1. Preliminary results In vitro expression of membrane proteins Vectorial incorporation of expressed proteins into cell membranes Protein diffusion not observed within minutes time scale Protein density increases with expression time First complete incorporations measurable after  10’ Broad distribution of local protein densities (clusters) Photo-bleaching of fluorescent antibody identified as major limitation of single molecule imaging ConclusionsOutlook + Excitation confined to the surface Optimize image correlation analysis + Analysis of CCD image series Increase robustness of the analysis – Non-uniform excitation intensity, photo-bleaching Test functionality of odorant receptors – CCD readout noise, artefacts Investigate ligand-receptor interactions Total internal reflection fluorescence imaging Spatial image correlation Background B Excitation E Intensity I = (I raw – B)/E Fluorescence images of odorant receptors C-terminal VSV tag (negative control) 60’ no antibody binding N-terminal VSV tag 15’ 30’60’ 90’ few spots more, brightermany, brightclusters References:1D. Lossner et al. (2006) Anal. Chem. 78: 4524–4533.3N. O. Petersen et al. (1998) Faraday Discuss. 111: 289–305. 2B. Wiltschi, W. Knoll, E.K. Sinner (2006) Methods 39: 134–146.4P. W. Wiseman, N. O. Petersen (1999) Biophys. J. 76: 963–977. Introduction Odorant receptors are an excellent example of natural superiority in binding specific small and hydrophobic molecules. Difficulties in expression, isolation and solubilisation of these receptors have been overcome recently by in vitro synthesis and incorporation of receptors into a planar lipid membrane [1,2]. The incorporation density and the mobility of the membrane embedded receptors were analyzed at the single-molecule level by means of image correlation spectroscopy [3,4]. Instead of a confocal scanning microscope for the image acquisition, we used our total internal reflection fluorescence microscope and an ultra-sensitive CCD camera. The non-uniform excitation intensity requested a modification of the image correlation analysis, i.e. a normalization of the image intensity with the excitation intensity distribution. Contact:Marcel Leutenegger, marcel.leutenegger@epfl.ch, Tel.: +41 21 693 78 21 / 77 19 Acknowledgements We gratefully acknowledge the Swiss National Science Foundation (SNSF) for financial support, André Galliker at Lot-Oriel for support with the ultra-sensitive CCD camera and Samuel Terrettaz at EPFL for his generous help in preparing gold coatings. Single molecule imaging of odorant receptors in supported planar lipid membranes M. Leutenegger 1, R. Robelek 2, E. K. Sinner 2, T. Lasser 1 1 Laboratoire d‘Optique Biomédicale, École Polytechnique Fédérale de Lausanne, Switzerland, http://lob.epfl.ch/ 2 Max-Planck Insitut für Polymerforschung, Mainz, Germany, http://www.mpip-mainz.mpg.de/ 10  m Cy5 / Antibody VSV tag Membrane Peptide linker Microscope slide Receptor   10  m G(x,y)G(x,y) 1.6 1.5 1.4 1.3 1.2 1.1 1.0 100 10 1 100 10 1 100  x [100nm]  y [100nm] Noise peak Fit Protein density [1/  m 2 ] ??? Fluorescence intensity [a.u.] SNR < 0.1