Determination Of Fluorescence Lifetimes Research Articles

A Multifrequency Phase Fluorometer Using the Harmonic Content of a Mode-Locked Laser

ABSTRACT We describe the construction and operation of a cross-correlation phase and modulation fluorometer which uses the harmonic content of a high repetition rate mode-locked laser as the excitation source. A mode-locked argon ion laser is used to synchronously pump a dye laser. The pulse train output from the dye laser is amplitude modulated by an acousto-optic modulator and then frequency doubled with an angle tuned frequency doubler. With the particular dye utilized in these studies, the ultraviolet light obtained was continuously tunable over the range 280-310 nm. In the frequency domain the high repetition rate pulsed source gives a large series of equally spaced harmonic frequencies. The frequency spacing of the harmonics is determined by the repetition frequency of the laser. Amplitude modulation of the pulse train permits variation of the frequency quasi-continuously from a few hertz to gigahertz. Use of cross-correlation techniques permits precise isolation of individual frequencies. The cross-correlation frequency required for the analysis of the phase delay and modulation ratio is obtained using coupled frequency synthesizers. In the present instrument three synthesizers are used. One drives the pump mode-locker head, a second drives the acousto-optic modulator and the third is used to modulate the response of the photomultiplier tubes which detect the signal. The accuracy, reproducibility and sensitivity of the instrumentation have been determined. Experimental data are provided to show use of this high frequency cross-correlation phase-modulation fluorometer for the determination of fluorescence lifetimes and rotational motions of tryptophan in solution and in proteins.

Novel Techniques for the Determination of Fluorescence Lifetimes

Abstract: Two novel techniques for the measurement of fluorescence lifetimes are discussed. The first method operates in the frequency domain, selectively amplifying the signals generated by the mode beats of a cw laser with a UHF television tuner. The second approach measures the fluorescence with simultaneous temporal and spectral resolution to simplify the characterization of complex samples.

Construction of an Inexpensive Subnanosecond Fluoriheter Using a Microcomputer

ABSTRACT We describe a simple construction of a data acquisition system using an APPLE 11+ Microcomputer. The system uses a commercially available plug-in Interface board containing an analog/digital converter, a digital/analog converter, and a user dedicated 6522 Versatile Interface Adaptor (VIA). The microcomputer controls the time base and digitizes the vertical output of a sampling oscilloscope through the Interface card. Software has been developed for the data acquisition and manipulation. The system was applied to the determination of subnanosecond fluorescence lifetimes. It is pointed out that the system can be very versatile as a data acquisition and manipulation system despite its lower cost.

Excitation pulse-shape mimic technique for improving picosecond-laser-excited time-correlated single-photon counting deconvolutions

The determination of accurate subnanosecond fluorescence lifetimes by the time-correlated single-photon counting technique is often limited by difficulties in obtaining the correct instrumental response function f(λ,t) to the excitation pulse. These difficulties are increased when a grating monochromator is used to disperse the emission due to the introduction of an additional temporal broadening of f(λ,t). A technique for determining the correct f(λ,t) at the wavelength of sample emission, λem, is described. The technique consists of using a very short-lived fluorophore to ‘‘mimic’’ the excitation pulse shape at λem, yielding the convoluted mimic decay function Cm(λem,t), and then to computationally extract f (λem,t) from Cm(λem,t). The technique is experimentally and computationally simple and yields the desired instrumental response function at λem which eliminates problems due to the sensitivity of f(λ,t) to λ (color shift artifact). The photomultiplier tube receives spatially equal illumination when both the sample decay and Cm(λem,t) are determined because both the mimic and sample emission are spectrally broad, eliminating the problem of the variation of f(λ,t) with position of incident light on the photomultiplier tube photocathode (targeting artifact). Artifacts due to monochromator temporal broadening are eliminated since both the mimic and sample emission are monitored at constant wavelength.

A LASER FLASH PHOTOLYSIS STUDY OF PYRENE‐1‐ALDEHYDE. INTERSYSTEM CROSSING EFFICIENCY, PHOTOREACTIVITY AND TRIPLET STATE PROPERTIES IN VARIOUS SOLVENTS

Abstract— Triplet‐and singlet‐related photoprocesses of pyrene‐1‐aldehyde (PA) in various solvents have been investigated in detail using 337.1 and 355 nm laser flash photolysis in conjunction with time‐correlated determination of fluorescence lifetimes (τF) and steady‐state photochemical and absorption‐emission spectral measurements. In benzene, the lowest triplet of PA (43 < ET < 46 kcal/mol) has a lifetime of about 50 µs (τT) and displays the absorption maximum at 443 nm with a maximum extinction coefficient (εmax) of 21000 M‐1cm‐1; the corresponding ketyl radical has a sharp absorption maximum at 428 nm (εmax≥ 25000 M‐1cm‐1). The quantum yields (φT) of lowest triplet occupation are high in nonprotic solvents (0.6–0.8), decrease in protic solvents (alcohols) as the polarity of the latter is increased, and maintain a complementary relationship with the quantum yields (φF) of fluorescence. Quantum yields (φPC) of loss of PA due to photoreactions in some solvents have also been determined under conditions of steady irradiation at 366 nm; φPC is in the range 0.1–0.2 in electron‐rich olefinic solvents such as cyclohexene and tetramethylethylene. These results concerning τF, τT, φF. φT and φPC as well as the effects of 1,2,4‐trimethoxybenzene and 2,5‐dimethyl‐2,4‐hexadiene as quenchers for fluorescence, triplet yield, and photochemistry are discussed in the light of possible state orders for PA in polar and nonpolar environments.

