Time-resolved surface-enhanced ellipsometric contrast imaging for label-free analysis of biomolecular recognition reactions on glycolipid domains.
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.
Department of Applied Physics, Division of Biological Physics, Chalmers University of Technology, SE-412 96 Göteborg, Sweden. anders.gunnarsson@chalmers.se
Anders
A
Gunnarsson
Marta
M
Bally
Peter
P
Jönsson
Nicolas
N
Médard
Fredrik
F
Höök
D000906:Antibodies / Q000276:immunology; D036681:Fluorescence Recovery After Photobleaching; D005699:Galactosylceramides / Q000737:chemistry / Q000276:immunology; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry*; D046210:Microfluidic Analytical Techniques; D008853:Microscopy; D010713:Phosphatidylcholines / Q000737:chemistry; D011485:Protein Binding; D013499:Surface Properties; D013997:Time Factors
D000906:Antibodies; D005699:Galactosylceramides; D008051:Lipid Bilayers; D010713:Phosphatidylcholines