Apparent Fluorescence Lifetime Research Articles

Collision-Free Lifetimes of SO(B3Σ-, v‘ = 0, 1, and 2) and Vibrational Level Dependence of Deactivation by He

Collision-free lifetimes and deactivation rate constants of SO(B3Σ-, v‘ = 0, 1, and 2) by collisions with He have been determined. SO(X3Σ-) was generated in the photolysis of SO2 at 193 nm (ArF laser) and excited to a single rovibrational level of B3Σ- with a pulsed ultraviolet laser. We recorded dispersed fluorescence spectra, and found that 0−12 (350.8 nm), 1−14 (368.5 nm), and 2−17 (401.2 nm) bands are suitable for detecting fluorescence from single vibrational level of interest. Time profiles of wavelength-resolved fluorescence were recorded at various buffer gas pressures (5−165 Torr of He). Deconvolution by the integrated phase plane method derives apparent fluorescence lifetime at a given pressure. Total pressure dependence of fluorescence decay rates leads to collision-free lifetimes and rate constants for deactivation of SO(B3Σ-, v‘ = 0, 1, and 2) by collisions with He. Lifetimes of SO(B3Σ-) have been determined to be (28 ± 2) ns (v‘ = 0), (30 ± 3) ns (v‘ = 1), and (27 ± 4) ns (v‘ = 2). Rate coef...

Frequency-domain fluorescence lifetime imaging for endoscopic clinical cancer photodetection: Apparatus design and preliminary results

We describe a new fluorescence imaging device for clinical cancer photodetection in hollow organs in which the tumor/normal tissue contrast is derived from the fluorescence lifetime of endogenous or exogenous fluorochromes. This fluorescence lifetime contrast gives information about the physicochemical properties of the environment which are different between normal and certain diseased tissues. The excitation light from a CW laser is modulated in amplitude at a radio frequency by an electrooptical modulator and delivered by an optical fiber through an endoscope to the hollow organ. The image of the tissue collected by the endoscope is separated in two spectral windows, one being the backscattered excitation light and the other the fluorescence of the fluorochrome. Each image is then focused on the photocathode of image intensifiers (II) whose optical gain is modulated at the same frequency as the excitation intensity, resulting in homodyne phase-sensitive images. By acquiring stationary phase-sensitive frames at different phases between the excitation and the detection, it is possible to calculate in quasi-real time the apparent fluorescence lifetime of the corresponding tissue region for each pixel. A result obtained by investigating the endogenous fluorochromes present in the mucous membrane of an excised human bladder is presented to illustrate this method and most of the optical parameters which are of major importance for this photodetection modality have been evaluated.

Enhanced excitation energy transfer in microdroplets — a study by time-resolved fluorescence microscopy

Time-resolved fluorescence microscopy was used to study the fluorescence quenching of rhodamine 6G (R6G) in single microdroplets with diameters equal to or larger than 2 μm. The microdroplets were prepared by dispersing ethylene glycol solutions of R6G in silicone oil. The fluorescence lifetime of R6G was found to be independent of the size of the droplet at low concentrations (1.0 × 10 −4 M or less; a value of 3.60 ± 0.02 ns was observed, which was measured for the bulk solution in a thin glass capillary, indicating that neither the radiative process nor the intramolecular radiationless processes of R6G were influenced by the size of the droplet for droplets greater than 2 μm in diameter. However, a significant size effect on the fluorescence decay was observed at higher concentrations of R6G. At the highest concentration of R6G (5.2 × 10 −3 M) examined in this work, the apparent fluorescence lifetime decreased from 3.60±0.02 ns for droplets larger than 20 μm in diameter to approximately 0.4 ± 0.07 ns for the smallest droplet of 2 μm in diameter. Despite such a significant decrease in the fluorescence lifetime, the decay curves could be analysed satisfactorily by fitting to single exponential functions. When malachite green (MG) was added as an acceptor to a solution of R6G (10 −4 M), a large enhancement of Förster-type energy transfer from R6G to MG, as compared with the bulk solution, was observed for droplets with diameters of less than 10 μm. In this case, all the decay curves obtained were well described by the function derived from the Förster-type energy transfer, but the effective critical distance increased by approximately a factor of two on going from a 10 μm droplet to a 2 μm droplet. On the other hand, the fluorescence quenching of R6G by potassium iodide (KI) showed only a small size effect on the quenching rate constant. The significant size effect observed for droplets of concentrated R6G solution can be interpreted in terms of enhanced Förster-type energy migration, followed by energy trapping by dimers of R6G which act as quenching centres.

Simultaneous dual-frequency phase-sensitive flow cytometric measurements for rapid identification of heterogeneous fluorescence decays in fluorochrome-labeled cells and particles.

