Development of a narrow-band tunable picosecond dye laser and its application to excited-state lifetime measurement of a chlorinated aromatic hydrocarbon.

The development of a distributed-feedback dye laser, with a pulse width and a line width of 25 ps and 8.78 pm, respectively, is described. Using this nearly Fourier-transform-limited pulse, we measured the first singlet-excited-state lifetime of 1,2,4-trichlorobenzene. The tunable picosecond dye laser developed herein has a potential for the lifetime measurement and the efficient multiphoton ionization of aromatic hydrocarbons with a larger number of chlorine atoms and shorter excited-state lifetimes.     

Enchanced fluoresence emission of croconate violet in ethanol containing poly(4-vinyl-pyridine)

When croconate violet 1. a dye with strong visible absorption and reversible redox behavior, is microeneaged by a polymer, its fluorescence quantum yield increases by a factor of 8; the fluorescence lifetime increases from <9 ps to 28 ps. The influence of the polymer [poly(4-vinyl-pyridine)] on the photochemical properties of the dye is discussed.     

The 1:1 host-guest complexation between cucurbit[7]uril and styryl dye

The photophysical properties of aqueous solution of styryl dye, 4-[(E)-2-(3,4-dimethoxyphenyl)ethenyl]-1-ethylpyridinium perchlorate (dye 1), in the presence of cucurbit[7]uril (CB[7]) was studied by means of fluorescence spectroscopy methods. The production of 1:1 host-guest complexes in the range of CB[7] concentrations up to 16 μM with K = 1.0 × 10(6) M(-1) has been observed, which corresponds to appearance of the isosbestic point at 396 nm in the absorption spectra and a 5-fold increase in fluorescence intensity. The decay of fluorescence was found to fit to double-exponential functions in all cases; the calculated average fluorescence lifetime increases from 145 to 352 ps upon the addition of CB[7]. Rotational relaxation times of dye 1 solutions 119 ± 14 ps without CB[7] and 277 ± 35 ps in the presence of CB[7] have been determined by anisotropy fluorescence method. The comparison of the results of quantum-chemical calculations and experimental data confirms that in the host cavity dye 1 rotates as a whole with CB[7].     

AN INSTRUMENT FOR SIMULTANEOUS ACQUISITION OF FLUORESCENCE SPECTRA AND FLUORESCENCE LIFETIMES FROM SINGLE CELLS

Abstract— An instrument is described that has the capability of acquiring both the spectrum and lifetime(s) of fluorescent species dispersed in biological cells. It operates at the single cell (or organelle) level and the spectral and temporal data collection can be performed simultaneously. A synchronously pumped, mode-locked dye laser provides the excitation light, time-correlated single-photon counting is used for lifetime measurements, and a diode array spectrograph is used for spectral work. Spatial resolution of sub-micrometer is obtained using a fluorescence microscope. The temporal resolution is better than 300 ps and wavelength resolution is less than 1 nm per channel. The instrument has been used for observing the spectral and temporal characteristics of hematoporphyrin in mouse myeloma cells.     

Picosecond multidimensional fluorescence spectroscopy: a tool to measure real-time protein dynamics during function

Advanced multidimensional time-correlated single photon counting (mdTCSPC) and picosecond time-resolved fluorescence in combination with site-directed fluorescence labeling are valuable tools to study the properties of membrane protein surface segments on the pico- to nanoseconds time scale. Time-resolved fluorescence anisotropy changes of protein bound fluorescent probes reveal changes in protein dynamics and steric restriction. In addition, the change in fluorescence lifetime and intensity of the covalently bound fluorescent dye is indicative of environmental changes at the protein surface. In this study, we have measured the changes in fluorescence lifetime traces of the fluorescent dye fluorescein covalently bound to the first cytoplasmic loop of bacteriorhodopsin (bR) after light activation of protein function. The fluorescence is excited by a picosecond laser pulse. The retinylidene chromophore of bR is light-activated by a 10 ns laser pulse, which in turn triggers recording of a sequence of fluorescence lifetime traces in the mdTCSPC-module. The fluorescence decay changes upon protein function occur predominantly in the 100 ps time range. The kinetics of these changes shows two transitions between three intermediate states in the second part of the bR photocycle. Correlation with photocycle kinetics allows for the determination of reaction intermediates at the proteins surface which are coupled to changes in the retinal binding pocket.     

