Problem in area measurement after threshold with Analyze Particles


I am trying to use the command analyze particles in a stack of photographs but it only shows me the area measured of the last slice of the stack as many times as slices I have. For instance, if I have a stack with 8 photographs, it gives me eight times the measurements of the last photograph. I don’t know what is the problem here or if I am doing something wrong.

My idea is doing something like this:

But instead of make everything binary, I use threshold to define the area I want to measure. Before using Analyze particle I already used thresholding and applied to the whole stack.

Hope you can help me with that, I really need it for my work.

Thank you for your time!

Hi Serafin_garcia_ferna,

it’s always easier for others to help if you could upload some example images. Then people can easier suggest macros, workflows or something else.


PS: Interesting problem btw!

Hi dieztzc,

Sorry I am new here. Firstly I did a merge with photoshop, with this result:

I want to measure the pinky part.

  • With Image-Stacks-Montage to stack I split the photograph in all the parts that I want. Let’s say I made 8 divisions, so in my stack I have 8 different photographs.

  • After that, using threshold I adjust using RGB my values. I used:

  • Red: 115-255
  • Green: 49-122
  • Blue: 0-255
  • Right now I press stack, so all the photographs in the stack are right now under this thresholding values.

  • Here comes my problem. Now I use Analyze-Analyze particles. I select show the outline so I can see what does it measure, I select a minimum particle size to take out the noise, display result and click ok. Fiji asks about using Analyze Particle over all the photographs. I click yes. Right now, Fiji shows the same measurement 8 times, instead of one per each photograph that there is in the stack. In the outline I have the same drawing 8 times.

Hope I explain everything better and hope you can help me out with this!


I’m afraid I can’t help you on the FIJI front, but hopefully someone else can? If you want we can try to create a KNIME workflow to solve your problem?!

Hi @serafin_garcia_ferna

I created this KNIME workflow

to measure the area of the big red segment.

Here you can download the workflow.

If you want to segment the red spots let me know and I can help you to adjust the workflow.

You don’t have to create an image stack as input. You can read each image as single file and KNIME creates for each image a row with the area of the segment.


Hi, I am really interested in this workflow, however I can no longer download it. As it appears the server wiped out the data after 3 days. Could you maybe send me a new version of this?

@tibuch I strongly second this—let’s please never use temporary file sharing services to share files on this forum. Best to attach directly to the thread if possible. If the forum settings do not allow the needed functionality, one of the admins here can investigate how to fix it.

Hi @Luuklag

Sorry about that. Here is a new download link (48.2 KB).

@ctrueden .zip is an invalid file upload extension. But I agree, it would be nice if I could attach the workflow directly to the thread.

Yes, that is what I meant by “one of the admins here can investigate how to fix it.”

I did so now, adding .zip as a valid extension. However, the max upload size is currently 3MB. Hopefully that is large enough for most things. We can increase it later if not. (I did not increase it yet because doing so is slightly more involved than just typing in a new number and hitting the save button.)

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Hm, the workflow @tibuch linked to has 12MB. But maybe it contains unnecessary image data that can be stripped?

I can upload the workflow without image data.

Here the workflow without image data (48.2 KB) :slight_smile:

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Thanks @tibuch for the smaller version.

However, it does seem like 3MB was going to become a frequent issue. So I upped the limit to 32MB.


Hi guys,

Sorry for all the delay, I was receiving all the replies in the spam mail. @tibuch what you did seems to be pretty much what I was looking for but I am pretty lost with KNIME, even for opening a folder.

After trying different things I found a way to do it just with ImageJ, later I will write step by step how to do it!

maybe the information on helps you getting started. Especially, the tutorials/example workflows and the video on youtube might help.

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Did you get down to writing how to do this in another thread, because i cant see it here
I have a similar problem and i think your procedure could be helpful.
I have got a stack of over 3000 images.