INFLUENCE OF THE TRIPLET EXCITED-STATE ON THE PHOTOBLEACHING KINETICSOF FLUORESCEIN IN MICROSCOPY

The investigation in this report aimed at providing photophysical evid ence that the long-lived triplet excited state plays an important role in the non-single-exponential photobleaching kinetics of fluorescein in microscopy. Experiments demonstrated that a thiol-containing reduci ng agent, mercaptoethylamine (MEA or cysteamine), was the most effecti ve, among other commonly known radical quenchers or singlet oxygen sca vengers, in suppressing photobleaching of fluorescein while not reduci ng the fluorescence quantum yield. The protective effect against photo bleaching of fluorescein in the bound state was also found in microsco py. The antibleaching effect of MEA led to a series of experiments usi ng time-delayed fluorescence spectroscopy and nanosecond laser flash p hotolysis. The combined results showed that MEA directly quenched the triplet excited state and the semioxidized radical form of fluorescein without affecting the singlet excited state. The triplet lifetime of fluorescein was reduced upon adding MEA. It demonstrated that photoble aching of fluorescein in microscopy is related to the accumulation of the long-lived triplet excited state of fluorescein and that by quench ing the triplet excited state and the semioxidized form of fluorescein to restore the dye molecules to the singlet ground state, photobleach ing can be reduced.