Script requirements

MATLAB Compiler Routine (MCR) 2014a (downloaded during the installation)
Minimum configuration of a PC: Windows XP or higher, 1 GHz processor, 512 MB RAM

Input format

ImageJ/MOSAIC plugin – plain text file with x,y,t coordinates of a particle
ICY/Spot Track plugin – XML file with x,y,t coordinates of a particle

Input parameters in the configuration file

video framerate (fps): the framerate of your video sequence
particle radius (µm): radius of particles used
pixel size (µm): the reslolution of your imaging system
minimum trajectory length: minimal number of consecutive video frames covered by trajectory.
minimum correlation coefficient: The square correlation coefficient in the linear regression. It allows to exclude MSD curves where the linear regression does not explain enough of experimental data points. E.g. the default value 0.93 (square root 0.96) indicates that 96% of experimental data points in a MSD curve are explained with the regression line.
maximum 3-σ test iterations: how many consecutive steps of 3-sigma tests are applied to exclude outliers.

Click here for an example of a configuration file

Workflow

• Reading configuration file
• Reading the data
• Application of the criterion of the minimum trajectory length in order to omit short trajectories.
• MSD curves are calculated for individual trajectories. Linear approximation is applied to each individual MSD curve and the slope and correlation coefficient are determined by means of linear regression statistics. The diffusion coefficient is calculated as 1/4 of the MSD slope. MSD curves which show a too low correlation coefficient are excluded.
• The distribution of the remaining diffusion coefficients is displayed and the operator may consider the iterative application of the 3-σ rule to exclude outlying data and/or changing the range manually. The latter allows the user to focus on a narrow subpopulation of diffusion coefficients.
• The viscosity is calculated out of the mean diffusion coefficient using the Stokes – Einstein formula for 2D space:
  
  where η [Pa s] is the dynamic viscosity, k_B [1.38065 m² kg s^-2 K^-1] is the Boltzmann constant, T [K] is thermodynamic temperature, r [m] is particle radius, and D [m²s^-1] is the diffusion coefficient.
• The value of the dynamic viscosity is displayed together with SD and a protocol showing the step-by-step processing of data. The protocol also contains information about the excluded trajectories.