Noncontact temperature measurement in microliter-sized volumes using fluorescent-labeled DNA oligomers

A new method of noncontact temperature measurement in microliter-sized volumes is demonstrated, based on the temperature sensitivity of the fluorescence lifetime of rhodamine-G when it is attached to a DNA oligomer. As temperature changes, the spacing between the fluorescent dye and a designed sequence of DNA bases is modulated by conformation changes of the DNA chain, and as a result the ability of dye molecules to fluoresce is also modulated according to differential quenching by bases on the DNA. In the system that we studied, the temperature sensitivity of the fluorescence lifetime was 36-42 ps/ degrees C depending on specific solution conditions. Although this strategy of temperature measurement is demonstrated using a specific sequence of DNA, it can also be generalized to a dye attached to any other intrinsic quencher of fluorescence whose conformation changes with temperature.     

FLUORESCENCE KINETICS OF SPINACH CHLOROPLASTS MEASURED WITH A PICOSECOND OPTICAL KERR GATE

Abstract. An overview of the reported chlorophyll a fluorescence lifetimes from green plant photosystems is presented and the problems encountered in the measurement of fluorescence lifetime using two currently available picosecond techniques are discussed.
The fluorescence intensity of spinach chloroplasts exposed to 10 ps flashes was measured as a function of time after the flash and wavelength of observation by the ultrafast Kerr shutter technique. Using a train of 100 pulses separated by 6ns and with an average photon flux per pulse of ˜2 times 10¹⁴ photons/cm², the fluorescence intensity at 685 nm (room temperature) was found to decay with two components, a fast one with a 56 ps lifetime, and a slow one with a 220 ps lifetime. The 730 nm fluorescence intensity at room temperature decays as a single exponential with a 100 ps lifetime. The 730 nm fluorescence lifetime was found to increase by a factor of 6 when the temperature was lowered from room temperature to 90 K while the lifetime of 685 and 695 nm fluorescence were unchanged. At room temperature, the fast and slow components at 685 nm are attributed to the emission from pigment system I (PS I) and PS II, respectively. It is likely that the absolute values of lifetimes, reported here, may increase if single ps low intensity flashes are used for these measurements.     

New phosphonate-substituted tricarbocyanines and their interaction with bovine serum albumin

New tricarbocyanine dyes with phosphonate groups (2, 3) were synthesized and their binding constants to bovine serum albumin (BSA) were determined. The binding constants of the synthesized tricarbocyanines and cardiogreen (1) (K_b ~ 10⁵ M^–1) are similar, indicating an insignificant contribution of the Coulomb interaction to the complex formation, which is determined by the polymethine chain interaction with BSA. The fluorescence lifetimes attest to the formation of two types of complexes: the lifetime of the dye complex with BSA with a major contribution (~80%) is 740–800 ps, and a lifetime of ~210 ps corresponds to the complex of dye aggregates with BSA.     

Near-infrared tetra-substituted aluminum 2, 3-naphthalocyanine dyes for optical fiber applications

The synthesis and spectral characterization of several tetra-substituted aluminum 2, 3-naphthalocyanine dyes for the determination of metal ions is reported. The synthesis is done by means of a homogeneous phase reaction, replacing the previously used heterogeneous method. The new scheme allows for improved product yields, higher purity, better product reproducibility and can be monitored at different stages using UV-Vis-near-infrared spectroscopy. The incorporation of electron-donating or -withdrawing groups was found to influence the product yield and to cause a shift in the absorbance maximum. The typical shift in the excitation maximum (of up to 27 nm) enables the dye to match the output of semiconductor laser diodes. In addition the tetra-substituted groups were capable of undergoing an ion-exchange process with the metal ions which produced a change in the fluorescence signal of the dye. Similar results were achieved using an optical fiber metal probe. The detection of metal ions using the near-infrared dyes was accomplished via steady-state fluorescence using both a commerically available instrument and a fiber optic system and also via the fluorescence lifetime technique.     

