# Photobleaching of fluorophores

1. May 7, 2014

### gkiverm

Can someone explain the cause behind photobleaching (http://en.wikipedia.org/wiki/Photobleaching) of fluorophores when they are continuously excited. The wikipedia article does not really explain why photobleaching occurs.

I tried looking it up online, and most explanations were too complicated for my background in physics / chemistry. I got into terminology with words like singlet and triplet state.

Thanks!

2. May 7, 2014

### Bashkir

It happens by photon-induced chemical damage and covalent modification. As I'm sure you might know, florescence is caused by the absorption of a photon, then the admittance of a florescent photon. Well, pretend we have two electrons in the ground state, one with a spin of $$+\frac{1}{2}$$ and the other with a spin of $$-\frac{1}{2}$$. The sum of their spins is 0. We call this state this singlet state. Well, when one electron absorbs a photon and gets promoted to a higher energy level one of three things can happen. It can keep its spin, either the $$+\frac{1}{2}$$ or $$-\frac{1}{2}$$ and the sum of their spins still be zero. This is called the excited singlet state. From here the electron can fall down back into the ground state and admit a fluorescent photon. The electron can also fall back to ground state without radiating anything. The other option is for the promoted electron to undergo something called Intersystem Crossing. This is where the spin of the electron is flipped in the transition and it is now the same as the as the electron in the ground state, a forbidden transition. This is now known as the excited triplet state. This triplet state is when the sum of their spins adds to 1. In this state they are incredibly reactive which allows them potentially interfere with bonds in the material. The chances of Intersystem Crossing happening aren't high, some factors can allow it to happen more frequently, but the more something is excited the more chances it has for this transition to happen.

Last edited: May 7, 2014
3. May 7, 2014

### Andy Resnick

Basically, photobleaching means the fluorophore has been (chemically) destroyed and can no longer fluoresce. When the fluorophore is in the excited state, after absorbing light, it can more easily undergo chemical reactions: oxidation, decomposition, etc.

Photobleaching is different than quenching: a reduction in fluorescence due to the non-radiative transfer of energy from the excited fluorophore to some other molecule. Quenching is reversible.

4. May 7, 2014

### gkiverm

Thanks for the reply. I have some few questions though,

1) how can the electron fall back to the ground state without radiating anything? If the explanation is too complicated I can just take this for granted.
2) Does intersystem crossing only occur if the electron falls back without radiating anything?

Edit: Also, how does this relate to the formation of oxygen atoms that are free radicals?

5. May 7, 2014

### Bashkir

1) Well, when an electron emits and absorbs a photon all it is doing is absorbing and re-emitting energy again. For a "radiationless de-excitation" as they are called, meaning they don't radiate a photon, the energy is just transferred to something else. This is normally done through some transference of vibrational energy that disperses as heat.

2) Yes, in a way! Intersystems crossing is a two-directional process that describes the radiationless transitions between a singlet state to a triplet state, and from a triplet state to a singlet state.

O2 naturally in its ground state due to its electron configuration is in the triplet state. Oxygen is incredibly reactive with the other triplet states which can cause oxygen to be reduced to its singlet state, forming free radicals.

What is your level of education in physics and chemistry? I have some reading for you on the subject if you would like, but I don't know what difficulty level to tailor it to.

6. May 7, 2014

### gkiverm

I have only taken chemistry I and II at the college level (introductory classes) and for physics I have taken a course on waves, oscillations, and quantum physics (which is a little above the level of an introductory college course).

7. May 7, 2014

### Bashkir

Introductory college course can be a little misleading. At my school there are 4 different "introductory" physics classes. There is physics for non-science majors (no math, all concepts), physics for life sciences (algebra based physics), physics for engineers (calculus based applications of physics), and then physics for majors (calculus based physics, application,and theory). If you are comfortable with calculus then this should give you some extra information.

http://www.wiley-vch.de/books/sample/3527316698_c01.pdf