Fluorescence of horse liver alcohol dehydrogenase using one- and two-photon excitation

We examined the steady-state and time-resolved emission of liver alcohol dehydrogenase resulting from one-photon and two-photon excitation. Previous studies with one-photon excitation revealed that the two nonidentical tryptophan residues display different emission spectra and decay times. The use of two-photon excitation resulted in similar emission spectra, multiexponential intensity decays, time-resolved emission spectra, and anisotropy decays as was observed for one-photon excitation. These results suggest that both nonidentical tryptophan residues are excited to a similar extent for one- and two-photon excitation. However, the limiting anisotropy (r ₀) with two-photon excitation from 585 to 610 nm is below 0.1 and appears distinct from that observed previously forN-acetyl-l-tryptophanamide.Abbreviations LADH liver alcohol dehydrogenase - -NAD⁺ -nicotinamide adenine dinucleotide - OPE one-photon excitation - OPIF one-photon induced fluorescence - TPE two-photon excitation - TCSPC time-correlated single photon counting - TPIF two-photon induced fluorescence     

Silver Fractal-Like Structures for Metal-Enhanced Fluorescence: Enhanced Fluorescence Intensities and Increased Probe Photostabilities

Substantial increases in fluorescence emission from fluorophore-protein–coated fractal-like silver structures have been observed. We review two methods for silver fractal structure preparation, which have been employed and studied. The first, a roughened silver electrode, typically yielded a 100-fold increase in fluorophore emission, and the second, silver fractal-like structures grown on glass between two silver electrodes, produced a 500-fold increase. In addition, significant increases in probe photostability were observed for probes coated on the silver fractal like structures. These results further serve to compliment our recent work on the effects of nobel metal particles with fluorophores, a relatively new phenomenon in fluorescence we have termed both metal-enhanced fluorescence [1] and radiative decay engineering [2,3]. These results are explained by the metallic surfaces modifying the radiative decay rate () of the fluorescent labels. We believe that this new silver-surface preparation, which results in ultrabright and photostable fluorophores, offers a new generic technology platform for increased fluorescence signal levels, with widespread potential applications to the analytical sciences, imaging, and medical diagnostics.     

Picosecond time resolved fluorescence spectroscopy study of aggregation of porphyrin derivative Mn(3,4,5-OMeTPP)Cl

The picosecond time-resolved fluorescence spectrum I(, t) of the porphyrin derivative Mn(3,4,5-OMeTPP)Cl is measured. Experiments show that the fluorescence relaxation of the concentrated sample solution is composed of a fast (ps), a moderately fast (100 ps) and a slow (ns) decay process. Furthermore, the fluorescence spectra for various times after excitation have different shapes. Detailed analysis shows that the fluorescence is composed of three components with different relaxation times and spectrum shapes. The three components are attributed to monomers, dimers, and aggregates respectively.     

Fluorescence anisotropy of indole molecules under two-photon excitation in the spectral range of 485–510 nm

Decay of polarized fluorescence in indole dissolved in propylene glycol under two-photon excitation by femtosecond laser pulses in the wavelength range of 485–510 nm has been studied. It is shown that under the experimental conditions used the fluorescence decay signal can be well described by a single excited state lifetime τ_f and a single rotation diffusion time τ_rot. By processing the data obtained, the times τ_f and τ_rot as well as anisotropy parameter r ₀ characterizing the symmetry of two-photon excitation of indole molecules have been determined. Decreasing of the anisotropy parameter r₀ down to zero under two-photon excitation energy higher than 5.1 eV has been observed. Interpretation of the obtained results have been done on the basis of ab initio quantum-mechanical computations. A model of energy relaxation under the condition of twophoton excitation of indole in a polar solvent has been discussed.     

