Fluorescence Phasor Plots Using Time Domain Data: Effect of the Instrument Response Function.
Liliana Martelo1, Alexander Fedorov1, Mário N Berberan-Santos1
1CQFM - Centro de Química-Física Molecular and IN - Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal.
The Journal of Physical Chemistry. B
|July 17, 2015
Summary
Phasor plots analyze fluorescence decay data, crucial for imaging and material characterization. This study details phasor plot construction and Fourier deconvolution, effectively correcting for instrument response in fluorescence lifetime measurements.
Area of Science:
- Biophysics
- Spectroscopy
- Microscopy
Background:
- Phasor plots are increasingly utilized for analyzing fluorescence intensity decay data.
- Applications span fluorescence lifetime imaging microscopy (FLIM) of biological samples and characterization of homogeneous systems.
Purpose of the Study:
- To discuss the construction of phasor plots from time-domain fluorescence data.
- To investigate the impact of the instrument response function (IRF) on phasor plots.
- To present a deconvolution method in Fourier space for correcting IRF effects.
Main Methods:
- Construction of phasor plots directly from time-domain fluorescence decay data.
- Application of a deconvolution method in the Fourier domain.
- Experimental validation using fluorescein in potassium iodide solutions.
Main Results:
- The study demonstrates how to construct phasor plots and highlights the significant influence of the IRF.
- The proposed Fourier space deconvolution method effectively corrects for the IRF.
- Successful application to fluorescein fluorescence decays, validating the method's predictions.
Conclusions:
- Phasor plot analysis is a powerful tool for fluorescence decay characterization.
- Fourier space deconvolution is effective for lifetimes significantly longer than the IRF width.
- This approach enhances the accuracy of fluorescence lifetime measurements in various applications.
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