Hi all,
I need to measure the fluorescence intensity for a given object (on two different channels) over time. The object moves (drifts) over time, so its position isn't fixed throughout the observation.
In addition, I don't want to measure the fluorescence of the whole field, but rather only of the fluorescent regions.
In short:
1. intensity of the fluorescent regions
2. changes in fluorescence over time for these defined regions
3. Be able to follow the object as it moves.
I here attach a representative image sequence.
Thanks a lot in advance.
Rafa
Hi Rafael, I would attack the tracking side of this first as the intensity measurement is fairly straight forward.
If you are trying FIJI then I would go for TrackMate as it works well in most use cases - https://imagej.net/TrackMate
If you are confident in python then try Trackpy - http://soft-matter.github.io/trackpy/v0.4.2/index.html#trackpy-fast-flexible-particle-tracking-toolkit
For the intensity measurement in Fiji - start with setting a threshold (image>adjust>threshold) that contains your signal, then measure the signal for individual particles (analyse>analyse particles...)
For the intensity measurement in Python - you can threshold the image in a similar way - take all pixels with an intensity over a given threshold and then use Regionprops to look at each individual cluster - https://scikit-image.org/docs/dev/api/skimage.measure.html
Dear Callum Arthurs , thank you for your reply.
I'm afraid the tracking plug-in might not work for me. If I understand it right, it tracks spots. I'm not sure you checked to video I uploaded, but I have a large object, and to make things a bit more complicated, these objects change in morphology too..
But I'll give it a try and see if I can still use it.
Thank you.
Agnishwar Girigoswami, a bit more details would be appreciated
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