Femtosecond fluorescence dynamics and molecular interactions in a water-soluble nonlinear optical polymeric dye

The excited state dynamics of a water-soluble polymeric dye poly(S-119) was investigated using femtosecond time-resolved fluorescence upconversion. Multi-exponential relaxation of fluorescence was observed for poly(S-119) in picosecond and sub-picosecond time ranges. The azo-chromophore of the functionalized polymeric dye Sunset Yellow was used as a model compound for detailed investigations of intermolecular interactions. Excited state decay of this azo-dye can be described by a two-exponential decay law with time-constants of 0.48 ps and 1 ps. Fluorescence anisotropy decay was investigated for both systems. The difference in excited state dynamics between the polymeric dye and the azo-chromophore is explained in terms of inter-molecular interactions resulting in intra-chain aggregate formation.     

Absorption and emission spectroscopic characterization of some azo dyes and a diamino-maleonitrile dye

A linear and nonlinear optical spectroscopic characterization is carried out on three azo dyes (Reactive orange 1, Reactive violet 8, and Acidproof purplish red), and on N-(p-hydroxybenzylidene)-diamino-maleonitrile. Fluorescence quantum distributions, fluorescence quantum yields, and fluorescence lifetimes are measured. The saturable absorption is studied by nonlinear transmission measurements with intense picosecond laser pulses. The ground-state absorption recovery is studied by picosecond time-resolved pump and probe measurements. Absolute ground-state absorption cross-sections, excited-state absorption cross-sections, and dye concentrations are extracted from saturable absorption studies. The azo dyes have fluorescence lifetimes and ground-state absorption recovery times of around 2 ps and their excited-state absorption cross-sections are small (measured at 527 nm) making them good mode-locking dyes for picosecond and femtosecond lasers. The investigated diamino-maleonitrile dye exhibits sub-picosecond fluorescence lifetime and slow ground-state absorption recovery (>1 ns).     

Observation of the diffusion-free intermolecular excimer of 9-methyladenine in aqueous solution by picosecond time-resolved spectroscopy

Diffusion-free intermolecular excimer formation in liquid solution is observed for an aqueous solution of 9-methyladenine using the synchroscan streak camera technique. The excimer fluorescence (330 ps lifetime) appears at 380 nm, at longer wavelength than the monomer fluorescence (5 ps lifetime). The excimer is formed via a weakly coupled stacking dimer composed of ground-state monomers.     

Photophysical behavior of a dimeric cyanine dye (BOBO-1) within cationic liposomes

This study is aimed at establishing optimal conditions for the use of 2,2'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl-1(4H)-pyridinyl-4-ylidenemethy-lidyne]]bis[3-methyl]-tetraiodide (BOBO-1) as a fluorescent probe in the characterization of lipid/DNA complexes (lipoplexes). The fluorescence spectra, anisotropy, fluorescence lifetimes and fluorescence quantum yields of this dimeric cyanine dye in plasmid DNA (2694 base pairs) with and without cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]), are reported. The photophysical behavior of the dye in the absence of lipid was studied for several dye/DNA ratios using both supercoiled and relaxed plasmid. At dye/DNA ratios (d/b) below 0.01 the fluorescence intensity increases linearly, whereas lifetime and anisotropy values of the dye are constant (tau approximately 2.5 ns and = 0.20). By agarose gel electrophoresis it was verified that up to d/b = 0.01 DNA conformation is not considerably modified, whereas for d/b = 0.05-0.06 a single heavy band appears on the gel. For these and higher dye/DNA ratios the fluorescence intensity, anisotropy and average lifetime values decrease with an increase in BOBO-1 concentration. When cationic liposomes are added to the BOBO-1/DNA complex, an additional effect is noticed: The difference in the environment probed by BOBO-1 bound to DNA leads to a decrease in quantum yield and average lifetime values, and a redshift is apparent in the emission spectrum. For fluorescence measurements including energy transfer (FRET), a d/b ratio of 0.01 seems to be adequate because no considerable change on DNA conformation is detected, a considerable fluorescent signal is still measured after lipoplex formation, and energy migration is not efficient.     

