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     

Coherence gating in optical heterodyne detection measurements of scattering and absorption in highly scattering media

Laser-Induced Fluorescence (LIF) from the S₁ state of acetone and 3-pentanone was studied as a function of temperature and pressure using excitation at 248 nm. Additionally, LIF of 3-pentanone was investigated using 277 and 312 nm excitation. Added gases were synthetic air, O₂, and N₂ respectively, in the range 0–50 bar. At 383 K and for excitation at 248 nm, all the chosen collision partners gave an initial enhancement in fluorescence intensity with added gas pressure. Thereafter, the signal intensity remained constant for N₂ but decreased markedly for O₂. For synthetic air, only a small decrease occurred beyond 25 bar. At longer excitation wavelengths (277 and 312 nm), the corresponding initial rise in signal with synthetic air pressure was less than that for 248 nm. The temperature dependence of the fluorescence intensity was determined in the range 383–640 K at a constant pressure of 1 bar synthetic air. For 248 nm excitation, a marked fall in the fluorescence signal was observed, whereas for 277 nm excitation the corresponding decrease was only half as strong. By contrast, exciting 3-pentanone at 312 nm, the signal intensity increased markedly in the same temperature range. These results are consistent with the observation of a red shift of the absorption spectra (9 nm) over this temperature range. Essentially, the same temperature dependence was obtained at 10 and 20 bar pressure of synthetic air. It is demonstrated that temperatures can be determined from the relative fluorescence intensities following excitation of 3-pentanone at 248 and 312 nm, respectively. This new approach could be of interest as a non-intrusive thermometry method, e.g., for the compression phase in combustion engines.     

Single fluorescence centers on the tips of crystal needles: First observation and prospects for application in scanning one-atom fluorescence microscopy

A scanning optical fluorescence microscope is suggested, whose active element (needle) is made of a crystal containing impurity ions or color centers subject to excitation by laser radiation. The excitation energy from a single impurity center at the very tip of the needle is transferred by the resonance dipole-dipole exchange mechanism to fluorescence centers on the surface of the sample under study. It is demonstrated that this approach can help attain a nanometer-high spatial resolution at a high sensitivity substantially exceeding in some cases the sensitivity of the standard near-field fluorescence microscopy technique. Various crystals and impurity centers, potentially most suitable for the implementation of the method under consideration, are briefly analyzed. Information is presented on the manufacture of sharp-pointed needles from LiF crystals containing F₂-centers and the first observation of single F₂-centers on their tips by the laser-photoelectron projection microscopy technique, which allows one to speak of the practical creation of the first active elements for the microscope suggested.Dedicated to Prof. F. P. Schäfer on the occasion of his 65th birthday.     

Dynamics of Trp Residues in Crystals of Human α1-Acid Glycoprotein (Orosomucoid) Followed by Red-Edge Excitation Spectra

The present work reports for the first time the development of a method that allowed us to obtain crystals of orosomucoid complexed to progesterone. Then we investigated the dynamics of the microenvironments of the two buried Trp residues in the crystals of protein, by the red-edge excitation spectra method. The fluorescence excitation spectrum of the crystals is characteristic of that known for Trp residues (_max = 290 nm and bandwidth = 38 ± 1 nm), indicating that the Trp residues are responsible for the fluorescence of the protein in the crystals. The position of the maximum and the bandwidth of the steady-state emission spectrum of the crystals (331 ± 1 and 43 ± 1 nm, respectively) are equal to those obtained in aqueous buffer for the orosomucoid–progesterone complex (330 ± 1 and 43 ± 1 nm) (_ex, 295 nm). Thus, the fluorescence of the crystals occurs from the Trp residues buried in the protein core. The red-edge excitation spectra studies indicate that the Trp residues are surrounded by microenvironments that display motions, a result identical to that observed in solution. Thus, the crystallization process does not modify the structure or the dynamics of orosomucoid core. The fluorescence intensities depend on the angular orientation of the crystals with respect to the polarization of the incident beam. The general feature of this dependence is identical at the three excitation wavelengths used (295, 300, and 305 nm). Our results confirm the fact that the local structure and dynamics are the key for any interpretation of tryptophan fluorescence parameters of orosomucoid.     

