FRAP analysis help please

[RUNinspired]

My biggest pondering related to FRAP deals with the following scenarios. I am dealing with FRAP analysis on moving axons in one instance or just moving puncta within them. Both shapes are very tricky compared to a classical textbook FRAP of a small rectangle within a cell. My axons are dynamic and in the process of recovering they change shape, length etc. The puncta as well goes up and down the axon.


I guess my first question is about the more simple scenario of the moving puncta. Before bleaching the puncta has an area X and after the recovery the area never reaches the size X and it always remains smaller. What area do I use to collect a mean value? I am afraid that by selecting just the area of the puncta I am artificially increasing the mean value of recovery.  After all a big and a small area can have very close mean values despite the fact that the smaller area has obviously lost part of its fluorescence. Do I use a selection from the first pre-bleach frame and keep using this selection, or do I manually go and track each frame since the puncta grows slowly over time. Is there any way to adjust for area-size? What about using total sum of pixels? I am attaching a little image. (http://i39.tinypic.com/2ir2a1c.jpg) In this example the puncta did not move much but very often they would traverse almost the full field of view. When I took this project I thought tracking puncta must be the simplest thing to do since they are well defined but I have not had much luck in automating FRAP analysis on this recovering moving puncta, Especially since tracking fails if there are higher number puncta and some are bleached and some are not.

-Summary: FRAP on puncta which are moving a lot and also growing over time after the recovery. What area do I use? If I use area in pre-FRAP, the recovering puncta never come back to the same big size. If I have different area for each frame (smaller area) the mean value is basically increased a lot since removing the empty volume around. 1 px of 1000 units has the same mean as 100px of 100 000 total units.

My second most important question is how should I go about measuring FRAP on the dynamic axons. I am attaching a sample image. The axons shorten, elongate, sometimes quite significantly. My previous question about the shrinking area is still valid. Very often the recovered axon area of signal is slightly thinner than the pre-bleach.  Is my best bet to basically manually trace out each individual frame and measure mean. Would it be possible to have any type of automation on such a problem? http://i41.tinypic.com/219vr5z.jpg

-Summary: Second question is very similar to the first as in dealing with a shape in FRAP that is changing size, shape, etc

THANK YOU!!!!

[Unruh, Jay-2]

From the images you uploaded it looks like the "area" changes after bleaching are simply due to saturation before bleaching and lack thereof afterwards.  All objects will look bigger in a saturated image.  Are you trying to measure exchange of the red label to and from this structure?  In that case, the best option is to measure the raw integrated density from the object (in a background subtracted sum projected z stack ideally) as a function of time during the recovery.  In this analysis, area doesn't matter because the recovery is measured in terms of total label, not the area that it occupies.  If you care about the fraction of mobile red label, make sure you account for the relative about of red label you bleached initially.  Also avoid saturation.

Judging from the size of your image, this object is approaching the resolution limit anyway in which case you really have no idea what the actual area is.

Alternatively you might want to measure the motion of the red label within the structure.  This is more difficult, especially for this small size structure.  In that case, you could bleach half of the structure (hopefully your scope is accurate enough to do this) and watch the decay of fluorescence from the unbleached part and the fluorescence increase in the bleached part.  In this case the area will change over time but it doesn't necessarily need to be measured in the analysis.

In the case of your whole-axon recovery experiments, I would recommend bleaching a much smaller portion of the axon initially.  That will decrease the recovery time so that you avoid morphological change.

Jay

-----Original Message-----
From: ImageJ Interest Group [mailto:[hidden email]] On Behalf Of RUNinspired
Sent: Friday, October 04, 2013 5:33 AM
To: [hidden email]
Subject: FRAP analysis help please

My biggest pondering related to FRAP deals with the following scenarios. I am dealing with FRAP analysis on moving axons in one instance or just moving puncta within them. Both shapes are very tricky compared to a classical textbook FRAP of a small rectangle within a cell. My axons are dynamic and in the process of recovering they change shape, length etc. The puncta as well goes up and down the axon.


I guess my first question is about the more simple scenario of the moving puncta. Before bleaching the puncta has an area X and after the recovery the area never reaches the size X and it always remains smaller. *What area do I use to collect a mean value? I am afraid that by selecting just the area of the puncta I am artificially increasing the mean value of recovery. * After all a big and a small area can have very close mean values despite the fact that the smaller area has obviously lost part of its fluorescence.* Do I use a selection from the first pre-bleach frame and keep using this selection, or do I manually go and track each frame since the puncta grows slowly over time. Is there any way to adjust for area-size?* What about using total sum of pixels? I am attaching a little image. ( http://i39.tinypic.com/2ir2a1c.jpg <http://i39.tinypic.com/2ir2a1c.jpg>  ) In this example the puncta did not move much but very often they would traverse almost the full field of view. When I took this project I thought tracking puncta must be the simplest thing to do since they are well defined but I have not had much luck in automating FRAP analysis on this recovering moving puncta, Especially since tracking fails if there are higher number puncta and some are bleached and some are not.

*-Summary: FRAP on puncta which are moving a lot and also growing over time after the recovery. What area do I use? If I use area in pre-FRAP, the recovering puncta never come back to the same big size. If I have different area for each frame (smaller area) the mean value is basically increased a lot since removing the empty volume around. 1 px of 1000 units has the same mean as 100px of 100 000 total units.*

My second most important question is how should I go about measuring FRAP on the dynamic axons. I am attaching a sample image. The axons shorten, elongate, sometimes quite significantly. My previous question about the shrinking area is still valid. Very often the recovered axon area of signal is slightly thinner than the pre-bleach.  Is my best bet to basically manually trace out each individual frame and measure mean. Would it be possible to have any type of automation on such a problem?
http://i41.tinypic.com/219vr5z.jpg <http://i41.tinypic.com/219vr5z.jpg>  

*-Summary: Second question is very similar to the first as in dealing with a shape in FRAP that is changing size, shape, etc*

THANK YOU!!!!



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