In frequency-domain lifetime spectroscopy, the apparent fluorescence lifetimes obtained from phase-shift measurements are independent of modulation frequency only in the special case of a single exponential fluorescence decay. For heterogeneous fluorescence decay, the apparent fluorescence lifetimes measured by the phase-shift methods are functions of the modulation frequency. This modulation-frequency dependent property of apparent fluorescence lifetimes may be used to identify heterogeneous fluorescence decays by measuring lifetimes at multiple frequencies. In this article we explore the requirements and experimental design considerations for making such measurements in flow. We report a phase-sensitive flow cytometric method that allows one to probe the excited state-lifetimes of labeled cells by using multiple simultaneous modulation frequencies. Application of this method is demonstrated by measuring fluorescence lifetimes of labeled cells at two frequencies simultaneously, using a continuous-wave, dual-frequency modulated excitation in flow. The dual-frequency method presented herein can be used to rapidly identify heterogeneity in the fluorescence decay on a cell-by-cell basis in real time. Information on the nature of the fluorescence decay is important in biological measurements because it can provide insight into intermolecular interactions at the subcellular level.

Fluorescence lifetime-based biosensing of zinc: Origin of the broad dynamic range

Fluorescence lifetime-based chemical sensors have recently been described for applications in medicine, environmental monitoring, and bioprocess control. These sensors transduce the level of the analyte as a change in the apparent fluorescence lifetime of an indicator phase. We have previously developed a wavelength-ratiometric fluorescence biosensor for zinc based on binding of zinc and dansylamide to apo-carbonic anhydrase which exhibited high sensitivity and selectivity. We demonstrate that the apo-carbonic anhydrase/dansylamide indicator system is very well suited for lifetime-based sensing, with a subnanomolar detection limit and greater than 1000-fold dynamic range. The theoretical basis for the wide dynamic range is discussed.

Fluorescence lifetime imaging of intracellular calcium in COS cells using Quin-2

We describe the first fluorescence lifetime images of cells. To demonstrate this new capability we measured intracellular images of Ca 2+ in COS cells based on the Ca 2+-dependent fluorescence lifetime of Quin-2. Apparent fluorescence lifetimes were measured by the phase-modulation method using a gain-modulated image intensifier and a slow-scan CCD camera. We describe methods to correct the images for photobleaching during acquisition of the data, and to correct for the position-dependent response of the image intensifier. The phase angle Quin-2 images were found to yield lower than expected Ca 2+ concentrations, which appears to be the result of the formation of fluorescent photoproducts by Quin-2. Fluorescence lifetime imaging (FLIM) does not require wavelength-ratiometric probes and appears to provide new opportunities for chemical imaging of cells.

Fluorescence lifetimes of the tryptophan residues in ornithine transcarbamoylase.

Multifrequency (2-230 MHz) phase-modulation fluorescence measurements and site-directed mutagenesis have been employed to assign fluorescence lifetimes, quantum yields, and emission maxima to the four tryptophans in the enzyme ornithine transcarbamoylase from Escherichia coli (OTCase) (Trp-125, -92, -233, and -243). OTCase displays two apparent fluorescence lifetimes, 7.2 and 3.2 ns. Results on specific mutants show that Trp-233 has a lifetime of 7.1 ns, while TRP-125, -192, and -243 have lifetimes of 4.0, 3.6, and 4.9 ns, respectively. Thus, the specific conformational changes of the polypeptide segment involving Trp-233 may be monitored conveniently in the wild-type enzyme. On the basis of quantum yield values, Trp-233 is calculated to contribute approximately 43% of the fluorescence intensity of the enzyme, while direct measurements of the enzyme show that up to 65% of the total intensity is really emitted by this tryptophan. The discrepancy may arise from energy transfer from Trp-125 to Trp-233, with an efficiency of 20%. Application of the assigned tryptophan lifetimes to probe ligand-induced protein conformational changes has also been demonstrated.

A time-resolved fluorescence study of electronic excitation energy transport in concentrated dye solutions

Singlet-state radiative and nonradiative energy transport among randomly distributed donor and acceptor molecules in solutions of ethylene glycol has been investigated by using time-resolved fluorescence spectroscopy. Radiative energy transfer in moderately concentrated solutions of rhodamine 6G contained in a 1 cm pathlength cuvette results in nonexponential fluorescence decay curves which can be described in terms of the relative fluorescence emission yield of the ith transfer process, φ i , the average number of transfer steps, < n >, and the apparent fluorescence lifetime, <τ >. The fluorescence decay profiles measured from thin films (⩽ 20 μm) of solutions of rhodamine 6G donors in the presence of malachite green acceptors are also nonexponential and at low donor concentrations (1.0 × 10 −4 M) the decay curves can be described by using Förster's dipole-dipole model for nonradiative energy transfer, where the value for the critical transfer distance, R 0, is calculated to be (5.9±0.1) nm. At higher donor concentrations (3.0 × 10 −3 M) the Förster model is inappropriate. However, the model proposed by Loring, Anderson and Fayer (LAF), which allows for the effects of nonradiative energy transfer among donors, provides excellent fits to the experimentally determined fluorescence decay curves for this donor concentration and results in a value of R 0 for nonradiative energy transfer between rhodamine 6G chromophores of (5.8±0.1) nm. The LAF model also provides a satisfactory description of the kinetics of the quenching by rhodamine 6G dimers of the fluorescence from thin films of highly concentrated solutions of rhodamine 6G. The value of R 0 for nonradiative energy transfer from monomer to dimer and the equilibrium constant for rhodamine 6G dimerization are calculated to be (3.2±0.1) nm and 19 M −1, respectively.

