Fluorescence lifetime images of reduced nicotinamide adenine dinucleotide (NADH) that is a key cofactor in cellular metabolism were obtained in a cell at various values of intracellular pH. The average fluorescence lifetime of NADH is found to become shorter monotonically with increasing pH, indicating that pH in a single cell can be determined by fluorescence lifetime imaging of NADH without adding exogenous fluorescent probes. The magnitude of the pH-induced lifetime change is higher in cells than that in buffer solution. The fluorescence lifetime of NADH is not uniform inside a cell; the fluorescence lifetime of nuclear NADH is shorter than that of mitochondrial NADH at each pH, and the magnitude of the pH-induced change is larger in nuclei than in other areas. The local electric field effect on the fluorescence lifetime is discussed since this effect may be one of the strong possibilities for the nonuniformity of the autofluorescence lifetime of NADH in cells.

Electroabsorption and electrofluorescence spectroscopies were conducted for tri-9-anthrylborane (TAB) doped in poly(methyl methacrylate) films (1.0 mol %) to reveal the spectroscopic and excited-state properties of the compound. TAB showed three distinct absorption bands: bands I [(19 - 25) x 10(3) cm(-1)], II [(25-31) x 10(3) cm(-1)], and III (>31 x 10(3) cm(-1)). The electroabsorption spectrum demonstrated that the electronic transitions in bands I and III accompanied electric dipole moment changes (Deltamu), while the change in the molecular polarizability contributed mainly to electroabsorption band II. Because of the similarities of the electroabsorption spectrum of band II with that of anthracene itself, band II was assigned to the electronic transition to the locally excited (LE) state of the anthryl group. On the other hand, bands I and III were best described by the electronic transitions to the excited charge-transfer (CT) states. The study demonstrated furthermore that the Deltamu value of TAB accompanied by the lowest-energy electronic transition was as large as 7.8 D, which agreed very well with that determined by the solvent dependences of the absorption and fluorescence maximum energies of TAB (approximately 8.0 D, ref 1): Deltamu = 7.8-8.0 D. The results proved explicitly that the excited state of TAB was localized primarily on the p orbital of the boron atom. Despite the dipole moment change (Deltamu = 7.8-8.0 D) for the lowest-energy electronic transition (band I), the electrofluorescence of TAB accompanied the change in the molecular polarizability. The spectroscopic and excited-state properties of TAB including the curious behavior of the electrofluorescence spectrum as mentioned above were discussed on the basis of theoretical considerations.

Stark absorption spectroscopy was applied to 6-hydroxyquinoline (6-HQ) doped in polymer films of poly(methyl methacrylate) (PMMA) and poly(vinyl alcohol) (PVA) at temperatures of 50-300 K. The electroabsorption (E-A) spectrum of 6-HQ markedly depends on temperature in a PMMA film. The polarization dependence as well as the temperature dependence of the E-A spectra reveals that 6-HQ is oriented along the direction of the applied electric field at room temperature in a PMMA film. As the temperature becomes lower, the field-induced orientation of 6-HQ is restricted, and only the Stark shift induced by a change in electric dipole moment and in molecular polarizability is observed. On the other hand, E-A spectra of 6-HQ doped in a PVA film are essentially independent of temperature, suggesting that 6-HQ is not oriented along the electric field even at room temperature in PVA. These results show that the molecular motion of 6-HQ in a polymer film is very sensitive to the microenvironment of the surrounding matrix.

No abstract

Fluorescence spectra and electrofluorescence spectra (plots of the electric field-induced change in fluorescence intensity as a function of wavelength) have been measured at different temperatures for pyrene butyric acid (PBA) in a PMMA film at different concentrations. At a low concentration of 0.5 mol % where fluorescence emitted from the locally excited state of PBA (LE fluorescence) is dominant, LE fluorescence spectra show only the Stark shift in the presence of an electric field (F), which results from the difference in molecular polarizability between the ground and emitting states. At a high concentration of 10 mol % where the so-called sandwich-type excimer fluorescence (EX(1)) is dominant, both EX(1) and LE fluorescence are quenched by F. Another fluorescence assigned to a partially overlapped excimer (EX(2)) also exists at room temperature, and this emission is enhanced by F. As the temperature decreases, three fluorescence emissions whose electric field effects are different from each other become clear besides EX(1) and LE fluorescence, indicating that at least five fluorescence components exist at high concentrations at low temperatures. At a medium concentration of 5 mol % where EX(1) is comparable in intensity to the LE fluorescence, the intensity of EX(1) is not affected by F at any temperature, but LE fluorescence and EX(2) are markedly influenced by F at room temperature, and four fluorescence emissions are confirmed at low temperatures.