Luminescence of Molecules with Internal Charge Transfer upon Longwave Excitation

We have studied the spectral properties of luminescence of laurdan molecules in glycerin upon excitation at the red edge of the absorption band at different temperatures. The most significant red-wave shift of the spectra (10 nm) for the longwave band of dual fluorescence is observed depending on the excitation wavelength at a low temperature of 260 K when a solvent forms a fairly rigid matrix. At the same time, at increased temperatures of up to 370 K a small bathochromic shift and a change in the shape of the luminescence bands are also recorded reliably. Changes in the excitation spectra were observed when luminescence was recorded in the bands of the LE- and CT states. The difference spectrum responsible for the additional absorption that does not make a contribution to the longwave luminescence component has been isolated. The decay kinetics of both luminescence components have been measured and their expansions in decay constants have been analyzed. The experimental dependences obtained point to the complex mechanism of inhomogeneous broadening of spectra.     

Femtosecond coherence in poly(p-Phenylene- vinylene) – polarization beatings and phase-relaxation probed by wavepacket fluorescence interferometry

Coherent, optical excitation transients have been measured in the bottom-states of the dynamically disordered multi-chromophore poly(p-phenylenevinylene) at low phonon temperatures. Interferometric femtosecond (fs) excitation/probing experiments using freely propagating 70 fs pulses have been employed to generate spatially overlapping electronic and nuclear wavepackets. Narrow-bandwidth detection tuned to two vibronic 01 S₁S₀ fluorescence transitions with discrete arrival-states , , respectively, projects-out two distinct site-subensembles from the pulsed target-area of site excitations. By monitoring the interferences of the fluorescence correlations for - and -photons on the detector, the coherent superposition can be probed as a typical polarization beating. The fluorescence interferograms show strong damping on an overall time-scale of 200 fs with typically, half-to-single cycle beating waves. The relaxation of coherence is indicative of a predominantly homogeneous site-dephasing and hence, gives rise to the observation of the structural equilibration of the initial Franck–Condon (FC)-excitation, i.e. the formation of the dressed state. PACS 78.47.+p; 42.50.Md; 42.25.Kb     

Spectral‐Luminescence Characteristics of Polyphenyls and Polyacenes in the Gas Phase in Electron‐Beam Excitation

The spectra of electronenergy loss, excitation functions, and fluorescence spectra in excitation of the vapor of polyphenyls and polyacenes by electron beams of different energies are determined. The influence of successive complication of the molecules under study on these spectralluminescence characteristics is tracked. Unlike the optical absorption spectra, in the spectra of electronenergy loss of all the substances studied one observes a band which is related to the singlettriplet transition S ₀–T ₁. The transitions up to S ₀–S ₅ are recorded in excitation of the molecules by highenergy electrons, including the region of vacuum ultraviolet. From the functions of fluorescence excitation the authors have determined the excitation thresholds that correlate with the energies of the S ₁ levels, except for pyrene in which the S ₀–S ₁ transition is forbidden and does not show up not only in photon excitation but also in electronbeam excitation, although the intercombination forbiddenness in the latter case is removed and the S ₀–T ₁ band is observed.     

Free induction decay with radiation damping I. General treatment

The problem of the free induction decay of a two-level spin system in the presence of a strong coupling between a resonant circuit and the system is discussed under different experimental conditions. It is shown that under certain conditions the shapes of these signals are profoundly modifled by this coupling. In some cases one obtains, after a pulse excitation of the system, several emission peaks of energy from the spin system having a population difference below inversion, creating some sort of photon-bottleneck or of coherent echo.     

Tyrosyl Fluorescence Decays and Rotational Dynamics in Tyrosine Monomers and in Dipeptides

