Transcript Diapositive 1

TIRF as a method for single particle tracking
Tutors:
Students:
Emmanuel Margeat
Eva Wegel
Patrice Rassam
Department of Genetics
Centre de Biochimie
Structurale
Montpellier, France
University of Cambridge, UK
Małgorzata Lichocka
Laboratory of Plant Pathogenesis
IBB PAS Warsaw, Poland
Our experiment: Membrane dynamics of a transmembrane
protein – tetraspanin CD9
Comparison between whole IgG and Fab fragment labelling
Single particle (dye) tracking: principle and an example
- Label a cellular (membrane) component of interest
Dual labelling of tetraspanin CD9 in living cells:
- Cy3B ensemble label (nM)
- Atto647N single molecule label (pM)
either with Fab fragments or whole IgG
- Observation of the movements of the probes via video-microscopy
Wide-field microscope
Excitation at 560 nm and 633nm
Objective 100x 1.46 NA
EM CCD Camera
- Analyze trajectories of individual molecules:
- Diffusion coefficient
- Type of movement
Image analysis with PaTrack software:
• Analysis of the mean squared displacements curve allows to
distinguish between diffusion modes:
- directed motion
- brownian motion (free diffusion)
- confined diffusion
• Different experimental conditions: labelling with IgG or Fab
fragments – influence on the diffusion coefficient:
SINGLE MOLECULE TRACKING
Frame rate
100ms
5µm
PaTrack cbs
Detect the position of each fluorescent spot
Today’s results:
IgG induces a reduction of the diffusion coefficient, and an increase
in the number of confined trajectories