Fluorescence enhancement induced by quadratic electric-field effects on singlet exciton dynamics in poly(3-hexylthiophene) dispersed in poly(methyl methacrylate).

The dynamics of the exciton generated by photoexcitation of a regioregular poly(3-hexylthiophene) (P3HT) polymer dispersed in a poly(methyl methacrylate) (PMMA) matrix was examined using electro-photoluminescence (E-PL) spectroscopy, where electric field effects on the photoluminescence (PL) spectra were measured. The quadratic electric-field effect was investigated using the modulation technique, with field-induced changes in the PL intensity monitored at the second harmonic of the modulation frequency of the applied electric field. Absorption and PL spectra indicated the formation of both ordered crystalline aggregates and amorphous regions of P3HT polymer chains. Although previous studies of electric field effects on π-conjugated polymers have generally shown that the PL intensity is decreased by electric fields, we report that the PL intensity of P3HT and PL lifetime increased with the quadratic electric-field effect. The magnitude of the change in PL intensity was quantitatively explained in terms of the field-induced decrease in the nonradiative decay rate constants of the exciton. We proposed that a delayed PL, originating from charge carrier recombination, was enhanced in the presence of electric fields. The rate constant of the downhill relaxation process of the exciton, which originated from the relaxation in distributed energy levels due to an inherent energetic disorder in P3HT aggregates, was implied to decrease in the presence of electric fields. The radiative decay rate constant and PL quantum yield of P3HT dissolved in solution, which were evaluated from the molar extinction coefficient and the PL lifetime, were compared with those of P3HT dispersed in a PMMA matrix.

Application of Fluorescence Lifetime Imaging (FLIM) to Measure Intracellular Environments in a Single Cell.

Fluorescence lifetime imaging (FLIM) has now been used in many bioscience fields, which comes from the quantification of fluorescence lifetime. The procedure for obtaining lifetime images is very similar to that used in fluorescence microscopy. However, obtaining reliable lifetime images requires an understanding of the theory of fluorescence lifetime, principle of FLIM systems, and evaluation procedure of intracellular environments. In this chapter, the materials, methods, and notes on FLIM measurements have been described, in conjunction with a brief explanation of the background of FLIM.

Stark Spectroscopy of Rubrene. II. Stark Fluorescence Spectroscopy and Fluorescence Quenching Induced by an External Electric Field.

We report Stark fluorescence spectroscopy investigation of rubrene dispersed in a poly(methyl methacrylate) film. The features of the fluorescence spectrum are analogous to those in solutions. In the Stark fluorescence spectrum, the decrease of the fluorescence quantum yield in the presence of an external electric field is observed. This result shows that the yield of nonradiative decay processes is increased by the application of an external electric field. It is known that the fluorescence quantum yield for rubrene, which is nearly unity at room temperature, depends on temperature, and a major nonradiative decay process in photoexcited rubrene is ascribed to a thermally activated intersystem crossing (ISC). Equations that express the field-induced fluorescence quenching in terms of the molecular parameters are derived from the ensemble average of electric field effects on the activation energy of the reaction rate constant in random orientation systems. The molecular parameters are then extracted from the observed data. It is inferred that the field-induced increase in the yield of other intramolecular and intermolecular photophysical processes in addition to the ISC should be taken into account.

Stark Spectroscopy of Rubrene. I. Electroabsorption Spectroscopy and Molecular Parameters.

Electroabsorption spectroscopy investigation and the determination of molecular parameters for rubrene dispersed in a poly(methyl methacrylate) (PMMA) matrix are reported. The features of the band system in the absorption spectrum in PMMA are analogous to those in solutions. The changes in the electric dipole moment and the polarizability between the excited and ground states are determined from analysis of the Stark effect in the absorption band. The change in the transition dipole moment in the presence of an external electric field is also observed. Although rubrene is predicted to be classified as a nonpolar molecule, there is a contribution of the difference in the electric dipole moment between the excited and ground states to the electroabsorption spectrum. The origin of the nonzero difference in the electric dipole moment is argued. Stark fluorescence spectroscopy investigation is reported in Part II of this series.

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.

External Electric Field Effects on Excited-State Intramolecular Proton Transfer in 4'-N,N-Dimethylamino-3-hydroxyflavone in Poly(methyl methacrylate) Films.

