R(0)-Values (FRET)

An updated table of the critical distance R₀ of energy transfer (FRET) between ATTO-dyes is now available for download.
 

R(0)-Values.pdf  (2018/11/13)

This table should help you to select a suitable dye pair for your FRET-application. The R₀-values in this table have been calculated according to the theory developed by Förster using spectral data obtained on dilute aqueous dye solutions. Random orientation of the dye molecules was assumed (κ² = 2/3), and a refractive index of 1.333 (water) has been used.

In the theory given by Förster dye molecules (i.e. the oscillators responsible for absorption and emission) are assumed to be so-called point-dipoles, i.e. they are small compared to the distance between the molecules. However, in reality the diameter of a typical dye chromophore is 10 – 15 Å, which compared to typical R₀-values of 50 – 70 Å is not small at all.

The assumed statistical orientation of energy-donor and acceptor is realized for dyes in solution. However, in dye conjugates (with proteins, DNA …) the relative orientation of donor and acceptor may deviate significantly from the statistical distribution. In extreme cases the orientation factor κ² can attain the values of 0 or 4. As a result the R₀-value can be zero or by a factor [4/(2/3)]^1/6 = 1.35 higher than that given in the table. In practice any value in between may be correct – depending on the particular orientation of donor and acceptor.

The refractive index of 1.333 used in the calculations is valid for ”normal” solutions of the dye in water. However, – due to the fact that proteins, DNA etc. have a higher polarizability – for FRET on dye conjugates a slightly higher value for the refractive index would be appropriate. As a result R₀ would be smaller depending on the particular case.

When calculating R₀-values one should be aware of the fact that the precision of fluorescence data (fluorescence quantum yield, fluorescence spectrum) is usually lower than commonly assumed. In addition all R₀-values given are calculated using the optical data of the dyes with free carboxy group. However, the absorption and emission properties might change once a dye is bound to a biomolecule, e.g. a protein. In particular the fluorescence quantum yield can be drastically reduced compared to the unconjugated dye. Taking all facts into account one hardly can expect an exact match of calculated R₀-values with those determined experimentally. – The values given in the table are rounded to full Å.