title: Noise-Corrected Principal Component Analysis of fluorescence lifetime imaging data.
abstract: Fluorescence Lifetime Imaging (FLIM) is an attractive microscopy method in the life sciences, yielding information on the sample otherwise unavailable through intensity-based techniques. A novel Noise-Corrected Principal Component Analysis (NC-PCA) method for time-domain FLIM data is presented here. The presence and distribution of distinct microenvironments are identified at lower photon counts than previously reported, without requiring prior knowledge of their number or of the dye's decay kinetics. A noise correction based on the Poisson statistics inherent to Time-Correlated Single Photon Counting is incorporated. The approach is validated using simulated data, and further applied to experimental FLIM data of HeLa cells stained with membrane dye di-4-ANEPPDHQ. Two distinct lipid phases were resolved in the cell membranes, and the modification of the order parameters of the plasma membrane during cholesterol depletion was also detected. Noise-corrected Principal Component Analysis of FLIM data resolves distinct microenvironments in cell membranes of live HeLa cells.
authors.affiliation: Department of Physics, King's College London, Strand, WC2R 2LS, London, United Kingdom.
authors.forename: Alix
authors.initials: A
authors.lastname: Le Marois
authors.affiliation: Institute Fresnel, Avenue Escadrille Normandie Niemen, 13013, Marseille, France.|Leibniz Institute of Photonic Technology, Albert-Einstein-Straße 9, 07745, Jena, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Labouesse
authors.affiliation: Department of Physics, King's College London, Strand, WC2R 2LS, London, United Kingdom.
authors.forename: Klaus
authors.initials: K
authors.lastname: Suhling
authors.affiliation: Leibniz Institute of Photonic Technology, Albert-Einstein-Straße 9, 07745, Jena, Germany.|Institute of Physical Chemistry, Abbe Centre of Photonics, Friedrich Schiller University Jena, Helmholtzweg 4, 07743, Jena, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Heintzmann
mesh_terms: D002462:Cell Membrane; D006367:HeLa Cells; D006801:Humans; D007089:Image Enhancement / Q000379:methods*; D008856:Microscopy, Fluorescence; D061848:Optical Imaging; D017785:Photons; D025341:Principal Component Analysis
keywords: Confocal fluorescence microscopy; Poisson noise correction; data processing; fluorescence lifetime imaging; global analysis; live cell imaging