From the basic understanding of the Jablonski diagram any exciting light should excite the molecule to a higher energy state, and subsequently the molecule would de-excite to the lowest excited state via non radiative relaxation and then to the ground state giving out fluorescence. And depending on the probabilities the molecule can undergo a intersystem crossing and give out phosphorescence. But what actually happens when a high intensity light is subjected on the fluorescence specimen? What happens at the molecular level ?
The photobleaching consists of two process:
1-Photolysis wherein the fluorophore is destroyed (its chemical structure is damaged).
2-Phototransformation, involves bonding of fluorophore with neighboring molecules.
Best regards
Mohammad
Dear Swaroop kumar Palai,
How a fluorescent molecule photo-bleaches is very much dependent on the molecule and its environment. However, there are some general aspects to always consider. Think of the absorption-emission process as a cycle. The number of cycles per second a molecule does will increase as the excitation power goes up. Then assuming that a molecule can only do a finite set of cycles it is easy to understand why increased excitation power will make your molecules bleach faster.
Now the question is why fluorescent molecules have a finite number of cycles? Well one general phenomenon is that of intersystem crossing to a triplet state, which of course will happen more often the more cycles per second your molecule does. In many molecules this triplet state is a reactive molecular state that can, for example, react with oxygen to generate singlet oxygen. This is both good and bad news to the fluorescent molecule. In many cases this reaction allows the molecule to leave the triplet state and become fluorescent again. However, the singlet oxygen can easily react with the fluorophore, irreversibly changing its structure and thus bleaching it.
There are many other reactions that happen in the triplet state (and other excited states) that can’t occur on the ground state. In general, those are the ones responsible for the photo-bleaching.
Best,
Increased excitation power can also lead to excitation to higher excited states from which 'Anti-Kasha' processes may occur. Photobleaching quantum yields have been shown to be power dependent implicating bleaching from higher states.
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