Nobuhiro Ohta

Application of fluorescence lifetime imaging of enhanced green fluorescent protein to intracellular pH measurements

We have shown that the intracellular pH of a single HeLa cell expressing the enhanced green fluorescent protein (EGFP) can be imaged using the fluorescence lifetime of EGFP, which can be interpreted in terms of the pH-dependent ionic equilibrium of the p-hydroxybenzylidene-imidazolidinone structure of the chromophore of EGFP.

Intracellular pH Sensing Using Autofluorescence Lifetime Microscopy

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.

Electric field effects on absorption and fluorescence spectra of pyrene doped in a PMMA polymer film

Electric field modulated absorption, fluorescence and fluorescence excitation spectra of pyrene doped in a PMMA polymer film were measured with excitation from 220 to 380 nm. The observed spectra of the field-induced change in absorption intensity or fluorescence intensity for each electronic transition agree well with the first derivatives of the corresponding absorption or fluorescence spectrum, indicating that the field effect results from the Stark shift induced by a change in molecular polarizability between the ground state and the excited state. The magnitude of the change in polarizability was evaluated for the first, S1(1Lb), the second, S2(1Ba), the third, S3(1Bb), and the fourth, S4(1La), optically spin-allowed absorption bands. The change in average polarizability following excitation is found to be in the order of S1<S4<S3<S2, and all the polarizabilities in the excited states are larger than in the ground state. The third nonlinear order susceptibility (χ(3)) of a pyrene-doped PMMA film was also calculated on the basis of the electroabsorption and electrofluorescence excitation spectra.

pH Dependence of the Fluorescence Lifetime of FAD in Solution and in Cells

We have studied physiological parameters in a living cell using fluorescence lifetime imaging of endogenous chromophores. In this study, pH dependence of the fluorescence lifetime of flavin adenine dinucleotide (FAD), that is a significant cofactor exhibiting autofluorescence, has been investigated in buffer solution and in cells. The fluorescence lifetime of FAD remained unchanged with pH 5 to 9 in solution. However, the fluorescence lifetime in HeLa cells was found to decrease with increasing intracellular pH, suggesting that pH in a single cell can be estimated from the fluorescence lifetime imaging of FAD without adding exogenous fluorescent probes.

Vibronic coupling and Raman intensity of naphthalene and anthracene

Abstract The intensities of Raman lines of naphthalene and anthracene have been measured with exciting light of various wavelengths in the pre-resonance region. Great Raman intensity enhancements are found for the b 3g Raman line at 1629 cm −1 and two totally symmetric Raman lines at 1579 and 1381 cm −1 of naphthalene and for the corresponding Raman lines of anthracene. It is found that the Raman intensities of the b 3g vibrations are mainly responsible for the vibronic coupling between two π,π * excited states 1 B 1u ( 1 L a ) and 1 B 2u ( 1 B b ) of these molecules. In the longer wavelength region, the b 3g Raman line of naphthalene exhibits an “intensity de-enhancement” in contrast to the great intensity enhancement in the shorter wavelength region. The observed “de-enhancement” is explained as the result of destructive interference of two different vibronic coupling mechanisms. The intensity enhancements of the a g Raman lines are found to be reflecting the geometrical structures of the molecules in their 1 B 1u ( 1 L a ) states.

Vibronic coupling and intramolecular dynamics of pyrene as revealed by the S0→S2 excitation spectrum in a supersonic jet

Abstract The fluorescence excitation spectrum of pyrene obtained in a supersonic jet for the S 0 →S 2 transition shows a complicated structure due to the interaction of discrete levels of S 2 with the quasi-continuous levels belonging to S 1 . The intensity distribution pattern in this region has been evaluated from quantum-mechanically calculated quantities, such as the vibronic coupling integrals, and Franck-Condon factors deduced from independent experiments. The dynamics in pyrene following excitation into S 2 are discussed.

Electronic spectra and intramolecular dynamics of phenanthrene in a supersonic free jet

The fluorescence excitation spectra and the fluorescence decays of phenanthrene-h10 and phenanthrene-d10 in a supersonic free jet have been measured with excitation across the S 0 → S 1 and S 0 → S 2 absorption regions. The fluorescence excitation spectra of the isolated phenanthrene molecule are compared with the absorption spectra in low temperature matrices. The presence of a 2-type out-of-plane vibrational bands in the S 0 → S 2 absorption spectra indicates that a 1 B 1(πσ*) state lies near the S 2 state. The S 2 → S 1 internal conversion rates at various vibronic levels of S 2 for both compounds have been evaluated from the analysis of profile and width of the individual vibronic bands in the excitation spectra. The excess vibrational energy dependence of the S 2 → S 1 internal conversion rate is the opposite of that shown by the total decay rate in S 1. The excess vibrational energy dependence of the electronic decay rate and the profile of the vibronic absorption bands belonging to the S 0 → S 2 tr...

Stark absorption spectroscopy of indole and 3-methylindole

Indole and 3-methylindole (3-MI) doped into a polymethylmethacrylate (PMMA) film are studied by the Stark absorption (electroabsorption) spectroscopy. The 1La and 1Lb absorption bands are distinguished and the change in permanent dipole moment on 1La excitation is determined by a model fit to the measured absorption and electroabsorption spectra. Analysis of the spectra, measured at normal incidence and magic angle conditions, proved the essential role of the electric-field-induced orientation/alignment effects for polar indole and 3-MI molecules in the PMMA environment at room temperature.

Time-resolved measurement system of electrofluorescence spectra

A time-resolved measurement system for electrofluorescence spectra (plots of electric-field-induced change in fluorescence intensity as a function of wavelength) has been constructed by combining the time-correlated single-photon-counting system for emission decay measurement with modulated electric field as a sample bias. Four fluorescence decays, which correspond to positive, zero, negative, and zero sample bias, respectively, can be measured simultaneously, and a small field-induced change in decay profile can be detected precisely by using the constructed system. Measurements of time-resolved electrofluorescence spectra have been performed in electron donor–acceptor compounds which show a photoinduced electron transfer.

Magnetic field effects on yields and decays of fluorescence of pyrimidine vapor: Excited rovibronic level dependence

External magnetic field effects on yields and decays of fluorescence of pyrimidine vapor on excitation into various rotational levels belonging to the vibrationless level or the 6a1 level of S1 have been studied in a supersonic jet or in a bulk gas at room temperature with a field strength of 0–150 G. The fast component of fluorescence is not affected by an external magnetic field, whereas the slow fluorescence is quenched by a field except for excitation at the R(0) line belonging to the 0–0 transition. The fluorescence quenching is more effective at the 6a1 level than that at 00, indicating that the level density of the triplet state coupled to the singlet state plays an important role in the magnetic mixing of the triplet spin sublevels, in terms of which the fluorescence quenching by a magnetic field is interpreted. The excited rotational level dependence of the fluorescence quenching by a magnetic field is attributed to K scrambling in the triplet manifold following intersystem crossing.