Ludwig Brand

Simultaneous analysis of multiple fluorescence decay curves: A global approach

Abstract A procedure is described for simultaneous analysis of multiple fluorescence decay curves. The advantage of this approach is that it exploits relationships between individual decays. Analysis of simulated and real fluorescence lifetime data indicate that this procedure results in increased model testing sensitivity and more accurate parameter recovery.

[17] Time-resolved fluorescence measurements

Nanosecond fluorescence measurements can now be routinely performed and the data can be analyzed in terms of specified decay laws. It is anticipated that nanosecond fluorometry will continue to aid in the elucidation of a variety of excited state mechanistic pathways. This in turn will result in the more sophisticated use of fluorescence probes in biochemistry and cell biology. At the beginning of this decade, Ware9 indicated that the ideal decay fluorometer should permit measurements with samples where the absorbance quantum yield product is 10−10, with high spectral resolution and picosecond time resolution over a wide spectral range. Many of the requirements which seemed so stringent then have now been met. It is likely that the next review on rapid decay techniques will be able to show that picosecond experiments can be carried out with the same degree of confidence that is now possible on the nanosecond time scale.

[87] Fluorescence measurements

Publisher Summary This chapter focuses on some of the possibilities and future potentials inherent in fluorescence methods as well as to some of the difficulties in making absolute measurements and obtaining corrected spectra. Fluorescence involves the absorption of energy by a molecule with a transition from the ground state to one of several excited states followed by rapid internal conversion to the lowest energy excited state and finally by a return to the ground state with the emission of light. Fluorescence measurements are usually more selective than spectrophotometry. The chapter also describes the instrumental aspects of fluorescence, a typical fluorometer, and detailed information about the components utilized in building such an instrument. The specific components are light sources, igniters, light-source housings, monochromators, and detectors. A number of commercial fluorometers and spectrophotofluorometers are also discussed. The potential procedure for obtaining corrected spectra involves the use of digital computing facilities. Although some of the instruments give partially corrected spectra, it is important to understand the errors involved in fluorescence measurements and the type of corrections that must be applied in most cases.

Fluorescence decay studies of anisotropic rotations of small molecules

The fluorescence decay and the decay of the emission anisotropy of perylene and 9‐aminoacridine in glycerol have been investigated at several excitation wavelengths over the temperature range 10 to 40 °C. Under these conditions the fluorescence lifetime of both compounds is essentially constant: about 4.6 ns for perylene and 12.8 ns for 9‐aminoacridine. The decay of the fluorescence emission anisotropy could be analyzed in terms of a double exponential function with rotational correlational times independent of excitation wavelength and dependent on temperature. The pre‐exponential terms are independent of temperature but do depend on the excitation wavelength as expected from geometrical considerations. The anisotropic character of the rotation of perylene is quite pronounced. Excitation of an absorption dipole perpendicular to the emission dipole gives rise to decay curves for the emission anisotropy showing unusual oscillatory behavior; these are predicted by theory. The rotational dynamics of perylene are consistent with those of a disk with the slipping boundary condition. The ratio of the diffusion coefficients about the axes perpendicular and parallel to the plane of the disk is 10±1, in agreement with previous estimates. The anisotropic character of the rotation of 9‐aminoacridine is less striking than that of perylene, but is clearly revealed by measurements at several excitation wavelengths. The rotational dynamics can be interpreted in terms of a hydrodynamic prolate ellipsoid of revolution with a ratio of diffusion coefficients about the major and minor axes of 1.4±0.1 and an axial ratio of about 1.5. Experimental criteria for detecting anisotropic rotations by means of polarized fluorescence are discussed.

Time-resolved fluorescence of the two tryptophans in horse liver alcohol dehydrogenase

