Applications of phasors to in vitro time-resolved fluorescence measurements

Abstract

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.

A fluorescent molecular rotor showing vapochromism, aggregation-induced emission, and environmental sensing in living cells

“…6 The sensing capability of TPAP is exploited by a new imaging approach, named phasor analysis, which relies on a simple graphic analysis of spectral and lifetime fluorescence images following a mathematical transformation. 18 Overall, our results do support the use of TPAP as an outstanding probe in the field of environmental sensors.…”

Rapid fluorescence lifetime estimation with modified phasor approach and Laguerre deconvolution: a comparative study

“…The phasor method is a fit-free method that has been shown to be a powerful tool for the analysis of fluorescence lifetime [20, 22, 31] or spectral images [28, 32, 33]. This method provides a graphical representation of the fluorescence decay that is comparatively simpler than other existing methods but needs prior calibration on well characterized samples in order to correct for the IRF effect and to obtain quantitative results [4].…”

Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis

“…A phasor approach to FLIM imaging along with SHG generation can quantify fibrosis. 15–18 The signal originating from SHG and short wavelength fluorescence can be separated based on the lifetime of a signal. 19 Fluorescence is a delayed emission and thus has a non-zero lifetime.…”