Picosecond time-correlated single-photon counting was used to measure fluorescence lifetimes and fluorescence anisotropy decays of tyrosine and the tyrosine–alanine and tyrosine–leucine dipeptides. After excitation of tyrosine at 287 nm two emitting species were observed, one at 303 nm with a lifetime of 3.3 ns and another at 340 nm with a lifetime of 360 ps. The rotational correlation time of tyrosine at 303 nm is 38 ps in water at pH 7 and depends linearly on viscosity with a slope of 44 ps/cP, consistent with Stokes–Einstein–Debye theory. We calculated a value of 45 ns for the radiative lifetime of tyrosine, yielding a fluorescence quantum yield of 0.07. The dipeptides Tyr–Ala and Tyr–Leu exhibit two- or three-exponential decays. The amplitudes of the decay components for three-exponential fits correlate closely with the populations of rotamers in these peptides as determined by NMR. The quenching of dipeptide fluorescence is shown to depend on the solvent polarity, strongly supporting the hypothesis that tyrosyl fluorescence in peptides is quenched by charge transfer. The rotational correlation times of tyrosine, Tyr–Ala, and Tyr–Leu increase linearly with the van der Waals volumes. However, rotational relaxation is somewhat faster than expected from Stokes–Einstein–Debye theory with stick boundary conditions.     

Kinetics of the Decay of Prolonged Luminescence of Disordered Chrysene under the Conditions of Nonequilibrium Transfer of Energy

We present the results obtained in investigation of the decay of the prolonged luminescence of disordered chrysene in a microsecond range. At the temperature of liquid nitrogen, a highly nonexponential decay of both phosphorescence and annihilation of delayed fluorescence is observed. The observed character of decay is due to relaxation of the energy of electronic excitation in a system of energydisordered centers, when the value of kT is much smaller than the width of the excitedstate energy distribution. At the same time, in the system investigated there is a classical relationship between phosphorescence and delayed fluorescence, i.e., the rate coefficient for the reaction of triplettriplet annihilation is a constant.     

Coherent motion of an exciton in a semi-infinite chain

An explicit analytic expression for the propagator of a coherent motion of an exciton in a semi-infinite linear chain is derived. The surface relaxation is studied and the probability of an exciton being captured at the surface is calculated. The possibility of using the exponential decay approximation and the generalization to the three-dimensional crystal are also discussed.     

Solvent Exchange in Excited-State Relaxation in Mixed Solvents

The fluorescence of styrylthiazoloquinoxaline (STQ) in the solvent mixture methanol and dichloromethane (DCM) and 2-octanol have many common characteristics: biexponential fluorescence decay, wavelength-dependent amplitudes, a negative amplitude for the short-lifetime component at long emission wavelengths, and a time-dependent red shift of the emission spectrum. In octanol, the fluorescence lifetime decreases with increasing temperature, whereas the lifetime increases with temperature in the methanol/DCM mixture. The fluorescence characteristics in 2-octanol ( = 7.29 cP) are readily explained by the conventional model of excited-state relaxation kinetics by solvent reorientation. This model is not applicable for low-viscosity ( = 0.455 cP) solvent mixtures. A model of excited-state relaxation kinetics involving solvent exchange (versus solvent reorientation in pure solvents) in the excited state is proposed for the solvent mixture. The model assumes that the solvent compositions around the solute are different in the ground and excited states and the solvent composition is temperature dependent.     

Solvent and Temperature Effects on Polymer-Coated Glass Fibers. Fluorescence of the Dansyl Moiety

E-type glass fibers were coated with poly(-aminopropyltriethoxysilane), treating them with a 1% (v/v) monomer aqueous solution. The fibers were labeled with a dansyl-sulfonamide conjugate by reaction of acetonitrile solutions of dansyl chloride with the amine groups immobilized on the glass fiber surface. Interactions of the labeled coating polymer with solvents of different polarities were estimated by measurements of the fluorescence band shifts of the label. It was found that for aprotic solvents, the solvent dipolar coupling relaxation mechanism is dominated by thermodynamic interactions of the solvent with the polymer matrix, while for protic solvents this mechanism is dominated by specific interactions between solvent molecules and the excited state of the chromophore. Different experimental excited-state dipole moments were obtained for nonpolar and polar solvents (^* _NP = 7.2 ± 1.6 D, ^* _P = 11.9 ± 1.5 D). Using the AM1 method, excited-state dipole moments for the first and second singlets were calculated and it was concluded that ^* _NP ^*2 ₁^1/2 and ^* _P ^*2 ₂^1/2. Accordingly, neither the glass support nor the coating polar influence the excited-state properties of dansyl. The temperature dependence of dansyl emission allows the determination of the relaxation temperature of the coating polymer, which was estimated as 175 K for the coating used.     

