High throughput FRET screening of the plasma membrane based on TIRFM.
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.
Paar
Christian
C
Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, Linz, Austria.
Paster
Wolfgang
W
Stockinger
Hannes
H
Schütz
Gerhard J
GJ
Sonnleitner
Max
M
Sonnleitner
Alois
A
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
D007473:Ion Channels; D011956:Receptors, Cell Surface