title: Monitoring of apoptosis in 3D cell cultures by FRET and light sheet fluorescence microscopy.
abstract: Non-radiative cell membrane associated Förster Resonance Energy Transfer (FRET) from an enhanced cyan fluorescent protein (ECFP) to an enhanced yellow fluorescent protein (EYFP) is used for detection of apoptosis in 3-dimensional cell cultures. FRET is visualized in multi-cellular tumor spheroids by light sheet based fluorescence microscopy in combination with microspectral analysis and fluorescence lifetime imaging (FLIM). Upon application of staurosporine and to some extent after treatment with phorbol-12-myristate-13-acetate (PMA), a specific activator of protein kinase c, the caspase-3 sensitive peptide linker DEVD is cleaved. This results in a reduction of acceptor (EYFP) fluorescence as well as a prolongation of the fluorescence lifetime of the donor (ECFP). Fluorescence spectra and lifetimes may, therefore, be used for monitoring of apoptosis in a realistic 3-dimensional system, while light sheet based microscopy appears appropriate for 3D imaging at low light exposure.
authors.affiliation: Institute of Applied Research, Aalen University, Anton-Huber Str. 21, 73430 Aalen, Germany. petra.weber@htw-aalen.de.
authors.forename: Petra
authors.initials: P
authors.lastname: Weber
authors.affiliation: Institute of Applied Research, Aalen University, Anton-Huber Str. 21, 73430 Aalen, Germany. sarah.schickinger@htw-aalen.de.
authors.forename: Sarah
authors.initials: S
authors.lastname: Schickinger
authors.affiliation: Institute of Applied Research, Aalen University, Anton-Huber Str. 21, 73430 Aalen, Germany. michael.wagner@htw-aalen.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Wagner
authors.affiliation: Cellendes GmbH, Markwiesenstr. 55, 72770 Reutlingen, Germany. brigitte.angres@cellendes.com.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Angres
authors.affiliation: Institute of Applied Research, Aalen University, Anton-Huber Str. 21, 73430 Aalen, Germany. thomas.bruns@htw-aalen.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bruns
authors.affiliation: Institute of Applied Research, Aalen University, Anton-Huber Str. 21, 73430 Aalen, Germany. herbert.schneckenburger@htw-aalen.de.
authors.forename: Herbert
authors.initials: H
authors.lastname: Schneckenburger
mesh_terms: D017209:Apoptosis; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018874:Spheroids, Cellular / Q000378:metabolism / Q000502:physiology
chemical_list: D049452:Green Fluorescent Proteins; D053148:Caspase 3