Characterization and performance of the femtosecond fluorescence up-conversion microscope

The characterization and performance of the femtosecond fluorescence up-conversion microscope is reported in this paper. This new fluorescence microscope is a combination of the frequency up-conversion technique and a confocal optical configuration, which simultaneously achieves femtosecond time and nanometer space resolution. The femtosecond time resolution was evaluated by measuring the rise up of time-resolved fluorescence from a dye molecule, and it was 520 fs and 460 fs with 100× (N.A.=1.3) and 40× (N.A.=0.75) objective lenses, respectively. The best transverse (XY) resolution was 0.34 m with the 100× objective lens for 400 nm excitation. An axial (Z) resolution as high as 1.1 m was obtained for 600 nm fluorescence detection with a 50 m pinhole and a 100× objective lens. The axial resolution was remarkably improved compared with ordinary confocal microscopes owing to the up-conversion process, which requires spatial overlap between the tightly focused gate and the fluorescence beams. Femtosecond time-resolved fluorescence measurements were performed for micro-meter sized particles in liquids, fluorescent beads and C519/toluene micro droplets, by using the laser trapping technique. The high potential of the fluorescence up-conversion microscope was demonstrated. PACS 78.47.+p; 87.64.-t; 82.53.-k     

The Photophysics of β-Tyrosine and Its Simple Derivatives

Synthesis and photophysical studies of (O-methyl)--tyrosine (-tyrosine; an analogue of tyrosine, in which the amino group is moved from the - to the -carbon, closer to the phenol ring) and its derivatives with a blocked amino and/or carboxyl group were performed to explain the nature of the fluorescence of tyrosine derived analogues. All -tyrosine derivatives, except Ac-Tyr(Me), displayed the monoexponential fluorescence decay. The biexponential fluorescence decay observed for Ac-Tyr(Me) is assumed to be the result of the presence of two low-energy conformations (extended and with an intramolecular hydrogen bond). Higher quenching of the fluorescence of -tyrosine derivatives by the N-acetyl group than by the N-methylamide group moved farther was found, contrary to the data found for the respective derivatives of natural tyrosine. The obtained photophysical data are discussed with theoretical calculations (AMBER, AM1) on the basis of the rotamer model.     

Calibration methods and avoidance of errors in measurement of intracellular pH (pHcyt) using the indicator bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in human platelets

Determinations of pH_cyt in suspensions of human platelets using BCECF [bis(carboxyethyl)-5(6)-carboxyfluorescein] can be seriously biased by leakage of the fluorescent indicator. Two methods (pH jump and Mn²⁺) are presented for determining the fraction of external indicator (B _ext) and eliminating this error. Both methods rely on rapid perturbations (pH jump or Mn²⁺ addition), which affect the fluorescence of the external dye immediately and the intracellular dye more slowly. Identical values ofB _ext are reported. Failure to correct for dye leakage can result in overestimation of pH_cyt by as much as 0.4 unit at physiological external pH (pH_ext). Two methods of calibration of the cytoplasmic signal were compared after correcting forB _ext: the digitonin lysis method and the nigericin calibration method. In the digitonin method the dye is released at the end of the experiment and the dependence of its fluorescence is determined as a function of pH. The method assumes that the fluorescence and titration characteristics of the dye in the cytoplasm are not different from those in solution. It gives pH_cyt=6.75±0.07 for pH_ext=7.3. In the nigericin method, 150 mM external K⁺ and 10 M nigericin are used for the purpose of setting pH_cyt=pH_ext to accomplish anin situ calibration. The method was complicated by extra leakage induced by nigericin. Assuming that the ionophore could equilibrate pH in the alkaline range, the fluorescence of the anionic form of BCECF in the cytoplasm would be 15% lower than in solution and pH_cyt would be 0.3 unit higher than presented above. A number of observations favor the digitonin lysis method of calibration. The fluorescence polarization of BCECF in platelets is small and indistinguishable from that in solution (0.000±0.022). The spectrofluorimetric characteristics of the intracellular dye are identical to those in solution (150 mM NaCl or KCl). There was no evidence for self-quenching or binding to cellular elements for cytoplasmic BCECF concentrations up to 1.8 mM. The following agents are capable of introducing error: (1) the Na⁺ substituteN-methyl-d-glucamine doubles theK _d and decreases by 13% the F _max of BCECF; (2) the Na⁺/H⁺ exchange inhibitor amiloride quenches BCECF fluorescence and is intrinsically fluorescent; and (3) bovine serum albumin (used to remove nigericin) quenches external BCECF with kinetics mimicking acidification of the cytoplasm.     

