Applications of phasor plots to in vitro protein studies

James, Nicholas G.; Ross, Justin A.; Štefl, Martin; Jameson, David M.

doi:10.1016/j.ab.2010.11.011

Cited by 52 publications

(43 citation statements)

References 65 publications

(65 reference statements)

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“…where  f (t) and  s (t) are the new fluorescence and solvation relaxation functions: [24,25]. The variety of possible Trp environments leads to the nonexponential decay of the excited population.…”

Section: Introductionmentioning

confidence: 99%

Resolving environmental microheterogeneity and dielectric relaxation in fluorescence kinetics of protein

Rolinski¹,

McLaughlin²,

Birch³

et al. 2016

Methods Appl. Fluoresc.

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The fluorescence intensity decay of protein is easily measurable and reports on the intrinsic fluorophore-local environment interactions on the sub-nm spatial and sub-ns temporal scales, which are consistent with protein activity in numerous biomedical and industrial processes. This makes time-resolved fluorescence a perfect tool for understanding, monitoring and controlling these processes at the molecular level, but the complexity of the decay, which has been traditionally fitted to multi-exponential functions, has hampered the development of this technique over the last few decades. Using the example of tryptophan in HSA we present the alternative to the con-

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Section: Introductionmentioning

confidence: 99%

Resolving environmental microheterogeneity and dielectric relaxation in fluorescence kinetics of protein

Rolinski¹,

McLaughlin²,

Birch³

et al. 2016

Methods Appl. Fluoresc.

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“…The phasor approach was originally conceived for analysis of FLIM data in the frequency domain. The equations for conversion of the intensity decay into phasor plots have appeared in several publications (Digman, Caiolfa, Zamai, & Gratton, ; James, Ross, Stefl, & Jameson, ; Malacrida et al, ; Malacrida, Gratton, & Jameson, ; Malacrida, Jameson, & Gratton, ; Stefl, James, Ross, & Jameson, ). Although these equations are deceptively simple, the conditions appropriate for the conversion of data obtained in the time domain (typically Time Correlated Single Photon Counting or TCSPC) to phasors should be carefully considered.…”

Section: Introductionmentioning

confidence: 99%

Differences between FLIM phasor analyses for data collected with the Becker and Hickl SPC830 card and with the FLIMbox card

Ranjit

Malacrida

Gratton

2018

Microscopy Res & Technique

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The phasor approach to FLIM (Fluorescence Lifetime Imaging Microscopy) is becoming popular due to the powerful fit free analysis and the visualization of the decay at each point in images of cells and tissues. However, although several implementation of the method are offered by manufactures of FLIM accessories for microscopes, the details of the conversion of the decay to phasors at each point in an image requires some consideration. Here we show that if the decay is not properly acquired, the apparently simple phasor transformation can provide incorrect phasor plots and the results may be misinterpreted. In particular, we show the disagreement in experimental data acquired on the same samples using the two cards (FLIMbox, frequency domain and Becker & Hickl BH 830, time domain) and the effect produced by using the BH 830 card with different settings. This difference in data acquisition translates to the assignment of phasor components calculated using different acquisition parameters. This effect is already present in the original data that are not acquired with the proper parameters for the phasor conversion. We also show that the difference in the resolution of components already exists in the data acquired in the time domain when used with settings that do not allow acquisition of the fluorescence decay on a sufficient large time scale.

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“…Recently, in vitro studies of protein systems and their analysis by phasor diagrams were presented, simplifying complex analysis of time-dependent data and making interpretation of often complex results accessible to non-experts (16,26). Phasor analysis has been used to enhance fluorescence microscopy and in vitro protein studies, whereas extension of the method into nucleic acid studies has yet to be described.…”

Section: Introductionmentioning

confidence: 99%

G-quadruplex structure and stability illuminated by 2-aminopurine phasor plots

Buscaglia

Jameson

Chaires

2012

Nucleic Acids Research

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The use of time-resolved fluorescence measurements in studies of telomeric G-quadruplex folding and stability has been hampered by the complexity of fluorescence lifetime distributions in solution. The application of phasor diagrams to the analysis of time-resolved fluorescence measurements, collected from either frequency-domain or time-domain instrumentation, allows for rapid characterization of complex lifetime distributions. Phasor diagrams are model-free graphical representations of transformed time-resolved fluorescence results. Simplification of complex fluorescent decays by phasor diagrams is demonstrated here using a 2-aminopurine substituted telomeric G-quadruplex sequence. The application of phasor diagrams to complex systems is discussed with comparisons to traditional non-linear regression model fitting. Phasor diagrams allow for the folding and stability of the telomeric G-quadruplex to be monitored in the presence of either sodium or potassium. Fluorescence lifetime measurements revealed multiple transitions upon folding of the telomeric G-quadruplex through the addition of potassium. Enzymatic digestion of the telomeric G-quadruplex structure, fluorescence quenching and Förster resonance energy transfer were also monitored through phasor diagrams. This work demonstrates the sensitivity of time-resolved methods for monitoring changes to the telomeric G-quadruplex and outlines the phasor diagram approach for analysis of complex time-resolved results that can be extended to other G-quadruplex and nucleic acid systems.

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Applications of phasor plots to in vitro protein studies

Cited by 52 publications

References 65 publications

Resolving environmental microheterogeneity and dielectric relaxation in fluorescence kinetics of protein

Resolving environmental microheterogeneity and dielectric relaxation in fluorescence kinetics of protein

Differences between FLIM phasor analyses for data collected with the Becker and Hickl SPC830 card and with the FLIMbox card

G-quadruplex structure and stability illuminated by 2-aminopurine phasor plots

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