Transcript FCS on Apertures

Single molecule imaging of odorant receptors
in supported planar lipid membranes
M. Leutenegger1, R. Robelek2, E. K. Sinner2, T. Lasser1
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/
1
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.
Spatial image correlation
Total internal reflection fluorescence imaging
Cy5 / Antibody
I ( x, y) I ( x  x, y  y)
 2 2
2 
G(x, y) 
 1  G0 exp  2 x  y 
I ( x, y) I ( x  x, y  y)
 w0

Receptor
VSV tag
Membrane
Background B
Peptide linker
Microscope slide
Noise peak
1.6
1.5
G(x,y)
Excitation E
Intensity I = (Iraw – B)/E
1.4
Fit
1.3
1.2
1.1
1.0
100
10
10mm
100
10
1
1
x [100nm]
10
10
100 100
6

60’
no antibody binding
5
4
3
2
1
0

60’
many, bright
500
???
30'
45'
60'
• Photo-bleaching of fluorescent antibody identified as major limitation of single
molecule imaging
90’
clusters
Conclusions
Outlook
+ 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
Acknowledgements
Contact:
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.
Marcel Leutenegger, [email protected], Tel.: +41 21 693 78 21 / 77 19
References:
0
90'
• 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)
10mm
30’
more, brighter
15' 22'
1000
• In vitro expression of membrane proteins
• Vectorial incorporation of expressed proteins into cell membranes
N-terminal VSV tag
15’
few spots
1500
SNR < 0.1
10mm
Protein density
[1/mm2]
C-terminal VSV tag (negative control)
2000
Fluorescence intensity
[a.u.]
Preliminary results
Fluorescence images of odorant receptors

y [100nm]
1
D. Lossner et al. (2006) Anal. Chem. 78: 4524–4533.
3
N. O. Petersen et al. (1998) Faraday Discuss. 111: 289–305.
2
B. Wiltschi, W. Knoll, E.K. Sinner (2006) Methods 39: 134–146.
4
P. W. Wiseman, N. O. Petersen (1999) Biophys. J. 76: 963–977.