title: High throughput FRET screening of the plasma membrane based on TIRFM.
abstract: Monitoring protein function with high throughput at individual cell level is of high interest both for basic research and diagnostic applications. For this, following the changes in fluorescence resonance energy transfer (FRET) between a donor/acceptor pair, genetically encoded in the proteins of interest, is a frequently used tool. As proteins attached to or located in the plasma membrane represent a considerable fraction of total proteins, there is a need for high throughput imaging techniques suited for observation of proteins in the cell membrane only. A system is presented, which allows rapid imaging of large areas via total internal reflection fluorescence microscopy (TIRFM) conditions, using a focus-hold system, multiwavelength excitation and dual color detection. The developed imaging system enables screening of large numbers of cells under TIRFM illumination combined with FRET imaging, thereby providing the means to record, e.g., FRET-efficiency of a membrane-associated protein labeled with a donor-acceptor pair. The capability of the system to perform live-FRET scanning with TIRFM on stoichiometric FRET constructs, reaching throughput of up to 1,000 cells/s at the optical resolution limit is demonstrated. A comparison with confocal microscopy shows that TIRFM offers a 4.2-fold advantage in our conditions over confocal microscopy in detecting contributions from membrane-localized proteins.
authors.affiliation: Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, Linz, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Paar
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Paster
authors.forename: Hannes
authors.initials: H
authors.lastname: Stockinger
authors.forename: Gerhard J
authors.initials: GJ
authors.lastname: Schütz
authors.forename: Max
authors.initials: M
authors.lastname: Sonnleitner
authors.forename: Alois
authors.initials: A
authors.lastname: Sonnleitner
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D007473:Ion Channels / Q000378:metabolism; D019169:Jurkat Cells; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D011956:Receptors, Cell Surface / Q000378:metabolism
chemical_list: D007473:Ion Channels; D011956:Receptors, Cell Surface