FRET answers

Question 1

what does FRET stand for

Answer 1

fluorescence Resonance Energy Transfer

Question 2

what is the principle of FRET (4)

Answer 2

1) transfer of excitation energy from a donor fluorophore to an acceptor fluorophore done
2) through a non radiative process
3) exchange done through long-range dipole-dipole interactions
4) the distance bewteen the flurophores should not exceed 10nm

Question 3

key factors of FRET

Answer 3

1) can only occur between 1-10 nm in distance
2) For FRET to happen efficiently the donor fluorescence emission spectrum must overlap with the acceptor excitation spectrum
3) when the distance between the donor and acceptor is equal to the Forster radius, the energy transfer efficiency is 50%

Question 4

distances below 1nm

Answer 4

collisions become dominant and FRET cannot occur

Question 5

distances above 10 nm

Answer 5

the donor molecule is too far away from the acceptor molecule to transefer the energy and therefore is likely to emit the energy

Question 6

donor molecules have a spectrum

Answer 6

at which the energy can be emitted

Question 7

acceptor molecules can

Answer 7

absorb light only at specific wavelengths

Question 8

50% of the excitation energy of the donor is transferred

Answer 8

to the acceptor and the other 50% is emitted

Question 9

Limitations of FRET (5)

Answer 9

1) fluorescent labels, used as the donor and acceptor, are sensitive to environmental factors such as changes in pH, ionic concentrations oxidation, temp, and refractive index (skewed measurements if environment isn’t optimal(2)
3) Low signal to noise ratio can effect FRET reading (weak signals are hard to distinguish(4))
5) The loss of energy associated with FRET process when transferring energy from donor to acceptor can result in weaker FRET signals affecting the accuracy of measurements