The investigation in this report aimed at providing photophysical evidence 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 reducing agent, mercaptoethylamine (MEA or cysteamine), was the most effective, among other commonly known radical quenchers or singlet oxygen scavengers, in suppressing photobleaching of fluorescein while not reducing the fluorescence quantum yield. The protective effect against photobleaching of fluorescein in the bound state was also found in microscopy. The antibleaching effect of MEA let to a series of experiments using time-delayed fluorescence spectroscopy and nanosecond laser flash photolysis. 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 photobleaching of fluorescein in microscopy is related to the accumulation of the long-lived triplet excited state of fluorescein and that by quenching the triplet excited state and the semioxidized form of fluorescein to restore the dye molecules to the singlet ground state, photobleaching can be reduced.

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The interactions of the Grst excited singlet state of 4-N,N-dimethylaminobenzonitrile (DMABN) and of 3,5-N,N-tetramethyl-4-aminobenzonitrile (3,5-TMABN) with polar molecules have been studied. Mixtures of cyclohexane and a small amount of polar liquid have been used as solvents. The anomalous fluorescence of DMABN originates from a 1 : 1 complex between excited solute and a polar solvent molecule. The fluorescence of 3,5-TMABN in polar solvents arises only from exciplexes. A model is presented which describes the binding in the exciplexes in terms of a localized interaction between lone pair electrons of the polar solvent molecule with lone pair electrons of the dimethylamho group of the excited solute. The solvent-induced wavelength shift of any particular exciplex fluorescence of the two compounds can be correlated with the dielectric properties of the medium. If the conformation of the amino group is fused as in 1-methyl-5-cyano-indoline (l-M-5-CI) and in l-methyl-6-cyano-l,2,3,4-tetrahydroquinohe (1-M-6-CTHQ) no formation of exciplexes with polar solvents is observed. The formation of exciplexes does not only lead to a new fluorescence, but also enhances the intersystem crossing in the two compounds, DMABN and 3,5-TMABN. There is no enhancement of the triplet quantum yield in polar solvents in the case of 1-M-6-CTHQ. The quantum yield of the normal fluorescence of both DMABN and 3,5-TMABN in polar solvents depends on the wavelength of excitation. The quantum yield of exciplex emission does not show this behaviour. These two observations are explained by taking the existence of weakly bound complexes of ground state soIutes as well as the high rate of exciplex formation into account.Solutions of dimethylaminobenzonitrile (DMABN ; I) in polar solvents are known to exhibit two fluorescence bands. At the short wavelength side a band appears around 340 nm. Depending on the solvent used, a second band is observed in the region between 420 and 520nm. In non-polar solvents only the short wavelength fluorescence is emitted.After the discovery of this phenomenon,l a variety of theories have been offered for the explanation. A number of discussions are based on reversal in the ordering of the excited levels Lb and La when solvent reorientation is completed after excitation in polar solutions. Using the solvent shift of the long wavelength fluorescence together with the shift of the first absorption band, the dipole moment of the La state is estimated to be 76.7 C m.I However, within the picture of reordered levels the adopted procedure for determining the excited state dipole moment is inapplicable. The objection concerns the fact that the position of the first absorption band depends on Apb, whereas the location of the long wavelength fluorescence should be a function of Apa.6 The quantities Ap, and Apb are defined as Ap, = p(La)-pg and Apb = p(&) -u,, where p(La), [p(&.,) and pg are the dipole moments, respectively, in the La, Lb and ground state.Other mechanisms have been proposed for the dual fluorescence of DMABN, ...