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The Phasor Approach to Fluorescence Lifetime Imaging Analysis

Michelle A. Digman ^*, Valeria R. Caiolfa  , Moreno Zamai   and Enrico Gratton ^*

^* Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California, Irvine, California; Department of Molecular Biology and Functional Genomics, San Raffaele Scientific Institute, Milan, Italy; and IIT Network Research, Unit of Molecular Neuroscience, San Raffaele Scientific Institute, Milan, Italy

Correspondence: Address reprint requests and inquiries to Enrico Gratton, E-mail: egratton22{at}yahoo.com.

Changing the data representation from the classical time delay histogram to the phasor representation provides a global view of the fluorescence decay at each pixel of an image. In the phasor representation we can easily recognize the presence of different molecular species in a pixel or the occurrence of fluorescence resonance energy transfer. The analysis of the fluorescence lifetime imaging microscopy (FLIM) data in the phasor space is done observing clustering of pixels values in specific regions of the phasor plot rather than by fitting the fluorescence decay using exponentials. The analysis is instantaneous since is not based on calculations or nonlinear fitting. The phasor approach has the potential to simplify the way data are analyzed in FLIM, paving the way for the analysis of large data sets and, in general, making the FLIM technique accessible to the nonexpert in spectroscopy and data analysis.

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