Spectroscopic Investigation of Anhydrous Solutions of Europium Perchlorate and Europium Nitrate in N, N‐Dimethylformamide

AbstractAnhydrous solutions of Eu(ClO4)3 and Eu (NO3)3 0.05m in N, N‐dimethyl formamide (DMF) are investigated by means of conductometric measurements, vibrational spectroscopy, electronic absorption and emission spectra, and fluorescence lifetime determinations. Eu (ClO4)3 is completely dissociated and no inner‐sphere interaction takes place between ClO4 and Eu3+ ions. The solvated species Eu (DMF) has a C2v‐symmetry and x is probably equal to 8.A more complicated situation occurs for Eu (NO3)3, the solutions of which contain at least three different solvates; the predominant species is [Eu (NO3)2 (DMF)x−4]+ (≈80% of the total Eu‐concentration) and it is more stable than the mononitrato complex [Eu (NO3) (DMF) X−2]2+; the neutral complex Eu (NO3)3 (DMF) x−6 is also present, as can be inferred from a high‐resolution analysis of the 5D0→7F0 emission band. The absence of emission from the excited 5D1‐level can be rationalized in terms of an efficient non‐radiative deexcitation path through a vibrational mode of the DMF‐molecules bonded to the central metal ion.

Application of laser induced saturation of molecular fluorescence for lifetime measurements

A technique for the determination of fluorescence lifetimes based on the laser induced saturation of molecular fluorescence is described. It allows lifetime measurements using exciting laser pulses longer than the lifetime under determination. The range of applicability of the technique is discussed and an experimental verification is carried out.

Correction of instrumental time response variation with wavelength in fluorescence lifetime determinations in the ultraviolet region

The successful ratio correction technique described earlier to account for and eliminate the instrumental time response variation with wavelength was limited to wavelengths in the visible region. In this paper we described a modification of the ratio correction technique which allows the extension of this technique into the ultraviolet region (<350 nm). The modified technique is shown to allow the confident determination of fluorescence decay parameters of several molecular systems which show either single- or multiexponential decay in the ultraviolet region.

Confidence in fluorescence lifetime determinations: a ratio correction for the photomultiplier time response with wavelength

The photomultipliers used in single photon counting fluorescence lifetime Systems have a time response function which varies with wavelength. This variation results in a reduced confidence in the kinetic parameters obtained after deconvolution of the lamp intensity decay profile measured at the excitation wavelength from the sample emission decay profile measured at the emission wavelength. Moreover in the case of multi-component decays, inaccurate kinetic parameters are calculated because of this PMT variation. Using weighted residual plots, we demonstrate how one may make a satisfactory subjective appraisal of the adequacy of the deconvolution and the physical model of the decay processes. We have developed a ratio correction technique which accounts for the PMT time response variation with wavelength. This technique requires no apriori assumption of the nature of the PMT effect. Only when the data are corrected using the ratio correction technique can sufficient confidence in the accuracy of the singlet excited state parameters and the validity of the physical model be obtained.

The Determination of the Luminescence Lifetime of Dyes

The investigation of the photochemical and photodynamic behavior of organic dyes is complicated by the fact that the fluorescence lifetime of many dyes is very short because of their high oscillator strength. The determination of fluorescence lifetime from the absorption spectrum and the quantum yield, e.g., described by Turro, leads to very inexact results.

Non-exponential decay of indole fluorescence — the red-edge effect

The fluorescence of indole in glycerol decays exponentially at both 22° and −80°C for 280 nm excitation. Under 296 nm excitation the 22°C emission decay is exponential, but at −80°C, a second shorter lived (1.3 ns) component is also detected. The relevance of this result to the determination of protein fluorescence lifetime is described.

Dépolarisation de fluorescence du conjugué trypsine bovine-diméthylaminonaphtalène-sulfonate

Summary The conjugate bovine trypsin-dimethylaminonaphtalene-sulfonate is studied by the fluorescence depolarization method. It is shown that the label is heterogeneous but one dansyl residue is strongly bound to the enzyme. After a first fluorescence lifetime determination we found that the trypsin possesses a non-rigid structure. The analysis of the fluorescence decay curve showed us that this decay can be described by a sum of two exponentials. The second order mean decay times gives a higher value for the rotational relaxation time of the protein. The new results are consistent with a rigid structure and between pH 6 and 8 trypsin can be assimilated to a rigid sphere.

A simple device for the determination of fluorescence lifetimes

Describes a simple modification to the phase-shift technique for determination of radiative lifetimes. The time delay between the exciting and emitted radiation is measured rather than the phase lag, and this removes the necessity for accurate knowledge of frequency and permits operation at relatively low modulation frequencies. A circuit is presented which provides a modulating and a delayed reference waveform and which compares the phase of a signal derived from the emitted radiation with the reference. Some preliminary tests of the system are described in which it is shown that the time resolution, for a modulation frequency of about 50 kHz, is roughly 5 ns (corresponding to a phase shift of less than 0.1 degrees ).