Phagosome membrane (tracking and measuring intensity)

Hi,

I’m new to FIJI and I’m trying to measure the fluorescence of a phagosome membrane as it changes with time. I’d like to track the bacteria (particle) within the phagosome as it moves through multiple z stacks. The bacteria are fluorescent red. I’d then like to measure the fluorescence of a region of interest around the bacteria (the phagosome membrane). The phagosome membrane is fluorescent green. I was thinking about using the TrackMate-extras plugin offered by FIJI. Is this the best tool?

Thanks.

Hi @Felicity

Depending upon what you want to do, you can probably do this with Trackmate (plus the extras package). One option would be to track the green channel (ie your phagosomes) then filter based on those with a red signal above a certain threshold (this should give you phagosomes containing bacteria).

The problem here is that you don’t get the phagosome before it has acquired your GFP marker. An alternative way to approach this is (IMHO) a bit more finickey, and that would be:

  • to track the bacteria, then either export the positions and use some custom scripts to measure an enlarged (or segmented) green ROI in the green channel at each position
  • re-run trackmate on the second channel after changing the radius (same idea as editing the XML file in this post).

I’m not sure either of these suggestions will do what you want, but are hopefully a good place to start. Providing some example data / screenshots of the data will usually give people more to work with (and thus get more replies on the forum).

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Just to add to that, if you were to use the second method, the string you need to edit in the trackmate XML is the one within each spot section labelled RADIUS="X.X" (just do a search and replace)

You can then run Plugins > Tracking > Load a trackmate file and recompute all features as mentioned in the linked post above.


I can confirm using the Trackmate example tracks, that this does in fact change the intensity values reported by the extras module. Obviously caveats abound regarding blindly creating measurement regions.

Hi dnmason,

Thanks for helping me out. I’ve ended up tracking the bacteria (red channel) with Trackmate (see attached image).
This then gave me a readout for the fluorescence intensity of the green channel within the same ROI (see attached image). I think this will do for now, but may well need some refinement.

Thanks again.

image

image

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