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TIRF as a method for s ingle particle tracking

TIRF as a method for s ingle particle tracking. Tutors : Emmanuel Margeat Patrice Rassam Centre de Biochimie Structurale Montpellier, France. Students : Eva Wegel Department of Genetics University of Cambridge, UK Małgorzata Lichocka

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TIRF as a method for s ingle particle tracking

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  1. TIRF as a method for single particle tracking Tutors: Emmanuel Margeat Patrice Rassam Centre de Biochimie Structurale Montpellier, France Students: Eva Wegel Department of Genetics University of Cambridge, UK Małgorzata Lichocka Laboratory of Plant Pathogenesis IBB PAS Warsaw, Poland Our experiment: Membrane dynamics of atransmembrane protein – tetraspanin CD9 Comparison between whole IgG and Fab fragment labelling

  2. 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

  3. 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:

  4. PaTrack cbs SINGLE MOLECULE TRACKING Frame rate 100ms 5µm Detect the position of each fluorescent spot

  5. Today’s results: IgG induces a reduction of the diffusion coefficient, and an increase in the number of confined trajectories

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