FRAP acquisition issues - ROI not bleaching fully

Software
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I am trying to do FRAP on my samples using LSM980 zeiss confocal microscope and zen3.4 blue edition software. My samples either autofluoresce in DAPI range or are stained with dyes in FITC range - they are solutions in PBS, so contained inside PDMS well on glass coverslips.

However when I try to photobleach them, the specified ROIs don’t go completely dark - is this due to some issue with my FRAP settings or my samples…? Please help!

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Can you provide some pictures so that people can see what you mean by “don’t go completely dark”? How does the intensity after bleaching compare to the same sample but without the FITC dye? In general I think it is difficult to bleach a sample to the point that it has no background at all.

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Thank you for the comment.

Here are some reference images I found online - you can see how highlighted region goes dark after photobleaching (sorry it seems I can attach only one image)

IMG_9793
IMG_9793579×572 54 KB

In my case, there is very little change in intensity for the selected region for interest so I was wondering why that could be…

4

A couple thoughts:

  1. Are you using a very slow scan speed for the Bleach event? This is the line after “Iterations” in the Bleach control window. I find it hard to bleach using a fast scan speed, but the default is the Scan Speed for the image collection. I typically use bleach speed 2 or 3 while the Scan Speed in the Aquisition Mode window is 6-9.
  2. Is the area you are bleaching connected to a pool of soluble fluorophore? If so, it might be replenishing faster than your imaging settings. You can test this a couple ways. (A) try to catch the bleach: Use a very small area and choose Zoom Bleach to make the time between the bleach event and the first image as small as possible. When Zoom Bleach is unchecked, the mirrors still scan the entire image, not just the bleach region, so it takes longer. Zoom Bleach has the scanners move only in the area of the region. (b) Use a very large bleach area and measure the intensity of an area far away. This will only work if the contiguous solution is a relatively small volume such that the large bleach area is a substantial portion of the population of fluorescent molecules.
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Thank you… I will give these a try!

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Excellent points by FishCsCells. When doing FRAP experiments, I was always advised to not bleach to 100% anyway, as doing do risks ‘going too far’ and using too much energy on the sample, causing harm that can negatively influence experimental outcome, etc. We’d alway bleach to 70-80% or so (20-30% of original signal remaining).