Photobleaching mechanism question

Posted by John Runions on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Photobleaching-mechanism-question-tp2334316.html


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.