Gunnarsson, A., Bally, M., Jönsson, P., Médard, N., & Höök, F
Department of Applied Physics, Division of Biological Physics, Chalmers University of Technology, SE-412 96 Göteborg, Sweden ‡Department of Chemistry and Materials Technology, SP Technical Research Institute of Sweden, SE-501 15 Borås, Sweden §Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, U.K. ∥Nanolane, Parc des Sittelles, F-72450 Montfort-le-Gesnois, France.
FRAP/FLIP and TIRF
We have applied surface-enhanced ellipsometry contrast (SEEC) imaging for time-resolved label-free visualization of biomolecular recognition events on spatially heterogeneous supported lipid bilayers (SLB). Using a conventional inverted microscope equipped with total internal reflection (TIR) illumination, biomolecular binding events were monitored with a lateral resolution near the optical diffraction limit at an acquisition rate of ~1 Hz with a sensitivity in terms of surface coverage of ~1 ng/cm(2). Despite the significant improvement in spatial resolution compared to alternative label-free surface-based imaging technologies, the sensitivity remains competitive with surface plasmon resonance (SPR) imaging and imaging ellipsometry. The potential of the technique to discriminate local differences in protein binding kinetics was demonstrated by time-resolved imaging of anti-GalCer antibodies binding to phase-separated lipid bilayers consisting of phosphatidylcholine (POPC) and galactosylceramide (GalCer). A higher antibody binding capacity was observed on the GalCer-diluted fluid region in comparison to the GalCer-rich gel phase domains. This observation is tentatively attributed to differences in the presentation of the GalCer epitope in the two phases, resulting in differences in availability of the ligand for antibody binding. The complementary information obtained by swiftly switching between SEEC and fluorescence (including TIR fluorescence) imaging modes was used to support the data interpretation. The simplicity and generic applicability of the concept is discussed in terms of microfluidic applications.
… “The samples are illuminated with a fiber-coupled illumination source (pE1, CoolLED Ltd., U.K.), with a wavelength of 525 nm, close to the optimal settings (550 nm) for the SEEC substrate”…
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pE-2: A repeatable, controllable modular system with 20 different LED peaks. Instant on/off and intensity (0-100%) control
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