Posted by
Guy Cox on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Photobleaching-mechanism-question-tp2334316p2338823.html
I realised on seeing the message on the list that it wasn't clear what was me and what was Mark.
Mark Cannell sent the following addition to my explanation but so far as I can see just to me, not the list. So I'll add it here:
I said:
Oxygen is naturally a triplet molecule. Triplet-triplet reactions are particularly likely to occur, and so a triplet excited state is more likely to get oxidised.
Mark adds:
Your answer is very close. The final part is that the triplet-triplet interaction produces two singlet molecules and singlet oxygen is very reactive. Since it is in close proximity to the fluorochrome it is likely to oxidize it.
[from here on it's me, simple dumb biologist, again]
As to bizarre explanations, I can't see the point. How do they help? Singlet just means all electrons are paired with ones of opposite 'spin' - think of them as tiny magnets, each one that has its north pole upwards is matched with one with its south pole upwards, so there's no overall magnetic field. Triplet means that there are unpaired electrons. Mark has explained the reason for the names, but they're not so important.
One possible decay route for an excited singlet molecule is to go into a triplet state. It's relatively unlikely for an electron to change its spin so usually not many will decay that way. But since we're exciting a lot of molecules some will chance to take that path. Again, getting from there back to a non-excited state means changing an electron's spin, and since this is an infrequent occurrence the molecule will remain in a triplet state for a while.
If nothing happens in this time the molecule will go back to the ground state, unbleached, but it is very vulnerable to oxygen attack while waiting in the triplet state.
None of this is difficult to follow. The explanation that 'It's easy to switch to the dark side' is simply wrong. It's quite hard to switch to the dark side, it's just that if you keep on exciting a molecule, over and over, eventually it'll end up there.
The best analogy I can think of is a fly-paper. It's quite unlikely that a fly will bump into the fly-paper, but when it does, it can't get off. So eventually you'll get rid of all the flies in the room.
Guy
Optical Imaging Techniques in Cell Biology
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Associate Professor Guy Cox, MA, DPhil(Oxon) Electron Microscope Unit, Madsen Building F09, University of Sydney, NSW 2006 ______________________________________________
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http://www.guycox.net-----Original Message-----
From: Confocal Microscopy List [mailto:
[hidden email]] On Behalf Of Tobias Baskin
Sent: Monday, 16 February 2009 11:47 PM
To:
[hidden email]
Subject: Re: Photobleaching mechanism question
John,
From the non physicist's point of view the answer could go something like this. If you have a power surge it can fry your computer. But if your computer is not plugged into the mains then it would take a very big power surge indeed to do the damage. A molecule in the ground state can of course be damaged by free radical attack but no more or less than other molecules. But once a molecule has absorbed a photon then it is not in the ground state any more. To continue my hoaky analogy, a chromophore in light is like your computer plugged in to the mains.
Hope this helps. The physicists (and musicians) can go for the triplets.
Tobias
>Hi Everyone, this question follows on from a helpful discussion that
>we had about photobleaching back in November. I have recently tried to
>explain to a group of colleagues about the mechanism of photobleaching.
>The answer is based on the transition of molecules from the excited
>singlet state (S1) to the triplet state (T1) which is long-lived and
>therefore more susceptible to bleaching by free radicals (my entire
>discussion of this is below).
>
>My question that arises from my attempted answer is: why are excited
>molecules more susceptible to oxidative attack than ground state
>molecules. I hope I'm not completely mucking up the mechanism here.
>Would the physicists out there please help.
>
>Thanks, John.
>
>The original answer: When excited, fluorophores generally transition
>from singlet ground state (S0) to singlet excited state (S1).
>Relaxation from S1 to S0 results in emission of heat and light
>(fluorescence). Lifetime in S1 is in the nano to pico second range and
>allows very little time for the excited molecule to interact with free
>radicals. Periodically, however, an excited molecule will do a
>transition from S1 to the triplet excited state (T1 - the physics of
>this is a bit difficult to understand). T1 is a very long-lived state -
>molecules can remain in T1 for up to the microsecond range - i.e. a
>thousand to a million times longer than for normal S1 state. It is
>during this long T1 state that molecules are attacked by free radicals
>and destroyed.
>
>--
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>(Sent from my cra%#y non-Blackberry electronic device that still has
>wires)
>
>
>
>*********************************
>John Runions, Ph.D.
>School of Life Sciences
>Oxford Brookes University
>Oxford, UK
>OX3 0BP
>
>email: <mailto:
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>phone: +44 (0) 1865 483 964
>
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http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html%21>Runions'
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>
>
>Visit <
http://www.illuminatedcell.com/ER.html>The Illuminated Plant
>Cell dot com Oxford Brookes Master's in
><
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