TIRF as a method for single particle tracking

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Presentation transcript:

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 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 Integration time: 100 ms. Detect the position of fluorescence spot and a Gaussian fit is applied to determine the center of each spot (20-30 nm accuracy) frame per frame. The Levenberg-Marquardt (LM) algorithm is an iterative technique that locates the minimum of a multivariate function that is expressed as the sum of squares of non-linear real-valued functions. Localization of the PSF (Point spread function) of the signal of each particle which can modeled as a 2D gaussian. Then you can fit this gaussian and get a lateral precision of 30 to 40nm and even less depending on your S/N ratio. The point spread function (PSF) describes the response of an imaging system to a point source or point object. A related but more general term for the PSF is a system's impulse response. 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