your response answers my questions. I am still exploring Imagej options.

3D information would be better for my analysis. At this time, I am trying to see how I can apply Imagej for 2D image. yes, your suggestion about comparable sections makes me explain my main task. I’d like to select equally sized vesicles, for example 10 micron diameter, and then perform 2D or 3D spatial intensity distribution on all the selected ones. Is there any option in Imagej to automate this process?

I’d like to choose a random areas within vesicles and also some specific area, for example periphery of vesicles where intensity is much higher than inner portion of vesicles as shown in figure

I think Fiji / ImageJ should be able to do that.

I would suggest you dive into image segmentation. Here is a very large list of topics:

You can also automate stuff relatively easily in Fiji using its macro language:
https://imagej.nih.gov/ij/developer/macro/macros.html

Both 2D and 3D analysis should be feasible.

Once you have segments you can also decide on subsegmentation by creating an euclidean distance map: https://imagej.nih.gov/ij/docs/guide/146-29.html#sub:Distance-Map

If you have specific problems you can always ask for solutions here in the forum.

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awesome! I really appreciate it.
Thank you for your time today

John

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