A121 - Phasor analysis of Fluorescence Lifetime Microscopy (FLIM)

Chiara Stringari (chiara.stringari@polytechnique.edu)
Bahar Asadipour (bahar.asadipour@polytechnique.edu)

Here we propose a workshop to learn the Phasor analysis of Fluoresce Lifetime Microscopy (FLIM) data. The phasor approach to visualize and analyze FLIM data has seen widespread use due to its ease of use and interpretation. Phasor analysis is a fit-free technique based on a FFT transformation of the intensity decay that provides a visual distribution of the molecular species, clustering pixels with similar lifetimes even when they are spatially separated in the image. Phasor analysis has also been increasingly used to map complex autofluorescence distributions and to perform metabolic analysis of NAD(P)H and FAD intrinsic biomarkers. In this workshop we propose to learn the principles of phasor analysis as well as to perform image analysis of FLIM microscopy data with this approach using (F)luorescence (L)ifetime (U)l(t)imate (E)xplorer (FLUTE) a new free and user-friendly GUI that we recently developed. Participants will learn the basis of FLIM Calibration, Phasor plotting, FLIM data interactive visualization and exploration, data interpretations and advanced phasor analysis and quantification. We also propose to explore and understand the impact of the most important parameters and factors of FLIM acquisition, such as the number of photons, as well as sources of bias such as the presence of background. The phasor analysis will be performed on a variety a FLIM data acquired in the time domain from different fluorophores, autofluorescence from zebrafish embryo and cells in culture. Example of functional metabolic images will be explored. It will also be possible to analyze FLIM data that users will bring from their labs and/or acquired with different FLIM commercial systems.