Instability in degenerate two-photon running wave laser

The stability of the homogeneously broadened and degenerate two-photon running wave laser is analysed by using the full set of matter-field equations. The stability depends on the relative size of the relaxation constants. For 2k>1+r(k=/,r=/; is the cavity loss of the field and , are the longitudinal and transversal decay constants, respectively) no stable lasing state exists. Forr<k<(1+r)/2 an instability occurs. With the decrease in pumping the stable lasing state loses its stability due to Hopf-bifurcation.     

The Influence of Solvent Viscosity on the Fluorescence Decay and Time-Resolved Anisotropy of Green Fluorescent Protein

This report describes fluorescence decay and time-resolved anisotropy studies of green fluorescent protein (GFP) in various environments. The addition of glucose and fructose, NaCl, or polyethylene glycol changes the viscosity of the medium surrounding the GFP. Both the time-resolved anisotropy and the fluorescence decay of GFP are measured and it is shown that only the time-resolved anisotropy of GFP is affected by the viscosity, but not its fluorescence decay.     

Wavelength-dependent rotation of dye molecules in a polar solution

Investigation of rotation movement of 3-amino-N-methylphthalimide in glycerol was carried out, taking into consideration the fluctuation of solvate structure. It was shown theoretically and experimentally that structural relaxation of the solvate shell, which follows excitation of the dye molecule, causes not only shift of the fluorescence spectrum in time but also additional rotation of the dye molecule. This effect, which may be called wavelength-dependent rotation, depends on the light frequency of both excitation and fluorescence. In particular, at excitation near the maximum of the absorption band, when the relaxation process is followed with the red shift of the fluorescence maximum, the anisotropy of fluorescence decreases faster in the red part of the fluorescence band than in the blue part. On the contrary, in the case of far anti-Stokes excitation, when the temporal shift of fluorescence is going to the blue, the anisotropy in the red part of the spectrum drops more slowly than in the blue part. Finally, there is a special excitation frequency which causes neither change of the fluorescence maximum nor acceleration of the rotational movement of the dye molecule. It is also shown that the temporal evolution of the spectrum and anisotropy of fluorescence in a polar dye solution may be quantitatively described using the socalled inhomogeneous broadening function (IBF). This function gives the distribution of dye molecules in a solution over frequencies of pure electronic transition due to fluctuations of the surrounding shell structure. Measurements of IBF changes in time carried out for 3-amino-N-methylphthalimide showed that during first 3 ns after excitation, the half-width of the IBF grows, and at the same time its maximum quickly shifts to the red. At the later time period there are only small changes of IBF position but considerable exponential decrease in its half-width. The IBF during this period preserves the Gaussian shape.     

Investigation of aggregation of porphyrin derivative Fe(TPP)Cl at low temperature by time-resolved fluorescence spectrum

The time-resolved fluorescence spectrum I(, t) of the porphyrin derivative Fe(TPP)Cl in solution is measured. Experiments show that when the temperature is at least 5 K above the freezing point of the solvent, the sample fluorescence does not change with temperature. But when the temperature approaches the freezing point of the solvent, the fluorescence relaxation of the high concentration sample solution is faster than that of the above normal one. Furthermore, fluorescence spectra for various times after excitation have different shapes. Analysis shows that in this case the fluorescence is composed of two components with different relaxations and spectra. The two components are attributed to monomer and aggregates respectively.