The external electric field effects on the steady-state electronic spectra and excited-state dynamics were investigated for 4'-N,N-(dimethylamino)-3-hydroxyflavone (DMHF) in a poly(methyl methacrylate) (PMMA) film. In the steady-state spectrum, dual emission was observed from the excited states of the normal (N*) and tautomer (T*) forms. Application of an external electric field of 1.0 MV·cm(-1) enhanced the N* emission and reduced the T* emission, indicating that the external electric field suppressed the excited-state intramolecular proton transfer (ESIPT). The fluorescence decay profiles were measured for the N* and T* forms. The change in the emission intensity ratio N*/T* induced by the external electric field is dominated by ESIPT from the Franck-Condon excited state of the N* form and vibrational cooling in potential wells of the N* and T* forms occurring within tens of picoseconds. Three manifolds of fluorescent states were identified for both the N* and T* forms. The excited-state dynamics of DMHF in PMMA films has been found to be very different from that in solution due to intermolecular interactions in a rigid environment.

Fluorescence characteristics and lifetime images of photosensitizers of talaporfin sodium and sodium pheophorbide a in normal and cancer cells.

Fluorescence spectra and fluorescence lifetime images of talaporfin sodium and sodium-pheophorbide a, which can be regarded as photosensitizers for photodynamic therapy, were measured in normal and cancer cells. The reduction of the fluorescence intensity by photoirradiation was observed for both photosensitizers in both cells, but the quenching rate was much faster in cancer cells than in normal cells. These results are explained in terms of the excessive generation of reactive oxygen species via photoexcitation of these photosensitizers in cancer cells. The fluorescence lifetimes of both photosensitizers in cancer cells are different from those in normal cells, which originates from the different intracellular environments around the photosensitizers between normal and cancer cells.

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.

Application of Nanosecond Pulsed Electric Field and Autofluorescence Lifetime Microscopy of FAD in Lung Cells.

Exposure of nanosecond pulsed electric fields (nsPEFs) to live cells is an increasing research interest in biology and medicine. Despite extensive studies, a question still remains as to how effects of application of nsPEF on intracellular functions are different between cancerous cells and normal cells and how the difference can be detected. Herein, we have presented an approach of autofluorescence lifetime (AFL) microscopy of flavin adenine dinucleotide (FAD) to detect effects of application of nsPEF having 50 ns of a pulse width, nsPEF(50), on intracellular function in lung cancerous cells, A549 and H661, which show nsPEF(50)-induced apoptosis, and normal cells, MRC-5, in which the field effect is less or not induced. Then, the application of nsPEF(50) is shown to increase the lifetime of FAD autofluorescence in lung cancerous cells, whereas the electric field effects on the autofluorescence of FAD was not significant in normal healthy cells, which indicates that the lifetime measurements of FAD autofluorescence are applicable to detect the field-induced change in intracellular functions. Lifetime and intensity microscopic images of FAD autofluorescence in these lung cells were also acquired after exposure to the apoptosis-inducer staurosporine (STS). Then, it was found that the AFL of FAD became longer after exposure not only in the cancerous cells but also in the normal cells. These results indicate that nsPEF(50) applied to lung cells induced apoptotic cell death only in lung cancerous cells (H661 and A549) but not in lung normal cells (MRC-5), whereas STS induced apoptotic cell death both in lung cancerous cells and in lung normal cells. The lifetime microscopy of FAD autofluorescence is suggested to be very useful as a sensitive detection method of nsPEF-induced apoptotic cell death.

Insulator-metal transitions induced by electric field and photoirradiation in organic Mott insulator deuterated κ-(BEDT-TTF)2Cu[N(CN)2]Br.

The Mott insulator-metal transition induced by an external stimulus such as electric field, pressure, chemical doping, or photoirradiation has received considerable attention because of the potential use in new optoelectronic functional devices. Here we report an abrupt Mott insulator-metal transition observed as a current jump in a molecular-based Mott insulator, namely, deuterated κ-(BEDT-TTF)(2)Cu[N(CN)(2)]Br, where BEDT-TTF = bis(ethylenedithio)tetrathiafulvalene, upon application of a pulsed voltage of certain magnitude (threshold voltage). Furthermore, the threshold voltage needed for the transition is shown to be reduced by photoirradiation. Thus, the Mott insulator-metal transition can be controlled by a combination of an external electric field and photoirradiation.

Pyrene-Based AIEE Active Nanoprobe for Zn2+ and Tyrosine Detection Demonstrated by DFT, Bioimaging, and Organic Thin-Film Transistor.

