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Proceedings Paper

A phasor approach analysis of multiphoton FLIM measurements of three-dimensional cell culture models
Author(s): P. H. Lakner; Y. Möller; M. A. Olayioye; S. Y. Brucker; K. Schenke-Layland; M. G. Monaghan
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Paper Abstract

Fluorescence lifetime imaging microscopy (FLIM) is a useful approach to obtain information regarding the endogenous fluorophores present in biological samples. The concise evaluation of FLIM data requires the use of robust mathematical algorithms. In this study, we developed a user-friendly phasor approach for analyzing FLIM data and applied this method on three-dimensional (3D) Caco-2 models of polarized epithelial luminal cysts in a supporting extracellular matrix environment. These Caco-2 based models were treated with epidermal growth factor (EGF), to stimulate proliferation in order to determine if FLIM could detect such a change in cell behavior. Autofluorescence from nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in luminal Caco-2 cysts was stimulated by 2-photon laser excitation. Using a phasor approach, the lifetimes of involved fluorophores and their contribution were calculated with fewer initial assumptions when compared to multiexponential decay fitting. The phasor approach simplified FLIM data analysis, making it an interesting tool for non-experts in numerical data analysis. We observed that an increased proliferation stimulated by EGF led to a significant shift in fluorescence lifetime and a significant alteration of the phasor data shape. Our data demonstrates that multiphoton FLIM analysis with the phasor approach is a suitable method for the non-invasive analysis of 3D in vitro cell culture models qualifying this method for monitoring basic cellular features and the effect of external factors.

Paper Details

Date Published: 14 March 2016
PDF: 10 pages
Proc. SPIE 9712, Multiphoton Microscopy in the Biomedical Sciences XVI, 97120X (14 March 2016); doi: 10.1117/12.2220048
Show Author Affiliations
P. H. Lakner, Research Institute for Women's Health, Eberhard Karls Univ. Tübingen (Germany)
Y. Möller, Institute of Cell Biology and Immunology, Univ. of Stuttgart (Germany)
M. A. Olayioye, Institute of Cell Biology and Immunology, Univ. of Stuttgart (Germany)
S. Y. Brucker, Research Institute for Women's Health, Eberhard Karls Univ. Tübingen (Germany)
K. Schenke-Layland, Research Institute for Women's Health, Eberhard Karls Univ. Tübingen (Germany)
Fraunhofer Institute for Interfacial Engineering and Biotechnology (Germany)
M. G. Monaghan, Research Institute for Women's Health, Eberhard Karls Univ. Tübingen (Germany)


Published in SPIE Proceedings Vol. 9712:
Multiphoton Microscopy in the Biomedical Sciences XVI
Ammasi Periasamy; Peter T. C. So; Karsten König, Editor(s)

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