Time domain fluorescence spectrometry offers a versatile and powerful approach to the analysis of heterogeneous emitting systems. In this paper we describe a new approach, based on software, to the acquisition of phase and modulation resolved spectra. Mixtures of fluorophores with different lifetimes can be analyzed in real time to give the individual excitation or emission spectra. Examples of two- and three-component mixtures are given and comparisons are made with the commercially available hardware approach. © 1985, American Chemical Society. All rights reserved.

Oxygen quenching experiments were carried out on zincprotoporphyrin IX reconstituted myoglobin (MbFe-->Zn) at different temperatures and two solvent viscosities. The data were fit to a dynamic model for quenching of fluorophores in protein interiors previously presented (Biophysical J., 45 (1984) 789-794). The parameters associated with the oxygen entry rate (k+), exit rate (k-), and migration rate (chi) in the protein were obtained at six temperatures and two viscosities (1 and 8 cp), along with the activation enthalpies associated with the above rates (k+ and k-). The partition coefficient (alpha) was calculated at each temperature along with the free energy, delta G0, associated with this partition. The rate parameters (k+, k-, chi) and the partition coefficient (alpha) have also been determined for the sample in 40% sucrose (8 cp), to evaluate the effect of bulk solvent viscosities on these values. The steady-state Stern-Volmer quenching plot was calculated using the rate parameters obtained from the analysis (of the dynamic model). Comparison of the Stern-Volmer points obtained using the dynamic model and those obtained experimentally showed excellent agreement.

Mammalian cell membranes have different phospholipid composition and cholesterol content, displaying a profile of fluidity that depends on their intracellular location. Among the dyes used in membrane studies, LAURDAN has the advantage to be sensitive to the lipid composition as well as to membrane fluidity. The LAURDAN spectrum is sensitive to the lipid composition and dipolar relaxation arising from water penetration, but disentangling lipid composition from membrane fluidity can be obtained if time resolved spectra could be measured at each cell location. Here we describe a method in which spectral and lifetime information obtained in different measurements at the same plane in a cell are used in the phasor plot providing a solution to analyze multiple lifetime or spectral data through a common visualization approach. We exploit a property of phasor plots based on the reciprocal role of the phasor plot and the image. In the phasor analysis each pixel of the image is associated with a phasor and each phasor maps to pixels and features in the image. In this paper the lifetime and spectral fluorescence data are used simultaneously to determine the contribution of polarity and dipolar relaxations of LAURDAN in each pixel of an image.

Solvent dipolar reorientations following excitation of DANCA in glycerol at various temperatures were investigated by using multifrequency-phase fluorometry. The time evolution of the emission spectrum was determined by performing lifetime measurements at a number of wavelengths across the emission band and then reconstructing the spectrum at selected times after excitation. At low temperatures, a large displacement of the time-resolved spectrum was apparent. The spectral center of gravity was observed to move faster at short times and then to exhibit a long decay. The spectral width was also observed to increase at short times and then to decrease at much longer times. Several models have been considered to account for the major features of our experimental results. A two-state model agreed well with the data only in the high-temperature regime; at lower temperatures, the data were not consistent with the characteristic features of the two-state model. In the high-temperature regime, we obtained a reorientation rate for the glycerol molecules using the two-state model. Other models which consider a continuous translation of the emission spectra as a function of time also failed to account for the experimental results. Instead, a good qualitative agreement was found with the model proposed by Weber in which the charge separation of the excited fluorophore is followed by reorientation of the solvent molecules around the individual monopoles. © 1987, American Chemical Society. All rights reserved.

In this note, we present a discussion of the advantages and scope of model-free analysis methods applied to the popular solvatochromic probe LAURDAN, which is widely used as an environmental probe to study dynamics and structure in membranes. In particular, we compare and contrast the generalized polarization approach with the spectral phasor approach. To illustrate our points we utilize several model membrane systems containing pure lipid phases and, in some cases, cholesterol or surfactants. We demonstrate that the spectral phasor method offers definitive advantages in the case of complex systems.

The unfolding and refolding of apohorseradish peroxidase, as a function of guanidinium chloride concentration, were monitored by the intrinsic fluorescence intensity, polarization, and lifetime of the single tryptophan residue. The unfolding was reversible and characterized by at least three distinct stages-the intensity and lifetime data, for example, were both characterized by an initial increase followed by a decrease and then a plateau region. The lifetime data, in the absence and presence of guanidinium chloride, were heterogeneous and fit best to a model consisting of a major Gaussian distribution component and a minor, short discrete component. The observed increase in intensity in the initial stage of the unfolding process is attributed to the conversion of this short component into the longer, distributed component as the guanidinium chloride concentration increases. Our results clarify and amplify previous studies on the unfolding of apohorseradish peroxidase by guanidinium chloride.

A description is given of 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. 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.

Time-resolved fluorescence determinations on biochemical samples are often complicated by contributions from background. In the time-correlated single-photon counting method, background subtraction is a routine procedure. A limitation of frequency domain fluorometry, however, arises from the difficulty of performing this operation. This limitation has become increasingly significant as frequency domain methods are being applied to evermore complex biological systems using the higher-frequency capabilities of modern instrumentation. We have devised a method for such a correction in the frequency domain, regardless of the complexity of the background decay, based on measurement of the background phasor and subsequent subtraction from the sample phasor. This method is applicable to both lifetime and dynamic polarization measurements, and it can be readily implemented on commercially available frequency domain fluorometers. Decay curves may be accurately recovered from samples containing background contributions ranging from less than 5% to greater than 90% of the total signal intensity.