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 a transmembrane 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. 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

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