Nanometer-scale probing of optical and thermal near-fields with an apertureless NSOM

We have used a home-made apertureless near-field scanning optical microscope (ANSOM) for mapping nanometric steps between SiC and gold regions under visible (λ=655 nm) and infrared (λ=10.6 μm) illumination. The images, obtained with a signal demodulation at the tip oscillation frequency and at higher harmonics, clearly show optical contrasts with a subwavelength resolution of about 30 nm. Other images recorded in the visible on a YBa₂Cu₃O₇ crystal indicate that the tip used in our experiments is able to reveal polarization effects. We also present a near-field thermal optical microscope (NTOM) which operates without any external illumination. In this new kind of microscope, the laser source which is usually used to excite the evanescent waves, is replaced by a simple heating of the sample. The electromagnetic radiation locally scattered by the tip comes from the thermal radiation. Our results with this new technique prove a 200 nm lateral resolution.     

Identification and imaging of hot O2 (v″=2, 3, or 4) in hydrogen flames using 193 nm- and 210 nm-range light

Some simple molecules can be excited by light within the tuning ranges of ArF (193–194 nm) and KrF (247.8–248.8 nm) excimer lasers and their fluorescence has been previously used for imaging. Additional wavelength ranges should become available by Raman shifting. As a demonstration, we present excitation and fluorescence spectra from hot O₂ obtained (a) with tunable 193 nm-range light and (b) with that light shifted into the 210 nm range. All measurements are via predissociative upper states. In the 193 nm range, results are compared with those of Andresen's group. In the 210 nm range, the light is tuned to various excitation wavelengths in the (5 3), (8 4), and (7 4) bands of the B–X transition. The (7 4) excitations are well separated and the (7 v) fluoresence spectrum is in accord with Franck-Condon calculations. The wavelengths tend to overlap for the (5 3) and (8 4) excitations and the fluorescence is weaker. State-specific two-dimensional fluorescence images of an H₂-O₂ flame are obtained in both wavelength ranges to illustrate the use of the method.We acknowledge the National Science Foundation and the Donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this research     

The Fluorosolvatochromism of Brooker's Merocyanine in Pure and in Mixed Solvents

The fluorescence-based solvatochromism (fluorosolvatochromism) of 4-[(1-methyl-4(1H)-pyridinylidene)-ethylidene]-2,5-cyclohexadien-1-one (Brooker's merocyanine) was studied. The results revealed that the fluorescence emission band of the dye was dependent on the medium ( nm in water and nm in DMF). The fluorescence quantum yields (φ _f) were calculated for the dye in the solvents investigated. Low φ _f values ( < 10%) were obtained for the dye and in order to better comprehend the radiative and nonradiative decay processes of this dye, its fluorescence lifetime in methanol was measured and was found to be very short (230 ps). The results suggest that the dye in the excited state decays rapidly through nonradiative processes. The behavior of the probe in binary mixtures including a hydrogen-bond accepting solvent (acetonitrile, N,N-dimethylformamide, and dimethylsulfoxide) and a hydroxylic solvent (water, methanol, ethanol, propan-2-ol, and butan-1-ol) was also investigated. All data were successfully fitted to a model based on solvent exchange equilibria, which allowed the separation of the different contributions of the solvent species in the solvation shell of the dye. The data obtained for the mixed solvents were explained based on solute–solvent and solvent–solvent interactions.     

Internal reflection mode scanning near-field optical microscopy with the tetrahedral tip on metallic samples

An internal reflection mode is introduced for scanning near-field optical microscopy (SNOM) with the tetrahedral tip. A beam of light is coupled into the tip and the light specularly reflected out of the tip is detected as a photosignal for SNOM. An auxiliary STM mode is used to control the distance during the scanning process and to record the topography of the sample simultaneously with the SNOM image. Images were obtained of different metallic samples which show a contrast in the order of 10% of the total reflected photosignal. In images of metallic samples an inverted contrast is consistently obtained compared to images previously obtained of comparable samples in a transmission mode. The contrast shows a pronounced dependence on the polarization of the incident beam with respect to the orientation of the edges of the tip. In the case of gold surfaces, the photosignal as a function of distance between the tip and the surface shows a pronounced peak in the near-field range of 0–20 nm which is tentatively attributed to the excitation of surface plasmons on the gold surface. The pronounced near-field effects and the strong contrast in the near-field images and the resolution well below 50 nm are an indication of a highly efficient coupling of the incident beam to a local excitation of the tip apex which is essential for the function of the tip as a probe for SNOM. Received: 17 May 1999 / Accepted: 18 May 1999 / Published online: 21 October 1999     

