Greetings everyone,
I'm currently working on my bachelor's thesis about thermic effects in FRAP(fluorescence recovery after photobleaching)-experiments when using a confocal laser scanning microscope (CLSM).
Right now I'm trying to figure out what causes these increases in intensity around the bleach-spot that starts to shows up when doing FRAP at a certain depth. For all my measurements I set the height as 0 μm where the excitation with the laser caused the highest intensity. The increases in intensity started showing up at a height of 20 μm and persisted up until 300 μm (I went this deep into the sample out of interest). My samples were solutions of rhodamine B in glycerol and sulforhodamine B in glycerol (both with a concentration of roughly 1 g/L).
My research so far didnt give me any possible explanation for this phenomenon. I'd be glad if someone in here could either give an explanation or provide me with literature on this subject. I will add 2 pictures so you know what im talking about if my explanation wasnt clear enough. If some vital information is missing, I'll provide it down below.
Until then and thanks in advance,
Florian J.
Hello Florian,
unfortunately i don't have a straight up answer to your question, but it could be interesting to know how wide that ring is. Are we talking several micrometers, or is that a diffraction limited bleaching spot, generated by a stationary beam?
In the second case maybe the appearance of the ring could be in some way related to spherical aberration, produced by the mismatch of index of refraction between your glycerol solution and the medium that the objective is supposed to work with (oil, water or air).
That would explain the appearance of the "ring" with increasing depth, but i don't really have a good explanation of the physics that would lead from a spherically aberrated bleaching beam to a bright ring in the images taken afterwards.
An EXTREMELY wild guess would be some form optical trapping on the molecules from the first ring of the aberrated point spread function, strong enough to trap molecules, but not bright enough to bleach them. I would be very, very surprised if i was right though...
I agree it is spherical aberration since it is changing as a function of depth. If you are using an oil immersion lens then try and keep the refractive index of the sample mounting medium as close as possible to that of the immersion oil (1.515). If the mismatch is severe then the spot at which the light is focused will become distorted very quickly as you focus deeper.
Hi Florian,
It's really quite an interesting a puzzling effect. I second to Paolo Pozzi and Christopher B O'Connell in that it is connected to spherical aberration which would be quite prominent at such high depths (unless you are using a glycerol immersion objective). What is the size of your bleached spot and how does the bright ring evolve during the post bleach (recovery) phase?
Hello everyone,
Sorry for late response, it's been some busy two weeks with other more pressing matters on hand. I'll do my best to answer the questions that came up tomorrow when I have access to my data again. (This is just a quick PSA so you dont think I didn't bother to check here again after getting an answer.)
Have a nice sunday and until then
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