Combining TIR and FRET: From Fluorescence Microscopy to a Multi-well Reader System

Peter Lanzerstorfer (Speaker), Weghuber, J. (Speaker), Herbert Schneckenburger (Speaker), Petra Weber (Speaker), Michael Wagner (Speaker)

Activity: Talk or presentationOral presentation

Description

Pharmaceutical agents or drugs often have some pronounced impact on protein-protein interactions in cells and, in particular, cell membranes. Changes of molecular conformations as well as of intermolecular interactions may affect dipole-dipole interaction between chromophoric groups, which can be proven by measurement of Förster Resonance Energy Transfer (FRET). If these chromophores are located within or in close proximity to the plasma membrane, they are excited preferentially by an evanescent electromagnetic wave upon Total Internal Reflection (TIR) of an incident laser beam. For TIR-FRET screening of larger cell collectives we set up a test system for probing the interaction between the Epidermal Growth Factor Receptor (EGFR) and the Growth factor receptor-bound protein 2 (Grb2) prior and subsequent to stimulation with EGF. After fusion of EGFR with Cyan Fluorescent Protein (CFP) and Grb2 with Yellow Fluorescent Protein (YFP) we measured FRET by spectral analysis and Fluorescence Lifetime Imaging (FLIM) in a fluorescent microscope as well as in a multi-well TIR fluorescence reader with simultaneous detection of a larger number of samples. The latter system appears appropriate for high content (HCS) or even high throughput screening (HTS), however, the lack of visual control and sub-nanosecond time shifts between optical excitation of individual wells are still limiting factors for application of this method. For validation of the reader system a HeLa cell line expressing a membrane associated Green Fluorescent Protein (HeLa hFR-GPI-GFP) was used together with the energy acceptor Nile Red.
Period24 Jun 2019
Event titleEuropean Conferences on Biomedical Optics: null
Event typeConference
LocationMunich, Germany