Fluorescence of the Schiff bases of pyridoxal and pyridoxal 5′-phosphate withl-isoleucine in aqueous solutions

The present study reports on the absorption and emission properties of the Schiff bases formed by pyridoxal and pyridoxal 5-phosphate withl-isoleucine in aqueous solutions. Species protonated at the imine and ring nitrogen are the most fluorescent in both Schiff bases with a quantum yield of 0.02, i.e., 20-fold the value found for species in alkaline solutions. In agreement with other studies, species protonated at the imine nitrogen shows an emission around 500 nm upon excitation at 415 nm. In contrast to previous observations on other PLP Schiff bases, emissions at 560 nm (PL-Ile) and 540 nm (PLP-Ile) are observed upon excitation at 365 and 415 nm, respectively. The emission at 470 nm found in PLP-Ile Schiff base upon excitation at 355 nm is ascribed to a multipolar monoprotonated species. An estimation for the pK _a of the imine in the excited state ( 8.5) for both Schiff bases is also reached. Our results suggest that fast protonation reactions on the excited state are responsible for the observed fluorescence. These effects, in which the hydrogen bond and the phosphate group seem to play a role, could be extended to understanding coenzyme environments in proteins.     

Fluorescence and Rotational Dynamics of Dityrosine

We have examined the lifetimes and rotational correlation times of dityrosine emission by time-correlated single-photon counting. We first noticed dityrosine fluorescence in samples of tyrosine and tyrosine dipeptides by its characteristic red-shifted emission at 400 to 430 nm. The longer rotational correlation time relative to tyrosine proved that this fluorescence emanated from a distinct species. Comparison with the fluorescence properties of synthesized dityrosine established the identity of the emitting species. Fluorescence intensity decays of dityrosine are generally characterized by two decay components, one with a lifetime in the range of 150 to 800 ps and another between 2.5 and 4.5 ns. We found no evidence for an excited-state reaction, since a rising phase (negative-amplitude component) was not observed. In the pH range from 4 to 10, two ground-state species exist in equilibrium with pK _a 7. Both species exhibit two fluorescence decays. The average fluorescence lifetime increases gradually with pH over the pH range from 4 to 10 and decreases at pH 2. Anisotropy decays were measured for dityrosine and the alanine–dityrosine–alanine and leucine–dityrosine–leucine dipeptides. The rotational correlation times of dityrosine and dityrosine dipeptides increase linearly with van der Waals volumes. The slope indicates a stronger solute–solvent interaction than predicted with stick boundary conditions. It is suggested that these interactions result from the presence of two zwitterionic pairs.     

Spectral-Kinetic Characteristics of Pyrene Fluorescence in Model Systems and Membranes of Etioplasts

The decay kinetics parameters of monomer and excimer fluorescence of pyrene in artificial galactolipid protein-free micelles and membranes of prolamellar bodies (PLB) of etioplasts have been determined. A complex law of probe fluorescence decay in artificial and native membranes has been discovered. It has been found that the addition of protochlorophyllide (Pd) to lipid micelles led to a considerable reduction in the lifetime of the long-lived component of the fluorescence-decay kinetics of the monomer form of pyrene (₂) and to the appearance of luminescence in the region of 640 nm in the stationary spectra of fluorescence at excitation of the pyrene molecules present in the bilayer. In native membranes of etioplasts, the monomer duration ₂ did not depend on the pigment content and the Pd fluorescence upon excitation through pyrene was absent. The membranes of etioplasts and the pigment-containing artificial micelles did not differ in the ₂ value of the excimer. They only differed in the contribution of the long-lived component to the total fluorescence (low in native membranes). The pigment-protein interaction in the etioplast membranes and the absence from them of pigment directly associated with lipids are discussed.     

