You can download the RET bio-lab simulation package from here: fretlab.pxp

Read the following introductions to learn about the usage and functionalities of the program:






README file
for FRETlab program

Author: Jan Junis Rindermann
Version: 1.1
Date: Dec 2010
Contact: junis.rindermann (at) soton.ac.uk
http://www.hybrid.soton.ac.uk/research/fret-lab


In our recent paper (J.J. Rindermann et al., "Gauging the flexibility of fluorescent markers for the interpretation of fluorescence resonance energy transfer"
J. Am. Chem. Soc. 133 (2), 279–285 (2011)) we have introduced a methodology to study the effect of fluctuations in the orientation of the dipole moment in fluorescent molecules on the energy transfer process between them.

This program gives an example for the implementation of angular fluctuations in the simulation of fluorescence resonance energy transfer (FRET) between two dipoles.

0. You need to install the Igor Pro software from Wavemetrics to open the file fretlab.pxp and run the program. More information and a free test version of the program is available on the Wavemetrics website: www.wavemetrics.com
This file requires version 6.2 of Igor Pro.

1. After you have opened the file fretlab.pxp with Igor Pro you will see this README file.

2. The Menu bar of the Igor Pro Window contains an item named "FRETlab". Choose this Menu and click on "Initialize user interface - 1min"

3. You will see how a window with some graphs builds up and the program will calculate a fluorescence decay curve for a donor with an acceptor in its vicinity. The default parameters are used and the data plotted is only sample data. This can take up to a minute on a slow computer, and only a few seconds on a standard desktop computer.

4. Make yourself familiar with the user interface and try changing some parameters. After changing the FRET parameters you have to click on "Calculate" to update the graph containing the simulated data.

5. What the program does: it calculates the fluorescence decay curve of a donor molecule which has an acceptor molecule in its vicinity. FRET is mediated via the dipole moments of the molecules. The dipole moments are in general not fixed in space but fluctuate due to movement of the molecule at room temperature under physiological conditions. Therefore the orientation factor and the FRET rate changes. The fluctuation of the dipole direction is described with a parameter sigma, which is the vector analogue to the width sigma of a gaussian distribution. Here the fluctuations are assumed to be slower than the ET process (static averaging regime). The dynamic averaging regime will be added in the next verison.

6. In order to use this program with your own data, load your data in the format of the example data into the Igor session. See the manual if you are not familiar with Igor Pro. Your data should be background corrected.

7. Parameters:
Numerical parameters:
Time window of interest:
Choose for how long after time zero the fluorescence decay data is analyzed.
Number of Sampling points:
The experimental data has far too many data points to calculate the simulated fluorescence decay for each point. Therefore only a number of points is picked out, starting with the point at t=0.
Number of points for box average:
When the representative points are picked from the experimental data, you can choose to take an average over the neighbouring points.

FRET parameters:
D-A distance:
Distance between donor and acceptor dipole in nm.

Förster Radius:
Distance at which the efficiency of FRET drops to 50%, assuming the orientation factor is kappa^2=2/3. The Förster radius is given in nm, and has to be calculated by the user or locked up in the literature. 
See Lakowicz, J. R. Principles of Fluorescence Spectroscopy. Springer, New York, USA, 392-393 (2006) for instructions on how to calculate the Föster radius.

Donor decay rate:
Decay rate of the donor in absence of the acceptor without FRET. The rate is the inverse of the lifetime of the donor and is given in units of per second.

sigmaD and sigmaA:
These are the width parameters of the Fisher-von Mises distributions describing the angular spread in the dipole distributions for the donor and acceptor dipole. The input is in degree. (The relation to the concentration factor c is: c=1/sigma^2, where sigma is given in radians.)

Spherical coordinates to describe the average dipole orientations:
The center vector c_D and c_A of the Fisher-von Mises distributions describing the donor and acceptor dipole fluctuations are defined in spherical coordinates. In these coordinate systems S_D and S_A the z-axes point along the vector from the donor to the acceptor. The x axes are parallel and point in an arbitrary direction. 
ThetaD and ThetaA are the angles between the z-axis and c_D and c_A respectively. Thus, Theta is the inclination angle in respect to the z-axis as zenith direction. 
PhiD (PhiA) is the angle between the projection of c_D (c_A) on the x-y plane and the x axis. Thus, Phi is the azimuth angle in respect to the x axis as azimuth direction.

The window "Mean Dipole Orientation" shows the relative dipole orientation for the mean directions from different perspectives. It also shows the orientation factor for the these two dipoles and the average in the ensemble.
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