Analysis of multi-component fluorescence emission by phase-sensitive detection using one modulation frequency

Keating-Nakamoto, Susan; Cherek, Henryk; Lakowicz, Joseph R.

doi:10.1016/0301-4622(86)80001-1

Cited by 9 publications

(8 citation statements)

References 28 publications

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“…Phase-resolved spectra can be quickly obtained for proteins or other samples, by use of a phase fluorometer at a single modulation frequency. Prior determination of the fluorescence lifetime(s) of the sample is generally needed [see however Keating-Nakamoto et al (1986)]. PRS measurements are analogous to decay-associated spectral (DAS) measurements but can be made more quickly.…”

Section: Discussionmentioning

confidence: 99%

“…With either the hardware or software method, only a single modulation frequency is required, but as with time-resolved methods, a significant difference between the decay times of components is required to achieve resolution. Independent determination of the decay times of components is generally required [or alternatively, independent knowledge of the subspectra is required to resolve the component decay times (Keating-Nakamoto et al, 1986)].…”

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confidence: 99%

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Phase-resolved spectral measurements with several two tryptophan containing proteins

Eftink

Wasylewski²,

Ghiron³

1987

Biochemistry

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We have used frequency domain fluorescence techniques to resolve the component emission spectra for several two tryptophan containing proteins (e.g., horse liver alcohol dehydrogenase, sperm whale apomyoglobin, yeast 3-phosphoglycerate kinase, apoazurin from Alcaligenes denitricans). We have first performed multifrequency phase/modulation measurements and have found the fluorescence of each of these proteins to be described by a double exponential. Then, using phase-sensitive detection and the algorithm of Gratton and Jameson [Gratton, E., & Jameson, D. M. (1985) Anal. Chem. 57, 1694-1697], we have determined the emission spectrum associated with each decay time for these proteins. We have compared these phase-resolved spectra with the fractional contributions of the component fluorophores determined by selective solute quenching experiments. Reasonably good agreement is seen in most cases, which argues that the individual Trp residues emit independently. In the case of apoazurin, however, a negative amplitude is seen for the phase-resolved spectrum of the short-lifetime component. This pattern is consistent with the occurrence of energy transfer from the internal Trp residue to the surface Trp of this protein. We also present multifrequency lifetime measurements, phase-resolved spectra, and solute quenching data for a few protein-ligand complexes, to illustrate the utility of this approach for the study of changes in the fluorescence of proteins.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Phase-resolved spectral measurements with several two tryptophan containing proteins

Eftink

Wasylewski²,

Ghiron³

1987

Biochemistry

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“…The method was used for suppression of background fluorescence in Raman spectroscopy (45,46), for studies of ligand binding to macromolecules (47,48), and for resolution of mixtures with more than two components (49,50). The method was also modified to allow decomposition of the emission spectra using known decay times (51) or to allow recovery of the decay times and fraction intensities of the components using the known shapes for the emission spectra (52,53).…”

Section: Introductionmentioning

confidence: 99%

Resolution of multicomponent fluorescence emission using frequency-dependent phase angle and modulation spectra

Lakowicz¹,

Jayaweera²,

Szmacinski³

et al. 1990

Anal. Chem.

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We describe a new fluorescence method that allows the resolution of both the decay times and emission spectra of mixtures of fluorophores. This method is completely general and does not require any assumptions or knowledge of the decay times or emission spectra of the individual fluorophores. We use the phase angle spectra and modulation spectra of the mixture, measured over a range of suitable light modulation frequencies and emission wavelengths. These data are analyzed by nonlinear least-squares analysis to recover the emission spectra and the associated decay times. The principle of the method and the nature of the data are illustrated by using two-component mixtures with increasing spectral overlap. We then demonstrate the recovery of minor components, of structure emission spectra, and of a three-component mixture with completed overlapping emission spectra. And finally, we describe the resolution of a two-component mixture with decay times of 0.8 and 1.4 ns using modulation frequencies up to 774 MHz.

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“…This technique, which relies on a difference in the lifetimes of the components, has been used to resolve multicomponent fluorescence decays (23)(24)(25)(26)(27)(28)(29)(30) to separate Raman, phosphorescence, and fluorescence components (31,32) and to study excited-state reactions which shift the emission spectra (33,34). We recently reported (35,36) a new approach to the measurement and analysis of phase sensitive (PS) data which led to the resolution of three-component mixtures by using data measured at only one frequency. This method appears to be the only one that permits resolution of three decay times using data from only one frequency.…”

mentioning

confidence: 99%

“…We use the procedure of nonlinear least squares, based on the Marquardt algorithm as described by Bevington (39). The analysis is a modification of that previously reported for analysis of single frequency PS data (35,36). It should be noted that the analysis assumes the decay times are constant for each component across the emission spectrum.…”

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confidence: 99%

Resolution of multicomponent fluorescence emission by phase sensitive detection at multiple modulation frequencies

Keating-Nakamoto¹,

Cherek²,

Lakowicz³

1987

Anal. Chem.

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Phase sensitive emission spectra recorded at multiple frequencies were used to determine the lifetimes and steady-state spectra of the fluorophores In two-component mixtures. This analysis does not require any previous knowledge of the lifetimes, fractional Intensities, or the emission spectra and is thus an Improvement over single frequency phase sensitive detection which requires known emission spectra or known lifetimes. Phase sensitive emission spectra are recorded at several frequencies and arbitrarily chosen detector phase angles. The data are analyzed by use of nonlinear least squares to recover the lifetimes and wavelength-dependent fractional Intensities. The latter values determine the emission spectrum and relative intensity of each component in the mixture. Using this technique, we resolved two-component mixtures of fluorescein and 9amlnoacrldlne, 2-(p-toluidinyl)-naphthalene-6-sulfonlc acid and 6-proplonyl-2-(dlmethylamlno)naphthalene, and N-acetyl-L-tyrosinamide and N-acetyl-L-tryptophanamide. In these mixtures the lifetimes differ by about 2-fold. Analysis of simulated data is presented to Illustrate the requirements for a satisfactory resolution. For simulated two-component mixtures, the components can be resolved If the lifetimes differ by 2-fold or greater, even with extensive overlap of the emission spectra. Registry No.

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Analysis of multi-component fluorescence emission by phase-sensitive detection using one modulation frequency

Cited by 9 publications

References 28 publications

Phase-resolved spectral measurements with several two tryptophan containing proteins

Phase-resolved spectral measurements with several two tryptophan containing proteins

Resolution of multicomponent fluorescence emission using frequency-dependent phase angle and modulation spectra

Resolution of multicomponent fluorescence emission by phase sensitive detection at multiple modulation frequencies

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