Cells intensity measurement on different channels of timelapse images (different stainings) with trackmate

I would like to measure the intensity of moving cells (that I previously tracked with trackmate) over time (I work on a timelapse file of around 150 frames that I split in 4 channels).
I tried to do that using the tools of trackmate but when I “copy the overlays” from one channel to another and that I look at the intensity table, values are the same for my 4 channels while they should be different. My four channels correspond to four stainings. They should appear at different moments in cells, and cell’s intensity should vary according to the channel. That’s why I would like to know if there is a way to measure channel-specific intensity…

Many thanks in advance for your help!!

Hi @veve, I’ll make the usual request to share an example dataset with the forum (which will vastly increase engagement with your question).

If I understand your question correctly, you’re expecting the overlays to update the measuremetns that are made through TrackMate which AFAIK will not work.

The best way in my experience to measure intensities from multiple channels in TrackMate is to install the TrackMate-extras package which then provide Intensities for output or filtering.

You will obviously have to be careful about your selection of channel on which to do your original tracking if the intensity varies.

Hope that helps!

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@dnmason, thanks for your answer.
Yeah that’s what I am trying to do. I already am using TrackMate-extras package but I am not using the tool “export current spots to IJ ROIs” since this action takes every single spot found by the tracker in the picture and put them in the ROI manager without order and not by track (which is what I’d like to have).
By copying the overlays from one channel to another, I thought I might keep the tracks found by the tracker and measure intensity of the same cells trough time for my 4 different channels.
I am trying to be clear but it is not easy for me to explain it, I am sorry for that.
When I read the intensity (from the “Analysis” button in Trackmate), the values do not change in this intensity column when I copy the overlays to another channel and click on Analysis again.


I’ve not used this part of the tool, but I also don’t know why you would if I understand your needs correctly.

What I was getting at was that once you’ve installed the extra tools, if you run your tracking, the intensity in each of the channels will be reported in the “Analysis” tables. See below the Spots in Tracks statistcs. Note the two columns Mean_Intensity01 AND Mean_Intensity02 for each of the two channels in the original dataset.

You will also get columns in the Tracks output for mean track intensity and the ability to filter on intensity during tracking. I have a feeling you may be looking at the MEAN_INTENSITY column which will only report the channel which was used for tracking.

Does that help?

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Yes thank you very much. I was splitting my channels before tracking while I should have left them as one merged file. Therefore, I only was measuring the intensity of my cells for the first channel.

Thank you again for your precious help, have a great afternoon!

Happy to help. The only change you will need to take note of for multichannel data is an extra slider in the feature detector window to select the segmentation channel.


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Oh right I didn’t know that … Thanks!

Hello !

I have a question about this part of TrackMate extra. It is possible to get the total intensity for each channel and not only the channel used for tracking ?

Thanks in advance for your help !

Hi @AlanVDB,

Yes, you will have access to all of the channels in the filter dialogs, and they will also be columns in the outputs files, whether they’re used for tracking or not.

Hope that helps.

Thanks for your answer !

So, in the outputs files I have the mean intensity and total intensity for channel 1 but I have only the mean intensity for channel 2 and I don’t know how to see the total intensity for channel 2 in the outputs files.

Apologies, I didn’t read your last post correctly. You are right that you only get integrated intensity for the tracking channel, the rest are provided as mean intensity.

has there been any update on this?
I am looking into a very similar problem, and would rather not have to calculate the total_intensity for the channels separately, then linking them by hand to the tracking Fiji has performed.

Thanks in advance!

Late to the party here, but I’ve been using a simple work-around for several years now.

  1. Save the .xml tracking file once you’re done tracking in one channel.
  2. Then open the xml file with your favorite text editor (I recommend SublimeText!) and navigate all the way to the end of the .xml file.
  3. You should see a clearly marked field that looks like: “filename=image001.tif, filelocation=/Users/data/”. (i’m not sure about exactly what the line is, but its within the last 100 lines of the file)
  4. Change the filename to the filename of your different channel image stack (note - calibrations must be the same obviously)
  5. open up trackmate → recompute all spots → and voila!