1 March 2009 Förster resonance energy transfer-based total internal reflection fluorescence reader for apoptosis
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Abstract
A fluorescence reader for the detection of Förster resonance energy transfer (FRET) on surfaces of living cells is described. The method is based on multiple total internal reflections (TIR) of an incident laser beam within a glass slide, such that individual samples on top of the glass slide are illuminated simultaneously by an evanescent electromagnetic field. Enhanced cyan fluorescent protein (ECFP) anchored to the inner leaflet of the plasma membrane is optically excited and transfers its excitation energy via the peptide linker Asp-Glu-Val-Asp (DEVD) to an enhanced yellow fluorescent protein. Upon apoptosis, DEVD is cleaved, and energy transfer is disrupted, as proven by an increase of fluorescence intensity as well as of fluorescence lifetime of the donor ECFP. Due to selective excitation of membrane-associated fluorophores, intracellular fluorescence and background luminescence from the surrounding medium are eliminated. Therefore, this test system appears to be a sensitive device for the detection of apoptosis and more generally for drug screening or in vitro diagnosis on a nanometer scale.
© (2009) Society of Photo-Optical Instrumentation Engineers (SPIE)
Thomas Bruns, Brigitte Angres, Heiko Steuer, Petra Weber, Michael Wagner, Herbert Schneckenburger, "Förster resonance energy transfer-based total internal reflection fluorescence reader for apoptosis," Journal of Biomedical Optics 14(2), 021003 (1 March 2009). https://doi.org/10.1117/1.3055622 . Submission:
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