Ultrafast photoinduced relaxation dynamics of the indoline dye D149 in organic solvents

The relaxation dynamics of the indoline dye D149, a well-known sensitizer for photoelectrochemical solar cells, have been extensively characterized in various organic solvents by combining results from ultrafast pump-supercontinuum probe (PSCP) spectroscopy, transient UV-pump VIS-probe spectroscopy, time-correlated single-photon counting (TCSPC) measurements as well as steady-state absorption and fluorescence. In the steady-state spectra, the position of the absorption maximum shows only a weak solvent dependence, whereas the fluorescence Stokes shift Δν?(F) correlates with solvent polarity. Photoexcitation at around 480 nm provides access to the S(1) state of D149 which exhibits solvation dynamics on characteristic timescales, as monitored by a red-shift of the stimulated emission and spectral development of the excited-state absorption in the transient PSCP spectra. In all cases, the spectral dynamics can be modeled by a global kinetic analysis using a time-dependent S(1) spectrum. The lifetime τ(1) of the S(1) state roughly correlates with polarity [acetonitrile (280 ps) < acetone (540 ps) < THF (720 ps) < chloroform (800 ps)], yet in alcohols it is much shorter [methanol (99 ps) < ethanol (178 ps) < acetonitrile (280 ps)], suggesting an appreciable influence of hydrogen bonding on the dynamics. A minor component with a characteristic time constant in the range 19-30 ps, readily observed in the PSCP spectra of D149 in acetonitrile and THF, is likely due to removal of vibrational excess energy from the S(1) state by collisions with solvent molecules. Additional weak fluorescence in the range 390-500 nm is observed upon excitation in the S(0)→S(2) band, which contains short-lived S(2)→S(0) emission of D149. Transient absorption signals after excitation at 377.5 nm yield an additional time constant in the subpicosecond range, representing the lifetime of the S(2) state. S(2) excitation also produces photoproducts.     

Confined ultrafast torsional dynamics of Thioflavin-T in a nanocavity

The influence of confinement in the supramolecular β-cyclodextrin nanocavity on the excited state torsional dynamics of the amyloid fibril sensor, Thioflavin-T, is explored using subpicosecond fluorescence up-conversion spectroscopy. In the presence of β-cyclodextrin, the emission intensity and the fluorescence lifetime of Thioflavin-T significantly increases, indicating the confinement effect of the nanocage on the photophysical behaviour of the dye. Detailed time-resolved fluorescence studies show an appreciable dynamic Stokes' shift for the dye in the β-cyclodextrin nanocavity. Analysis of the time-resolved area normalized emission spectra (TRANES) indicates the formation of an emissive TICT state. The rate of formation of the TICT state, as calculated from the time dependent changes in the peak frequency and the width of the emission spectra, is found to be substantially slower in the β-cyclodextrin nanocavity compared to that in bulk water. Present results indicate that ultrafast torsional motion in Thioflavin-T is significantly retarded due to confinement by the β-cyclodextrin nanocavity.     

