r0ForFRET

Written by Peter Nagy.
Web: peternagy.webs.com
Email: [email protected]

The program calculates the overlap integral and the R0 value for a given donor-acceptor FRET pair. The R0 value gives the distance at which the FRET efficiency is 50% for a given donor-acceptor pair, and it can be calculated according to the following equation:

where ΦD is the fluorescence quantum yield of the donor in the absence of the acceptor (i.e. in the absence of FRET);

n is the index of refraction of the medium in which FRET takes place;

κ2 is the orientation factor;

J is the overlap integral characterizing the overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, and it can be calculated according to the following equation:

where FD is the normalized emission spectrum of the donor. Normalization means that the area under the curve of the emission spectrum is normalized to 1.

fD is the unnormalized emission spectrum of the donor.

εA is the molar absorption coefficient of the acceptor;

λ is the wavelength.

The constant in the equation defining R0 depends on the unit of the wavelength scale in the overlap integral. If the constant is 0.021, the unit of the wavelength scale in the overlap integral must be nm, and R0 will also be calculated in nm.

Syntax for the GUI mode: just type "r0ForFret" at the command prompt.

By hovering over the input fields tooltips are displayed about what kind of input is required.

Emission spectrum of donor: a variable storing the emission spectrum of the donor in a 1-D array. The spectrum does not have to be normalized since the program performs normalization.

Absorption spectrum of acceptor: a variable storing the absorption spectrum of the acceptor in a 1-D array. If the absorption spectrum is normalized (typically to a maximum value of 1 or 100), the "Normalized absorption spectrum of acceptor" check box must be checked. If the absorption spectrum is normalized, the "λ [nm] where ε is given" and the "ε" fields are activated and must be filled in.

Normalized absorption spectrum of acceptor: the check box must be checked if the spectrum does not contain molar absorption coefficients, but normalized absorption values. This is usually the case for spectra available on the internet.

λ [nm] where ε is given: only available if "Normalized absorption spectrum of acceptor" is checked. A numeric value giving the wavelength where the molar absorption coefficient is given in the "ε" field.

ε: only available if "Normalized absorption spectrum of acceptor" is checked. A numeric value giving the molar absorption coefficient at the wavelength given in the "λ [nm] where ε is given" field.

Wavelength scale for donor [nm]: a variable (1-D array) containing the wavelength scale of the "Emission spectrum of donor". The unit of the wavelength scale has to be nm.

Wavelength scale for acceptor [nm]: a variable (1-D array) containing the wavelength scale of the "Absortption spectrum of acceptor". The unit of the wavelength scale has to be nm.

Index of refraction (n): a numeric value giving the index of refraction of the medium in which FRET takes place. The index of refraction of water is 1.33. But for biological experiments an index of refraction of 1.4 is typically used.

κ2: a numeric value which gives the orientation factor defining the relative orientation of the donor and the acceptor. In many cases the donor and the acceptor are assumed to rotate rapidly during the excited state lifetime of the donor sampling all possible orientations of the donor and the acceptor. This case is called dynamic averaging. If the assumption of dynamic averaging holds, a value of 2/3 can be used for κ2.

Quantum yield of donor in the absence of acceptor: a numeric value giving the fluorescence quantum yield of the donor in the absence of the acceptor.

Calculations are performed upon pressing the "Calculate" button.

The calculated overlap integral and R0 are displayed in the blue fields designated "Overlap integral (J)" and "R0 [nm]", respectively.

If you would like to export the calculated overlap integral and R0 to the Matlab workspace upon pressing the "Return" button, specify variables in the yellow fields designated "Variable for J" and "Variable for R0".

Optionally, you can run the program in non-GUI (command prompt) mode if you provide input parameters. Help about this syntax is available by typing "help r0ForFret" at the command prompt.