Laser-induced fluorescence of the SiH 2 radical

The laser-induced fluorescence of the SiH 2 radical was observed in the photolysis of phenylsilane by an ArF excimer laser in the 550–650 nm range. The apparent fluorescence lifetime is 60 ± 5 ns at 580.1 nm. The bending vibrational spacing in the ground state was found to be 990 ± 20 cm −1 from the dispersed fluorescence spectrum.

Synthesis and Characterization of a Fluorescence Probe of the Phase Transition and Dynamic Properties of Membranes.

We describe the synthesis and characterization of a new fluorescence probe whose emission spectra, anisotropies, and wavelength-dependent decay times are highly sensitive to the phase state of phospholipid vesicles. This probe is 6-palmitoyl-2-[[2-(trimethylammonio)ethyl]methylamino] naphthalene chloride (Patman). The emission maximum of Patman shifts from 425 to 470 nm at the bilayer transition temperatures. The spectral properties of Patman reveal nanosecond time-dependent spectral shifts, which are the result of membrane relaxation around the excited state of Patman. The apparent fluorescence lifetimes of Patman are strongly dependent upon the emission wavelength, and the fluorescence phase and modulation data prove that the spectral shifts are due to an excited-state process, and not ground-state heterogeneity. As expected, the fluorescence anisotropies reflect the phase transitions of the bilayers. In addition, the anisotropies are dependent upon the emission wavelength because the duration of the excited state varies across the emission spectrum. The different apparent lifetimes across the emission spectrum allow the relaxed and unrelaxed emission spectra to be resolved by phase-sensitive detection of fluorescence. Also, the emission spectra of Patman show marked shifts to longer wavelengths as the excitation wavelength is increased. These red-edge excitation shifts are sensitive to the temperature and phase state of the bilayers.

Tryptophan fluorescence of human hemoglobin. I. Significant change of fluorescence intensity and lifetimes in the T − R transition

The fluorescence spectra and fluorescence lifetimes due to tryptophan residues in HbA, Hb Chesapeake, NES-des-Arg Hb and Hb Kempsey were determined at room temperature. The fluorescence intensity and apparent fluorescence lifetimes decrease when the deoxy or T structure in HbA changes to the oxy or R structure, while no significant difference was observed in Hb Kempsey. The difference of fluorescence behavior was ascribed to the quaternary conformational transition of T- and R-states.

Kinetics of Nitrogen Dioxide Fluorescence

The fluorescence lifetime and intensity of gas-phase nitrogen dioxide (2B1) have been measured as a function of excitation wavelength, fluorescence wavelength, and pressure (0.5–50 mtorr). The phase-shift method was used; this technique allows lifetime measurements to be obtained with signal intensities of 100 counts per sec and lower. The excitation source, tunable throughout the visible region, had a half-width bandpass as low as 15 Å. Fluorescence wavelength separation was accomplished with 15 interference filters between 400 and 8000 Å. The radiative lifetimes range from 55 to 90 μsec for excitation from 3980 to 6000 Å and tend to increase with excitation wavelength; however, the lifetimes exhibit considerable variation within a narrow excitation region. The fluorescence sample was contained in a 33-cm-diam spherical bulb; apparent fluorescence lifetimes in smaller cells were reduced because of migration of excited molecules (under collision-free conditions) and wall quenching. In order that the measured lifetimes exhibit no more than 5% error, observation must be extended beyond the excitation region to a distance five times the product of the lifetime by the most probable velocity. The Stern–Volmer analysis of fluorescence kinetics has been generalized to a multilevel system under conditions of modulated excitation and phase-sensitive detection. Analysis of fluorescence data in terms of this mechanism yields the product of energy-transfer rate constants times efficiencies (amount of energy lost by the excited polyatomic molecule per effective collision). This analysis implies the loss of at least one vibrational quantum (average value 1230 cm−1) per gas-kinetic collision over the entire range from 12 500–25 000 cm−1 energy above the ground state. This result indicates that molecules in the B12 excited electronic state are rapidly interconverted to the high vibrational levels of the ground electronic state.