Dual fluorescence and laser emissions from fluorescein-Na and eosin-B

Dual laser emissions were observed from fluorescein-Na and eosin-B in ethanolic solutions individually in the concentration range from 10⁻² to 10⁻³ mol dm⁻³ under N₂ laser excitation. The first compound was found to lase at two distinct regions with wavelength maxima around 540, 550 nm, while the second one around 558, 574 nm. Steady-state absorption, fluorescence excitation, fluorescence polarization, fluorescence emission and decays of the dyes in various solvents under varying conditions of excitation and detection systems were carried out to identify the nature of the emitting species responsible for laser emissions in two distinct regions. Both the dyes exhibited concentration and excitation wavelength dependence of fluorescence and the effects were found to be more pronounced in binary solution. The fluorescence decays of dyes were monoexponential in ethanol, while in some other solvents used, the decays showed biexponential behavior. The absorption and excitation studies using thin layers of solutions revealed the formation of dimers with the dye concentration around 1×10⁻³ mol dm⁻³. Fluorescence polarization and decay studies confirmed the presence of dimers. The two laser bands observed in the shorter and longer wavelengths were respectively ascribed to monomeric and dimeric species.     

Steady-State and Time-Resolved Spectroscopic Characteristics of Novel Silicon-Containing Organopolymers

The spectroscopic characteristics of novel -conjugated polymers containing four-coordinated silicon, acetylene groups and either 1,4-biphenylene or 2,7-fluorene in the main chain were investigated by steady-state and picosecond laser spectroscopy. The spectral features of absorption, fluorescence excitation spectra, fluorescence lifetime, and fluorescence polarization were explained by the existence of two kinds of inhomogeneously broadened electronic states formed in the disordered polymeric chain. The dynamics of photoinduced absorption was measured in the 400–900 nm spectral range with picosecond time resolution. The long-wavelength band with _max 710 nm was ascribed to excited-state absorption from higher-lying electronic states created in short polymeric segments with essential conformational distortion of the subunits. The short-wavelength band with _max 580 nm and a shoulder at 500 nm was interpreted as photoinduced absorption from a lower-lying state arisen in more planar, longer -conjugated segments populated via direct excitation and energy migration between disordered segments of the polymeric chain. For the fluorene-containing polymer, the smaller Stokes shift and the greater degree of fluorescence polarization are consistent with more extensive electron delocalization along the backbone.     

Fluorescence and attenuation properties of Er3+ -doped phosphate-glass fibers and efficient infrared-to-visible up-conversion

Fluorescence spectra of phosphate glasses with different erbium doping have been measured. The flourescence intensity reaches its maximum at an Er³⁺-doping concentration of 0.75 mol%. When the Er³⁺ doping exceeds 0.75 mol%, the fluorescence intensity decreases due to concentration quenching. The attenuation at 1.53 µm of the fiber is 12.8 db/m. The fluorescence up-conversion of 1.064 µm Nd:YAG laser pulses into intense green 547 and 667 nm light in the fiber has been measured. The fluorescence output power of green (547 nm) and red (667 nm) light is 178 and 42 W, respectively with an excitation power of 1 W. The two signals are referred to as⁴ S _3/2 ⁴ I _{1 5/2} and⁴ F _9/2 ⁴ I _{1 5/2} transitions through two-photon absorption fluorescence.     

Nature of 2-Aminoxanthone Fluorescence

Absorption and fluorescence spectra of 2-aminoxanthone in solutions of different types at 77–350 K were studied. The existence of three bands (_{1 max} = 417 nm, ₁ = 14 ns; _{2 max} = 528 nm, ₂ = 13 ns; and _{3 max} = 565 nm, ₃ = 6 ns) in fluorescence of 2-aminoxanthone solutions has been established. It was shown that the first short-wave band was determined by deactivation of singlet excitation of an aminoxanthone molecule. The band with _{3 max} = 565 nm (depending on the concentration) is connected with excimer-type aggregates, which are formed by aminoxanthone molecules grouped with the help of dipole molecules of solvent or by weak hydrogen bonds between aminoxanthone molecules. The emission in band _{2 max} = 528 nm is caused by reversible changes in the 2-aminoxanthone molecule and probably is connected with an intramolecular proton transfer.     

Effect of Deuteration on Quasiline Electronic‐Vibrational Spectra of Phthalocyanine

Quasiline electronicvibrational spectra of fluorescence and absorption (excitation of fluorescence in selective recording) of the molecules of phthalocyanine deuterated around the periphery of benzene rings (H₂Phcd ₁₆) and the center of the macrocycle (D₂Phc) are obtained. The vibrational frequencies of the ground state are almost insensitive to this deuteration (except for vibrations with the participation of angular deformations). In excitation spectra, changes in deuteration are more pronounced due to the effects of nonadiabatic vibronic interaction of the vibrational sublevels of the S ₁ state and of the purely electronic level S ₂.     