The development of aggregation-induced emission enhancement (AIEE) active nanoprobes without any synthetic complication for solution-state and organic thin-film transistor (OTFT)-based sensory applications is still a challenging task. In this study, the novel pyrene-incorporated Schiff base (5-phenyl-4-((pyren-1-ylmethylene)amino)-4H-1,2,4-triazole-3-thiol; PT2) with an AIEE property was synthesized via a one-pot reaction and was reported for detecting Zn2+ and tyrosine in the solution state and OTFT. In the AIEE studies of PT2 (in CH3CN) at various water fractions (fw: 0-97.5%), the existence of J-aggregation, crystalline changes, and nanofibers formation was confirmed by ultraviolet absorption/photoluminescence (UV/PL) spectroscopy, powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), and dynamic-light scattering (DLS) techniques. Similarly, PT2-based Zn2+ detection and sensory reversibility with tyrosine were demonstrated by UV/PL studies with evidence related to crystalline/nanolevel changes in PXRD, SEM, TEM, AFM, and DLS data. Distinct decay profiles associated with the AIEE and sensory responses of PT2 were observed in time-resolved photoluminescence spectra. From the standard deviation and linear fittings of PL titrations, detection limits (LODs) of the Zn2+ with PT2 and the tyrosine with PT2-Zn2+ were estimated as 0.79 and 45 nM, respectively. High-resolution mass and 1H NMR results confirmed 2:1 and 1:1 stoichiometry and binding sites of PT2-Zn2+-PT2* and tyrosine-Zn2+ complexes. Moreover, the values of association constants determined by linear fittings were 4.205 × 10-7 and 1.73 × 10-8 M-2, correspondingly. Optimization via the density functional theory disclosed the binding sites and suppression of twisted intramolecular charge transfer/photoinduced electron transfer (TICT/PET) as well as the involvement of restricted intramolecular rotation in the AIEE and PET "ON-OFF-ON" mechanisms in the Zn2+ and tyrosine sensors. Results from the B16-F10 cellular and zebrafish imaging of AIEE, Zn2+, and tyrosine sensors further attested the applicability of PT2 in biological samples. Finally, the PT2 and pentacene-incorporated OTFT devices were fabricated. The devices displayed more than 90% change in drain-source current when reacted with Zn2+ with an LOD of 5.46 µM but showed no response to tyrosine, thereby confirming the reversibility. Moreover, the OTFT devices also demonstrated Zn2+ ion detection in tap water and lake water samples.

Characterization of endogenous fluorescence in nonsmall lung cancerous cells: A comparison with nonmalignant lung normal cells.

Monitoring fluorescence properties of endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in normal and cancerous cells provide substantial information noninvasively on biochemical and biophysical aspects of metabolic dysfunction of cancerous cells. Time-resolved spectral profiles and fluorescence lifetime images of NADH and FAD were obtained in human lung nonsmall carcinomas (H661 and A549) and normal lung cells (MRC-5). Both fluorophores show the fast and slowly decaying emission components upon pulsed excitation, and fluorescence spectra of NADH and FAD show blue- and red-shifts, respectively, during their decay. All identified lifetime components of NADH and FAD were found to be shorter in cancerous cells than in normal cells, no matter how they were measured under different extra-cellular conditions (cells suspended in cuvette and cells attached on glass substrate), indicating that the changes in metabolism likely altered the subcellular milieu and potentially also affected the interaction of NADH and FAD with enzymes to which these cofactors were bound. The intensity ratio of NADH and FAD of cancerous cells was also shown to be larger than that of normal cells.

Diversiform Nanostructures Constructed from Tetraphenylethene and Pyrene-Based Acid/Base Controllable Molecular Switching Amphiphilic [2]Rotaxanes with Tunable Aggregation-Induced Static Excimers.

