Phase and Modulation Fluorometry

Teale, F. W. J.

doi:10.1007/978-1-4757-1634-4_3

Cited by 8 publications

(7 citation statements)

References 36 publications

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“…In the case of a system with multiple lifetimes, the measured τ p will be less than the measured τ m . The relationship between φ and squared amplitude (M 2 ) for a mixture of sinusoidal components are described below (the derivations can be found in [23,39]).…”

Section: Methodsmentioning

confidence: 99%

Applications of phasors to in vitro time-resolved fluorescence measurements

Štefl

James

Ross

et al. 2011

Analytical Biochemistry

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The phasor method of treating fluorescence lifetime data provides a facile and convenient approach to characterize lifetime heterogeneity and to detect the presence of excited state reactions, such as solvent relaxation and Förster Resonance Energy Transfer. The method utilizes a plot of M sin(Φ) versus M cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. A principle advantage of the phasor method is that it provides a model-less approach to time-resolved data, amenable to visual inspection. Although the phasor approach has been recently applied to Fluorescence Lifetime Imaging Microscopy it has not been extensively utilized for cuvette studies. In the present study we explore the applications of the method to in vitro samples. The phasors of binary and ternary mixtures of fluorescent dyes demonstrates the utility of the method for investigating complex mixtures. Data from excited state reactions, such as dipolar relaxation in membrane and protein systems and also energy transfer from the tryptophan residue to the chromophore in EGFP, are also presented.

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Section: Methodsmentioning

confidence: 99%

Applications of phasors to in vitro time-resolved fluorescence measurements

Štefl

James

Ross

et al. 2011

Analytical Biochemistry

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“…It was only in Urbana, however, in the mid-1960s that Weber, together with his graduate student Richard Spencer, succeeded in constructing a highly versatile phase and modulation fluorometer utilizing the principle of cross-correlation [79]. (For an excellent overview of the early history of phase fluorometry the reader is referred to an article by F. W. John Teale [80].) Their cross-correlation method proved to be the key to modern phase fluorometry and is still used universally today.…”

Section: Phase Fluorometrymentioning

confidence: 98%

The Seminal Contributions of Gregorio Weber to Modern Fluorescence Spectroscopy

Jameson¹

2001

New Trends in Fluorescence Spectroscopy

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Gregorio Weber is acknowledged to be the person responsible for many of the more important theoretical and experimental developments in modern fluorescence spectroscopy. In particular, Weber pioneered the application of fluorescence spectroscopy to the biological sciences. His list of achievements includes:-The synthesis and use of dansyl chloride as a probe of protein hydrodynamics -The extension of Perrin's theory of fluorescence polarization to fluorophores associated with random orientations with ellipsoids of revolution and to mixtures of fluorophores -The first spectral resolution of the fluorescence of the aromatic amino acids and of intrinsic fluorescence of proteins -The first demonstration that both FAD and NADH make internal complexes -The first report on aromatic secondary amines, which are strongly fluorescent in apolar solvents, but hardly in water, the most spectacular case being the anilino-naphthalene sulfonates CANS) -The first description of the use of the fluorescence of small molecules as probes for the viscosity of micelles, with implications for membrane systems -A general formulation of depolarization by energy transfer -The discovery of the "red-edge" effect in homo-energy transfer -The development of modern cross-correlation phase fluorometry -The development of the excitation-emission matrix method for resolving contributions from multiple fluorophores -The synthesis of several novel fluorophores, including pyrenebutyric acid, IAEDANS, bis-ANS, PRODAN, and LAURDAN, designed to probe dynamic aspects of biomolecules In addition to these seminal contributions, Gregorio Weber also trained and inspired generations of spectroscopists and biophysicists who went on to make important contributions in their fields, including both basic research as well as the commercialization of fluorescence methodologies and their extension into the clinical and biomedical disciplines. OverviewDuring the last few decades, fluorescence spectroscopy has evolved from a narrow, highly specialized technique into an important discipline widely utilized in the biological, chemical, and physical sciences. Fluorescence methodologies have also assumed an increasingly important role in the clinical and medical sciences. There are now world-renowned centers for fluorescence spectroscopy, highly successful commercial enterprises specializing in fluorescence instrumentation, fluorescence based clinical instruments in virtually all hospital laboratories, and thousands of practitioners worldwide. As in all scientific disciplines, the development of modern fluorescence spectroscopy has benefited

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“…Thus, at high enough fa photon noise will dominate and the signal-to-noise ratio will decrease. As shown in Table I, however, the precision and accuracy are rather stable over a wide range in 2x/r0 (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Therefore, method 2 is remarkably forgiving as to choice of operating f. This analysis ignores nonstatistical noise (e.g., source fluctuations, convection, and photochemistry) that would tend to delay the onset of the plateau with increasing /• Comparison of Methods.…”

Section: T H I S C O N T E N T Imentioning

confidence: 99%

“…THEORY If a sample is excited with a sinusoidally varying source, the sample emission will vary sinusoidally. The emission will, however, be phase shifted from the excitation, and the degree of modulation of the emission will be lower than the degree of modulation of the excitation source (6)(7)(8)(9)(10)(11)(12). These differences arise from the inability of the excited-state concentration to follow the excitation.…”

mentioning

confidence: 99%