PICOSECOND FLUORESCENCE STUDY OF PHOTOSYNTHETIC MUTANTS OF Chlamydomonas reinhardii: ORIGIN OF THE FLUORESCENCE DECAY KINETICS OF CHLOROPLASTS

Abstract— The fluorescence decay kinetics of photosynthetic mutants of Chlamydomonas reinhardii which lack photosystem II (PS II), photosystem I (PS I), and both PS II and PS I have been measured. The PS II mutant strain8–36C exhibits fluorescence decay lifetime components of 53, 424 and 2197 ps. The fluorescence decay of a PS I mutant strain12–7 contains two major fluorescence decay components with lifetimes of 152 and 424 ps. The fluorescence decay of mutant strain C2, which lacks both PS II and PS I, is nearly single exponential with a lifetime of 2561 ± 222 ps. In simulations in which it is assumed that wild-type decays are a simple sum of the major decay components of the isolated parts of the photosynthetic unit as measured in the mutants, curves are obtained that fit the wild-type C. reinhardii fluorescence decay data when the absorption cross-sections of PS II and PS I are weighted approximately equally. The 89 ps lifetime component in the wild-type is an average of 53 and 152 ps components arising from excitation transfer to and trapping in PS I and PS II. The single step transfer time in PS I is estimated to be between 100 and 700 fs depending on assumptions about array size. We find that between two and four visits to the PS I reaction center are required before final trapping.     

Near-Infrared Fluorescence Lifetime Imaging of Biomolecules with Carbon Nanotubes**

Single-walled carbon nanotubes (SWCNTs) are versatile near infrared (NIR) fluorescent building blocks for biosensors. Their surface is chemically tailored to respond to analytes by a change in fluorescence. However, intensity-based signals are easily affected by external factors such as sample movements. Here, we demonstrate fluorescence lifetime imaging microscopy (FLIM) of SWCNT-based sensors in the NIR. We tailor a confocal laser scanning microscope (CLSM) for NIR signals (>800 nm) and employ time correlated single photon counting of (GT)₁₀-DNA functionalized SWCNTs. They act as sensors for the important neurotransmitter dopamine. Their fluorescence lifetime (>900 nm) decays biexponentially and the longer lifetime component (370 ps) increases by up to 25 % with dopamine concentration. These sensors serve as paint to cover cells and report extracellular dopamine in 3D via FLIM. Therefore, we demonstrate the potential of fluorescence lifetime as a readout of SWCNT-based NIR sensors.     

Fluorescence ratiometry and fluorescence lifetime imaging: using a single molecular sensor for dual mode imaging of cellular viscosity

Intracellular viscosity strongly influences transportation of mass and signal, interactions between the biomacromolecules, and diffusion of reactive metabolites in live cells. Fluorescent molecular rotors are recently developed reagents used to determine the viscosity in solutions or biological fluid. Due to the complexity of live cells, it is important to carry out the viscosity determinations in multimode for high reliability and accuracy. The first molecular rotor (RY3) capable of dual mode fluorescence imaging (ratiometry imaging and fluorescence lifetime imaging) of intracellular viscosity is reported. RY3 is a pentamethine cyanine dye substituted at the central (meso-) position with an aldehyde group (CHO). In nonviscous media, rotation of the CHO group gives rise to internal conversion by a nonradiative process. The restraining of rotation in viscous or low-temperature media results in strong fluorescence (6-fold increase) and lengthens the fluorescence lifetime (from 200 to 1450 ps). The specially designed molecular sensor has two absorption maxima (λ(abs) 400 and 613 nm in ethanol) and two emission maxima (in blue, λ(em) 456 nm and red, 650 nm in ethanol). However it is only the red emission which is markedly sensitive to viscosity or temperature changes, providing a ratiometric response (12-fold) as well as a large pseudo-Stokes shift (250 nm). A mechanism is proposed, based on quantum chemical calculations and (1)H NMR spectra at low-temperature. Inside cells the viscosity changes, showing some regional differences, can be clearly observed by both ratiometry imaging and fluorescence lifetime imaging (FLIM). Although living cells are complex the correlation observed between the two imaging procedures offers the possibility of previously unavailable reliability and accuracy when determining intracellular viscosity.     

