Medium effects on the fluorescences of some probes of biological systems

A detailed investigation of the solvent off sets on the fluorescence properties of 1-aminonaphtholene, NN' dimethyl-1-aminonaphthalene.1 methyl indole, and 3 methyl indale, has been undertaken. The above named molecules represent the basic chromophores of two important classes of fluorescent probes of biological material: the dansyl and the tryptophan probes. As the polarity of the medium is increased the following effects are observed: the absorption spectra exhibit a loss of resolvable structure: the fluorescence spectra exhibit a shift to lower energy, this behaviour being somewhat different to that predicted by the various theories of solvent relaxations the radiative lifetimes (and the S1 -, So transition moments) increase (decrease).The temperature dependence of the emission spectra, in n-butan-1-ol, have also been measured. These measurements show that the effects observed on increasing the polarity of the solvent are also observed a) when the viscosity of the medium changes from a glassy to a fluid one, and b) as a function of time after excitation. All of these effects are explicable on the basis of a solvent stabilisation of internal charge transfer (CT) states. We conclude that the emitting state in non polar media (and polar glasses) is a somewhat perturbed 1Lb state, while that in fluid polar (especially polar protic) media is best described as a 1L./CT state. Measurements on hydrophobic derivatives of aminonaphthalenes and indoles incorporated into lipid bilayer systems are discussed in terms of environment relaxation and multiple siting phenomena. Measurements of solvent dependent radiative decay rates are liable to a number of distortions. For this reason we give a detailed description of an integrating sphere spectrofluorimeter, and a time correlated single photon counting instrument. Experimental and data analysis procedures are fully described, as are methods for the construction of time resolved emission spectra. These instruments have been carefully calibrated with the following standards: 9.10-diphenyl anthracene in non polar solvents: quinine bisulphate in sulphuric acid solutions: 2 aminopyridine in sulphuric acid solutions. From our calibration measurements we reached the following conclusions: 1) the measured radiative lifetime requires a refractive index squared (n2) correction. 2) the geometrical n2 correction, used in right angle viewing fluorimeters is valid, 3) the fluorescence standard quinine bisulphate exhibits a non exponential fluorescence decay, 4) the fluorescence decay of 2 aminopyridine exhibits a strong temperature dependence; these observations are discussed.