The tryptophan fluorescence decay of horse liver alcohol dehydrogenase, at 10 degrees C in 0.1 M pH 7.4 sodium phosphate buffer, with excitation at 295 nm, is a double exponential with time constants of 3.8 and 7.2 ns. Within experimental error, the two lifetimes remain constant across the emission spectrum. Only the 3.8-ns lifetime is quenched in the NAD+-pyrazole ternary complex, and only the 7.2-ns lifetime is quenched by 0-0.05 M KI. On the basis of these results, we assign the 3.8-ns lifetime to the buried tryptophan, Trp-314, and the 7.2-ns lifetime to the exposed tryptophan, Trp-15. The steady-state lifetime-resolved emission spectrum of Trp-15 has a maximum at approximately 340 nm and that of Trp-15 is at approximately 325 nm. The total time-resolved emission, after 40 ns of decay, has a maximum between 338 and 340 nm and is primarily due to the Trp-15 emission. As a consequence of the wavelength dependence of the preexponential weighting factors, there is an increase in the average lifetime from the blue to the red edge of the emission. This increase reflects the change in the spectral contributions of Trp-15 and Trp-314. Consideration of the spectral overlap between the emission spectra of the two tryptophans and the absorption due to formation of the ternary complex, as well as the distances between the two residues and the bound NAD+, shows that the selective fluorescence quenching in the ternary complex can be accounted for entirely by singlet-single energy transfer. The decay of the fluorescence anisotropy was measured as a function of temperature from 10 to 40 degrees C and is well described by a monoexponential decay law. Over this temperature range the calculated hydrodynamic radius increases from 33.5 to 35.1 A. Evidently, the indole groups of Trp-15 and Trp-314 rotate with the protein as a whole, and there is some expansion of the protein matrix as the ambient temperature is increased.

Analysis of fluorescence decay curves by means of the Laplace transformation.

A computational procedure is described for the analysis of fluorescence decay data convolved with a lamp flash of finite width. The computer program calculates the ratio of the Laplace transforms of the decay and the lamp flash for different values of s to give the transforms of the impulse response for each value of s. These are set equal to the analytical Laplace transforms of the decay law involved. Solution of the nonlinear simultaneous equations yields the desired decay parameters. The method can be modified to analyze data that contains a component due to scattered light and can also provide essential information regarding transit time changes of the photomultiplier with changes in emission wavelength. The method was tested by the analysis of real and simulated data. The accuracy of the analysis depends on the degree of correlation among the parameters.

Global analysis of fluorescence decay surfaces: excited-state reactions

Abstract A procedure is proposed for the analysis of fluorescence decay data for systems undergoing excited-state reactions. The rate constants describing the excited-state reactions as well as the spectra associated with each emitting state are recovered in a single analysis step. The method incorporates the simultaneous nonlinear analysis of a multidimensional data surface of time, multiple wavelenghts and concentrations. Complex excited-state reaction schemes can be tested and highly overlapping spectra can be resolved.

Fluorescence decay studies of reduced nicotinamide adenine dinucleotide in solution and bound to liver alcohol dehydrogenase.

The monophoton counting technique was used to obtain the fluorescence decay kinetics of NADH (dihydronicotinamide adenine dinucleotide) bound to LADH (HORSE LIVER ALCOHOL DEHYDROGENAS). It was found that the fluorescence decay of the enzyme complex did not follow a single exponential decay law but that the data could be well described as a sum of two exponentials. The decay parameters of the enzyme complex do not depend on the degree of binding-site saturation. These results are interpreted in terms of a reversible excited-state reaction forming a nonfluorescent product. Fluorescence decay kinetics are also reported for NADH and related molecules in solution. The decay parameters, fluorescence emission maxima, and fluorescence intensities depend on solvent polarity and viscosity.

Global and Target Analysis of Complex Decay Phenomena

ABSTRACT: Methodologies for the simultaneous analysis of multiple total-intensity, anisotropy, and excited-state reactions are developed. Alternatives to the standard sums-of-exponentials fitting are proposed for both anisotropy and excited-state reaction data. Both simulation studies and real-data examples show these methods to be superior to single curve analysis.

Nanosecond decay studies of a fluorescence probe bound to apomyoglobin.

Excited state interactions of N-(p-tolyl)-2-aminonaphthalene-6-sulfonate (2, 6 p-TNS) bound to apomyoglobin were studied by nanosecond time-resolved emission spectroscopy. A dynamic interaction of the excited dye molecule with its binding site, associated with a significant change in the emission energy with time, was observed. The decay kinetics were found to be complex and consistent with the kinetic model for solvent relaxation as proposed by Bakhshiev et al. (Opt. Spectrosc. 21:307. 1966). The behavior of 2, 6 p-TNS bound to apomyoglobin was found to be qualitatively similar to that of the dye dissolved in a viscous solvent such as glycerol or adsorbed to egg lecithin vesicles. The detailed information obtained by following the changes in emission spectra of fluorescent probes on the nanosecond time scale leads to a better understanding of their interactions with biological systems.