Dual-emissive tetraphenylethene (TPE) and pyrene-containing amphiphilic molecules are of great interest because they can be integrated to form stimuli responsive materials with various biological applications. Herein, we report the study of mechanically interlocked molecules (MIMs) with aggregation-induced static excimer emission (AISEE) property through a series of TPE and pyrene-based amphiphilic [2]rotaxanes, where t-butylcalix[4]arene with hydrophobic nature was used as the macrocycle. Evidently, by adorning TPE and pyrene units in [2]rotaxanes P1, P2, P1-b, and P2-b, they display remarkable emission bands in 70% of water fraction (fw) in tetrahydrofuran (THF)/water mixture, which could be attributed to the restricted intramolecular rotation of phenyl groups, whereas prominent blue-shifted excimer emission of pyrene started to appear as fw reached 80% for P1 and 90% for P1-b, P2, and P2-b, which was ascribed to the favorable π-π stacking and hydrophobic interactions of the pyrene rings that enabled their static excimer formation. The well-defined distinct amphiphilic nanostructures of [2]rotaxanes including hollowspheres, mesoporous nanostructures, spheres, and network linkages can be driven smoothly depending on the molecular structures and their aggregated states in THF/water mixture. These fascinating diversiform nanostructures were mainly controlled by the skillful manner of reversible molecular shuttling of t-butylcalix[4]arene macrocycle and also the interplay of multinoncovalent interactions. To further understand the aggregation capabilities of [2]rotaxanes, the human lung fibroblasts (MRC-5) living cell incubated with either P1, P2, P1-b, or P2-b was studied and monitored by confocal laser scanning microscopy. The AISEE property was achieved at an astonishing level by integrating TPE and pyrene to MIM-based reversible molecular switching [2]rotaxanes; furthermore, distinct nanostructures, especially hollowspheres and mesoporous nanostructures, were observed, which are rarely reported in the literature but are highly desirable for future applications.

Novel rhodamine probe for colorimetric and fluorescent detection of Fe3+ ions in aqueous media with cellular imaging.

A novel rhodamine-pyridine conjugated spectroscopic probe RhP was synthesized and its X-ray single crystalline properties were revealed with tabulation. The RhP displayed a distinct pale-pink colorimetric and "turn-on" fluorescent response to Fe3+ in aqueous media [H2O:DMSO (95:5, v/v)] than that of other interfering ions. During the Fe3+ recognition, the absorption (UV-Vis) and photoluminescence (PL) spectral studies revealed new peaks at 561 and 592 nm, respectively. The 1:1 stoichiometry and binding sites were verified by Job's plot, ESI-mass, and 1H NMR titrations. Subsequently, LOD and binding constant for RhP + Fe3+ complex were estimated as 102.3 nM and 6.265 × 104 M-1 from linear fitting and Benesi-Hildebrand plots, correspondingly. Sensor reversibility of RhP + Fe3+ by EDTA was demonstrated by UV/PL and TRPL investigations. Moreover, the photoinduced energy transfer mechanism and band gap changes were established from the DFT interrogations. Lastly, cellular imaging studies were carried out to authenticate the real applicability of RhP in Fe3+ detection.

Synergy effects of electric and magnetic fields on locally excited-state fluorescence of photoinduced electron transfer systems in a polymer film.

Photoluminescence of electron donor-acceptor pairs that show photoinduced electron transfer (PIET) has been measured in a polymer film under simultaneous application of electric field and magnetic field. Fluorescence emitted from the locally excited state (LE fluorescence) of 9-methylanthracene (MAnt) and pyrene (Py) is quenched by an electric field in a mixture of 1,3-dicyanobenzene (DCB) with MAnt or Py, indicating that PIET from the excited state of MAnt or Py to DCB is enhanced by an electric field. Simultaneous application of electric and magnetic fields enhances the reverse process from the radical-ion pair produced by PIET to the LE fluorescent state of MAnt or Py. As a result, the electric-field-induced quenching of the LE fluorescence is reduced by application of the magnetic fields. Thus, the synergy effect of electric and magnetic fields is observed on the LE fluorescence of MAnt or Py. Exciplex fluorescence spectra resulting from PIET can be obtained by analyzing the field effects on photoluminescence spectra, even when the exciplex fluorescence is too weak to be determined from the steady-state or time-resolved photoluminescence spectra at zero field.

Comparative study of electroabsorption spectra of polar and nonpolar organic molecules in solution and in a polymer film.

Electroabsorption (EA) spectra of polar and nonpolar molecules of coumarin 153 (C153) and pyrene in solution and in a polymer film of polymethylmethacrylate (PMMA) have been measured in the UV-visible region at room temperature. The shape of the EA spectra of C153 in benzene, 1,4-dioxane, or monochlorobenzene remarkably depends on the angle between the polarization direction of the absorption light and the applied electric field, whereas the EA spectra of C153 doped in PMMA show only the Stark shift and the field-induced change in spectral shape is negligible. These results demonstrate that C153 is reoriented by application of electric fields in solution, but the molecules are immobilized in a PMMA film. Based on the EA spectra, electric dipole moments both in the ground state and in the excited state have been evaluated for C153 in different solvents. In the EA spectra of pyrene, only the Stark shift is observed both in solution and in PMMA, indicating that the field-induced molecular reorientation does not occur both in solution and in PMMA. The change in dipole moment of C153 as well as the change in molecular polarizability of pyrene following absorption is much larger in solution than that in PMMA.