PICOSECOND FLUORESCENCE KINETICS IN SPINACH CHLOROPLASTS AT LOW TEMPERATURE

Abstract— At 77 K the fluorescence from spinach chloroplasts excited using picosecond mode-locked laser pulses at 620 nm is made up of 5 separate kinetic components. Three of these are predominant at short wavelengths. between 650 and 690 nm, and they appear to correspond to the 3 decay phases seen at room temperature. The 2 new components. a 100 ps rise and a 3000 ps decay, characterize the longer (730–770 nm) wavelength fluorescence. The temperature dependence of the kinetic components of the long wavelength fluorescence shows that the 3000 ps decay accounts for essentially all of the large increase in fluorescence yield observed at low temperature. Furthermore, it appears that this increase does not result entirely from an increase in the fluorescence lifetime, as has been proposed. The dependences of these 2 new components (the 100 ps rise and 3000 ps decay) on emission wavelength and temperature are similar enough to suggest they have a common origin, presumably the chlorophyll pigment component C705. The amplitudes (yield/lifetime) of these 2 phases are approximately equal, and they are opposite in sign. Thus. we see evidence of time-resolved excitation transfer from those pigment molecules that absorb the 620 nm radiation to those that give rise to the long wavelength fluorescence at low temperature.     

Luminescence properties of the mixed J-aggregate of oxacyanine dye and thiacyanine dye. Formation of a persistence-type aggregate

The monolayer assemblies incorporating the J-aggregates of oxacyanine dye, N,N'-dioctadecyloxacyanine perchlorate (S9), and thiacyanine dye, N,N'-dioctadecylthiacyanine perchlorate (S11), S9(J) + S11(J), have been fabricated by the Langmuir-Blodgett (LB) technique. The mole fraction X of S11, X = [S11]/([S9] + [S11]), was varied from 0 to 1. Steady-state absorption spectra, fluorescence spectra, and picosecond fluorescence decay curves of the monolayer assemblies have been measured. Spectroscopic properties of the monolayer assemblies incorporating the individual dye aggregates, S9 J-aggregate (S9(J), X = 0) or S11 J-aggregate (S11(J), X = 1), are characterized by a distinct J-band and resonance fluorescence at lambda(ab) = 403 nm and lambda(em) = 403 nm for S9(J) and lambda(ab) = 456 nm and lambda(em) = 463 nm for S11(J). On the other hand, absorption spectra of the S9(J) + S11(J) assemblies for X = 0.1-0.9 display two absorption bands, a shorter wavelength one and a longer wavelength one, whose peak positions are blue-shifted from those of the corresponding J-bands of the S9 J-aggregate and the S11 J-aggregate, respectively. Furthermore, fluorescence spectra are characterized by a single band (longer wavelength fluorescence) which is somewhat blue-shifted from the resonance fluorescence of the S11 J-aggregate. The fluorescence lifetimes of the S11 J-aggregate and isolated S11 molecules in LB films appear to be tau = 110 and 1900 ps, respectively, while the fluorescence lifetime of the longer wavelength fluorescence of the S9(J) + S11(J) assemblies takes practically a constant value of tau = 170-180 ps for X = 0.2-0.8. These observations would indicate that S9 and S11 molecules in the S9(J) + S11(J) assembly can form a specific mixed aggregate distinct from the individual S9 and S11 J-aggregates. From detailed considerations of the former works on luminescence properties of the S9 J-aggregate doped with isolated S11 molecules, as well as the mosaic-type mixed J-aggregate (M-aggregate) composed of a certain thiacyanine dye, 3,3'-disulfopropyl- 5,5'-dichlorothiacyanine sodium salt, and thiacarbocyanine dye, meso-substituted 3,3'-disulfopropyl-5,5'-dichlorothiacarbocyanine potassium salt, it is suggested that S9 and S11 can form a homogeneous aggregate of the persistence type (HP-aggregate). The HP-aggregate is distinguished from the M-aggregate because it is characterized by homogeneous mixing of two component dyes and persistence of two absorption bands.