Correction for contaminant fluorescence in frequency-domain fluorometry

Lakowicz, Joseph R.; Jayaweera, Ranjith; Joshi, Nanda B.; Gryczynski, Iganzy

doi:10.1016/0003-2697(87)90078-9

Cited by 23 publications

(20 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Scattering is manifest as a systematic deviation in the frequency dependence of the phase angle and modulation ratio from their true values. 81 Numerical simulations of anisotropy data demonstrate that the deviations so produced are in the correct direction to qualitatively reproduce the density-dependent behavior of the differential polarized phase and polarized modulation ratio. 82 Confirmation of this hypothesis awaits further frequency-domain experiments, which we hope to undertake in the near future.…”

Section: ∆Fmentioning

confidence: 82%

Rotation of Aromatic Solutes in Supercritical CO₂: Are Rotation Times Anomalously Slow in the Near Critical Regime?

Heitz

Maroncelli

1997

J. Phys. Chem. A

View full text Add to dashboard Cite

Picosecond fluorescence anisotropy decay measurements are used to examine the rotational dynamics of three solutes, 1,3,6,8-tetraphenylpyrene ("TPP"), 9,10-bis(phenylethynyl)anthracene ("PEA"), and N,N′-bis-(2,5-di-tert-butylphenyl)-3,4,9,10-perylenedicarboximide ("BTBP") in supercritical CO 2 (35°C ) T c + 4°C) and in a variety common liquid solvents. In liquids the rotation times of all three probes show an approximate proportionality to solvent viscosity, in rough agreement with simple hydrodynamic theories. In supercritical CO 2 two of the probes, TPP and BTBP, are found to exhibit rotation times consistent with the extrapolation of the hydrodynamic trends found in liquid solvents. In the case of BTBP, these results disagree with recently published reports of very long rotation times near the critical point [Heitz and Bright J. Phys. Chem. 1996, 100, 6889]. However, the rotation times of PEA deviate significantly from hydrodynamic predictions based on the viscosity of the supercritical fluid for near critical densities. In this case, it appears that local density augmentation leads to increased rotational friction on the solute compared to what would be expected on the basis of the bulk solvent properties. Using the observed rotation times, an effective density that is 50-100% greater than the bulk density is estimated for reduced densities (F/F c ) of 0.8-1. Similar estimates of the extent of local density augmentation are also obtained from the behavior of the electronic frequency shifts of this solute.

show abstract

Section: ∆Fmentioning

confidence: 82%

Rotation of Aromatic Solutes in Supercritical CO₂: Are Rotation Times Anomalously Slow in the Near Critical Regime?

Heitz

Maroncelli

1997

J. Phys. Chem. A

View full text Add to dashboard Cite

show abstract

“…One of the difficulties shared by the background correction methods described by Lakowicz et al [1] and Swift and Mitchell [2] is proper determination of the uncertainty associated with the phase and modulation recovered after correction. Since our procedure operates on-line by alternating between sample and blank cuvettes, and hence performs multiple determinations of the corrected signals at each frequency, the standard errors of the corrected signals are directly observed.…”

Section: Discussionmentioning

confidence: 99%

“…One can therefore continue acquisition until the standard error of the corrected signal falls within acceptable limits (which conforms to the customary way frequency domain data are currently collected). In other words, the user does not have to enter into an a posteriori evaluation of the error as required by the method described by Lakowicz et al [1].…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A method for on-line background subtraction in frequency domain fluorometry

et al. 1991

View full text Add to dashboard Cite

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 ihe total signal intensity.KEY WORDS: Frequency domain; background; lifetime; dynamic polarization. INTRODUC~ONPhase fluorometry has become a widely utilized technique for performing time-resolved fluorescence measurements. As this technique becomes increasingly applied to complex samples at low concentrations, notably biological samples, unavoidable background fluorescence has come to significantly limit its overall sensitivity. Fluorescent contaminants in reagents, parasitic light, and scattered light (Rayleigh and Raman) are among the most common sources of background. 153background is attributable to a single exponential decay, then a multicomponent analysis, in which one component can be clearly assigned to the background, is adequate to recover the sample's time-resolved parameters. Usually background contributions are more complex, though, rendering this approach inappropriate.A method to correct frequency domain data for background signals, whether they originate from contaminant fluorescence or scattered light, has been reported by Lakowicz et al. [1]. In their method, the fraction of the total steady-state signal attributable to the background must be determined in a separate experiment under identical instrument conditions. This measurement is necessitated by the fact that, in their method, the signal acquisition is based on phase and modulation data alone, so that the AC contributions of the background must be calculated a posteriori from the independently determined DC values. Moreover, in their approach a rigorous error propagation analysis cannot be achieved during data acquisition but must be performed sepa-1053-0509/91/0900-0153506.50/0 9 1991 Plenum Publishing Corporation 154 Reinhart, Marzola, Jameson, and Gratton rately. Consequently the method is not a true on-line procedure.Another approach to blank correction was recently proposed by Swift and Mitchell [2] specifically for the case of parallel data acquisition. In parallel phase fluoro...

show abstract

“…The optical density of the sample was maintained at Aa60=0.1. Corrections for background fluorescence and/or light scattering were made by performing the frequency-domain measurements with phospholipids without proteins as control sample and then correcting the data as in [17]. Notably, the addition of nystatin in DMSO resulted in an appearance of a much higher background signal in the control samples (liposomes without proteins PL and PE), which required the above correction.…”

Section: Dynamic Fluorescence Measurements and Data Analysis 281 Fmentioning

confidence: 99%

Possible nystatin‐protein interaction in yeast plasma membrane vesicles in the presence of ergosterol. A Förster energy transfer study

et al. 1996

View full text Add to dashboard Cite

Correction for contaminant fluorescence in frequency-domain fluorometry

Cited by 23 publications

References 12 publications

Rotation of Aromatic Solutes in Supercritical CO₂: Are Rotation Times Anomalously Slow in the Near Critical Regime?

Rotation of Aromatic Solutes in Supercritical CO₂: Are Rotation Times Anomalously Slow in the Near Critical Regime?

A method for on-line background subtraction in frequency domain fluorometry

Possible nystatin‐protein interaction in yeast plasma membrane vesicles in the presence of ergosterol. A Förster energy transfer study

Contact Info

Product

Resources

About

Correction for contaminant fluorescence in frequency-domain fluorometry

Cited by 23 publications

References 12 publications

Rotation of Aromatic Solutes in Supercritical CO2: Are Rotation Times Anomalously Slow in the Near Critical Regime?

Rotation of Aromatic Solutes in Supercritical CO2: Are Rotation Times Anomalously Slow in the Near Critical Regime?

A method for on-line background subtraction in frequency domain fluorometry

Possible nystatin‐protein interaction in yeast plasma membrane vesicles in the presence of ergosterol. A Förster energy transfer study

Contact Info

Product

Resources

About

Rotation of Aromatic Solutes in Supercritical CO₂: Are Rotation Times Anomalously Slow in the Near Critical Regime?

Rotation of Aromatic Solutes in Supercritical CO₂: Are Rotation Times Anomalously Slow in the Near Critical Regime?