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.     

Fluorescence of the C60-pyridine system in electrochemistry reduction processes

Three groups of strong fluorescence peaks of C₆₀, centered at 440, 575 and 700 nm, are observed in the supersaturated C₆₀-pyridine solution, and the onsite fluorescence spectra of this C₆₀-pyridine system in the first and second electrochemistry reduction processes are obtained for the first time. The results indicate that the fluorescence peaks centered at 440 nm originate from C₆₀ molecules dissolved in supersaturated C₆₀-pyridine solution, while fluorescence bands centered at 575 and 700 nm are emitted from undissolved C₆₀. It is also proved that a strong charge transfer interaction, which is significant for the symmetry increase of , as well as the formation of a transmission channel of energy, occurs between and pyridine, as is supported by surface-enhanced Raman scattering (SERS) spectra as well.     

Strong Surface Enhanced Florescence of Carbon Dot Labeled Bacteria Cells Observed with High Contrast on Gold Film

Strong surface (metal) enhanced fluorescence (SEF or MEF) is observed from clusters and single E coli bacteria cells labeled with Carbon nanodots (CDs), which were synthesized from date pits. The enhancement factor (EF) for SEF of the cell clusters were close to 50 for both 533 and 633 nm laser excitation wavelength. Those EFs are ratios of emission peak areas from CD labeled cell clusters on gold film to the peak areas of the same batch cell clusters on glass substrate. SEF with 633 nm excitation performed better than SEF with 532 nm excitation, achieving higher fluorescence intensity and much higher contrast. The contrast as high as 66 for cell clusters on gold film is a ratio of fluorescent emission peak area measured at the CD labeled cell clusters to the fluorescent peak area measured at unlabeled cell clusters (autofluorescence) on the same substrate. The contrast with the background (S/N) or the ratio of fluorescent peak area measured at bacteria cells to area measured at bare substrate was as high as 200. This report may pave a way for the broader application of surface enhanced fluorescence and especially metal enhanced fluorescence imaging of CD labeled cells and other biological objects.

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Graphical abstract Carbon dots, synthesized from dates, are used for direct staining of E coli cells. Emission fluorescent spectroscopy of those CD labelled cells on gold film and glass, demonstrated enhancement factor about 50 for emission on gold as compared to glass, Excitation at 633 nm appears far superior to excitation at 532 nm in terms of contrast (up to 67) with unlabeled cells /control due to decrease in auto fluorescence of cells. Maximum Signal to noise ratio is 200.

     

Fluorescence and picosecond induced absorption from the lowest singlet excited states of quercetin in solutions and polymer films

The spectroscopic and photophysical properties of the biologically important plant antioxidant quercetin in organic solvents, polymer films of polyvinyl alcohol, and a buffer solution at pH 7.0 are studied by stationary luminescence and femtosecond laser spectroscopy at room temperature and 77 K. The large magnitude of the dipole moment of the quercetin molecule in the excited Franck–Condon state μ _e ^FC = 52.8 C m indicates the dipolar nature of quercetin in this excited state. The transient induced absorption spectra S ₁→S _n in all solvents are characterized by a short-wave band at λ _abs ^max = 460 nm with exponential decay times in the range of 10.0–20.0 ps. In the entire spectral range at times of >100 ps, no residual induced absorption was observed that could be attributed to the triplet–triplet transitions Т ₁ → Т _k in quercetin. In polar solvents, two-band fluorescence was also recorded at room temperature, which is due to the luminescence of the initial enol form of quercetin (~415 nm) and its keto form with a transferred proton (550 nm). The short-wave band is absent in nonpolar 2-methyltetrahydrofuran (2-MTHF). The spectra of fluorescence and fluorescence excitation exhibit a low dependence on the wavelength of excitation and detection, which may be related to the solvation and conformational changes in the quercetin molecule. Decreasing the temperature of a glassy-like freezing quercetin solution in ethanol and 2-MTHF to 77 K leads to a strong increase in the intensity (by a factor of ~100) of both bands. The energy circuits for the proton transfer process are proposed depending on the polarity of the medium. The main channel for the exchange of electronic excitation energy in the quercetin molecule at room temperature is the internal conversion S ₁ ? S ₀, induced by the state with a proton transfer.