Will FRET be effected by polar solvent ? Suppose fluorophore is non-polar, if it is dissolved in polar protic solvent (like EtOH, MeOH) along with potential quencher. How will the FRET efficiency vary ?
Thanks
The Forster theory for FRET gives R0, the distance at which the efficiency of energy transfer is 50%, by the formula shown at this web page:
http://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook/technical-notes-and-product-highlights/fluorescence-resonance-energy-transfer-fret.html
There are several factors in this formula that are affected by the solvent, including the emission spectrum of the donor, the absorbance spectrum of the acceptor, the quantum yield of the donor, and the refractive index. All of these must be measured under the actual conditions of the experiment to get an accurate value of R0.
The Forster theory for FRET gives R0, the distance at which the efficiency of energy transfer is 50%, by the formula shown at this web page:
http://www.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook/technical-notes-and-product-highlights/fluorescence-resonance-energy-transfer-fret.html
There are several factors in this formula that are affected by the solvent, including the emission spectrum of the donor, the absorbance spectrum of the acceptor, the quantum yield of the donor, and the refractive index. All of these must be measured under the actual conditions of the experiment to get an accurate value of R0.
I agree with the answer by Shapiro. I would add that in most FRET analysis in cell biology the refractive index is completely ignored and as it is elevated to the 4th power this is a major mistake. In addition, and this is not mentioned by Shapiro, the dipole-dipole orientation is not adequately dealt with, and also this may be modified by the solvent.
The dipole-dipole orientation factor kappa-squared is generally assumed to be 2/3, which is based on assuming rapid rotation of the donor and acceptor relative to each other on the time scale of the excited state lifetime. There is no experimental method to measure the value, but it is possible to put upper and lower limits on it based on measurements of fluorescence polarization.
There is a way to calculate the kappa squared distribution by incorporation of your donor/acceptor dye pair to a rigid scaffold, thus making the distance between the dyes constant (except to their movements due to the rotational freedom of the linkers). For many years people looked for truly rigid scaffolds. Lately, researchers started to use the versatility and specificity of DNA origami scaffolds (for preparation details, see the works from Eyal Nir) which have been proven to be very rigid in 2D. You can design the DNA origami scaffold to already include the labeled oligos so that the positions of the donor and acceptor will be known from design, but the dyes will be still freely rotating owing their linkers.
Then you can measure FRET of the constant distance between the dyes, in different solvents. The methodology of measurement will be time-resolved FRET in bulk in which you will analyze the kappa-squared distribution out of the donor fluorescence decay - you have to measure a Donor-Only reference fluorescence decay.
The idea is that you already know the distance, it should be distributed around a mean distance with a very narrow distribution, so that whatever different FRET rates you identify, they will reflect the kappa squared distribution.
The base model to be fitted can be derived from Helmut Grubmuller's works which performed a lot of Molecular Dynamics works simulating FRET on true simple molecules.
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