Takakazu Nakabayashi

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.

Ultrafast excited-state dynamics in photochromic N-salicylideneaniline studied by femtosecond time-resolved REMPI spectroscopy.

Ultrafast processes in photoexcited N-salicylideneaniline have been investigated with femtosecond time-resolved resonance-enhanced multiphoton ionization spectroscopy. The ion signals via the S(1)(n,pi( *)) state of the enol form as well as the proton-transferred cis-keto form emerge within a few hundred femtoseconds after photoexcitation to the first S(1)(pi,pi( *)) state of the enol form. This reveals that two ultrafast processes, excited-state intramolecular proton transfer (ESIPT) reaction and an internal conversion (IC) to the S(1)(n,pi( *)) state, occur on a time scale less than a few hundred femtoseconds from the S(1)(pi,pi( *)) state of the enol form. The rise time of the transient corresponding to the production of the proton-transferred cis-keto form is within 750 fs when near the red edge of the absorption is excited, indicating that the ESIPT reaction occurs within 750 fs. The decay time of the S(1)(pi,pi( *)) state of the cis-keto form is 8.9 ps by exciting the enol form at 370 nm, but it dramatically decreases to be 1.5-1.6 ps for the excitation at 365-320 nm. The decrease in the decay time has been attributed to the opening of an efficient nonradiative channel; an IC from S(1)(pi,pi( *)) to S(1)(n,pi( *)) of the cis-keto form promotes the production of the trans-keto form as the final photochromic products. The two IC processes may provide opposite effect on the quantum yield of photochromic products: IC in the enol form may substantially reduce the quantum yield, but IC in the cis-keto form increase it.

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.

Low-frequency Raman spectra of crystalline and liquid acetic acid and its mixtures with water.: Is the liquid dominated by hydrogen-bonded cyclic dimers?

Abstract Low-frequency Raman spectra of crystalline and liquid acetic acid, and acetic acid–water binary solutions with varying mole fractions of acetic acid ( x A ), are presented in the form of the relative scattering activities, R( ν ) spectra. The spectrum of scattered light I( ν ) from crystalline acetic acid at 285 K shows five distinctive bands. Upon the melting, however, the liquid exhibited conspicuous difference in its appearance, although the R( ν ) spectrum shows two peaks and a shoulder at similar wavelengths to those of the crystalline bands. The spectral change does not directly support the dominance of the cyclic dimers in the liquid. R( ν ) spectra of acetic acid–water binary solutions suggest a three-state model composed of acetic acid associates, binary associates and water associates for the local structure of the mixtures.

Fluorescence decay dynamics of flavin adenine dinucleotide in a mixture of alcohol and water in the femtosecond and nanosecond time range.

Fluorescence decays of flavin adenine dinucleotide (FAD) that is a typical autofluorescent species in cells and tissues have been measured in a mixture of alcohol and water in the femtosecond and nanosecond time range. The fluorescence lifetimes of both the stacked conformation between the isoalloxazine and adenine moieties in close proximity and the extended open conformation in water are affected by the addition of alcohol. The nanosecond fluorescence lifetime of the open conformation increases with decreasing dielectric constant of the medium, contributing to the enhancement of the fluorescence intensity of FAD in less dielectric media. The fluorescence lifetime of the open conformation is also affected by medium viscosity, suggesting that the photoexcited open conformation is quenched by the dynamic interaction between the two aromatic rings. The fluorescence component decaying in tens of picoseconds is attributed to the stacked conformation that shows the efficient fluorescence quenching due to the intramolecular electron transfer. The picosecond fluorescence lifetime of the stacked conformation increases with decreasing dielectric constant, suggesting the shift of the distribution of the stacked conformation to a longer intramolecular distance between the two aromatic rings in less dielectric media. The pre-exponential factor of the picosecond decaying component relative to that of the nanosecond one decreases with the increase of the alcohol concentration in the femtosecond time-resolved fluorescence, which demonstrates the increase in the population of the open conformation with the reduction of the dielectric constant. The possibility to evaluate the polar environment in a cell by the fluorescence lifetime of FAD is discussed based on the results obtained.

Redox sensor proteins for highly sensitive direct imaging of intracellular redox state

Intracellular redox state is a critical factor for fundamental cellular functions, including regulation of the activities of various metabolic enzymes as well as ROS production and elimination. Genetically-encoded fluorescent redox sensors, such as roGFP (Hanson, G. T., et al. (2004)) and Redoxfluor (Yano, T., et al. (2010)), have been developed to investigate the redox state of living cells. However, these sensors are not useful in cells that contain, for example, other colored pigments. We therefore intended to obtain simpler redox sensor proteins, and have developed oxidation-sensitive fluorescent proteins called Oba-Q (oxidation balance sensed quenching) proteins. Our sensor proteins derived from CFP and Sirius can be used to monitor the intracellular redox state as their fluorescence is drastically quenched upon oxidation. These blue-shifted spectra of the Oba-Q proteins enable us to monitor various redox states in conjunction with other sensor proteins.

Stress-induced environmental changes in a single cell as revealed by fluorescence lifetime imaging.

The fluorescence lifetime image of HeLa cells expressing an enhanced green fluorescent protein (EGFP)-fusion protein changes under stress, which allows noninvasive determination of the status of individual cells.

Fluorescence lifetime images of green fluorescent protein in HeLa cells during TNF-α induced apoptosis

Fluorescence lifetime images of HeLa cells expressing enhanced green fluorescent protein (EGFP) have been measured as apoptosis is induced by tumor necrosis factor-alpha (TNF-alpha) in combination with cycloheximide. The fluorescence lifetime of EGFP is found to decrease after the induction of apoptosis, indicating that the change in environment occurs around the chromophore of EGFP with the apoptosis process. The fluorescence lifetime imaging technique can be used to perform in vivo observation of cell death processes. Fluorescence lifetime measurements are useful to examine the induction of the apoptosis process, even when a morphological change of each cell cannot be observed because of a low spatial resolution.

pH-dependent network formation of quantum dots and fluorescent quenching by Au nanoparticle embedding.

A simple approach to the creation of colloidal assemblies is in high demand for the development of functional devices. Here, we present the preparation of CdTe-QD (quantum dot) networks in as little as 1 day simply by pH modification without the use of oxidants. The QD network was tractable in water and casting from a droplet produced a porous networked film on both hydrophobic and hydrophilic solid substrates. Further, we found that citrate-protected gold nanoparticles (AuNPs, d = 5 nm) could be incorporated into the QD networks to afford a QD/Au composite network, and that the fluorescence from the QDs was largely decreased by the addition of a small proportion of AuNPs (QD:AuNP = 99.4:0.6), probably due to the efficient charge transfer through the network. These data indicate that our method is suitable for application to the creation of metal/QD hybrid materials that can be integrated into wet-based processes.