Theoretical quest for photoconversion molecules having opposite directions of the electric dipole moment in S0 and S1 states.

Ab initio calculations at the level of CASPT2 with Dunning's correlation consistent cc-pVXZ (X=D, T, Q) basis sets have been carried out for pyrimidine, quinoxaline, phthalazine, and their substituted compounds to find candidates that show a change in the direction of the electric dipole moment for the S(0)-->S(1) transition. The present calculations reveal that 6,7-difluorophthalazine and 6,7-dichlorophthalazine are strong candidates having a large and clear change in the direction of the electric dipole moment on the S(0)-->S(1) transition.

Enhanced Dissociation of Hot Excitons with an Applied Electric Field under Low-Power Photoexcitation in Two-Dimensional Perovskite Quantum Wells.

The dependence of photoluminescence (PL) on excitation power and the effect of an external electric field have been studied for a two-dimensional (2D) perovskite (C4H9NH3)2PbI4 thin-film sample. The efficiency of dissociation of hot excitons to produce free carriers was enhanced with a small excitation power because the relaxation of hot excitons to cold emissive excitons was slow, indicating that the thermal energies of hot carriers can be utilized in solar cells under weak photoirradiation. The dissociation was notably enhanced with an applied electric field, resulting in efficient field-induced quenching of the PL. The present results shed light on an application of 2D perovskite materials to photovoltaic (PV) devices with dim radiation, e.g., for indoor PV applications; the concept of electric field-assisted solar cells might be applicable to next-generation solar cells.

Magnetic field effect on fluorescence in a mixture of N-ethylcarbazole and dimethyl terephthalate in a polymer film in the presence of electric fields.

Magnetic field effects on the fluorescence spectrum and on the electrofluorescence spectrum (plots of the electric field-induced change in fluorescence intensity as a function of wavelength) have been examined in electron donor and acceptor pairs of N-ethylcarbazole (ECZ) and dimethyl terephthalate (DMTP) in polymer films at different ratios of donor/acceptor concentration. In the mixture having a high concentration of ECZ, electric field-induced quenching of the exciplex fluorescence originating from the photoinduced electron transfer becomes less efficient in the presence of a magnetic field. In the mixture having a low concentration of ECZ, on the other hand, no magnetic field effect was observed in the electrofluorescence spectrum, indicating that the hole carrier plays an important role in synergy effects of magnetic and electric field effects on exciplex fluorescence. In the absence of the applied electric field, the magnetic field does not affect either exciplex fluorescence with a peak at 450 nm or LE fluorescence emitted from the locally excited state of ECZ but enhances the broad emission with a peak at approximately 380 nm, probably assigned to the fluorescence of another type of exciplex between ECZ and DMTP. Thus, two kinds of magnetic field effects on fluorescence have been observed in a mixture of ECZ and DMTP in a polymer film.

Electric field effects on absorption and fluorescence spectra of trimethylsilyl- and trimethylsilylethynyl-substituted compounds of pyrene in a PMMA film.

External electric field effects on absorption and fluorescence spectra of 1,3,6,8-tetrakis(trimethylsilyl)pyrene and 1,3,6,8-tetrakis(trimethylsilylethynyl)pyrene (TMSPy and TMS(E)Py, respectively) have been examined in a poly(methyl methacrylate) (PMMA) film at various concentrations at various temperatures. TMS(E)Py preferentially forms an aggregate in a PMMA film, as the concentration increases, indicating that the acetylenic groups enhance the pi-pi interactions between pyrene molecules. The change in molecular polarizability following excitation has been determined both for the monomer and for the aggregate, based on the electroabsorption spectra. The change in molecular polarizability following emission has also been determined in both compounds, based on the electrofluorescence spectra. TMSPy exhibits two excimer fluorescence emissions at high concentrations which are ascribed to the partially overlapping excimer and the sandwich-type excimer, respectively, besides the monomer fluorescence emitted from the locally excited state. The sandwich-type excimer fluorescence as well as monomer fluorescence is quenched by an electric field, whereas the fluorescence of the partially overlapping excimer is enhanced by an electric field. Excimer fluorescence of TMS(E)Py, which arises from the sandwich-type excimer, is quenched by an electric field at any temperature. Only one species of the partially overlapping excimer is confirmed in TMSPy, while no partially overlapping excimer is confirmed in TMS(E)Py.