Lambert Instruments

FRET can be measured in several ways, like the ratiometric method FRET and the FLIM-FRET method. Both methods measure the FRET efficiency, i.e. the efficiency in which the donor fluorophores transfer their energy non-radiatively to the acceptor fluorophores that can be excited by this transferred energy.

FLIM-FRET method
The FLIM-FRET method measures the change in lifetime of the donor fluorophore after FRET and is explained in the section measurement methods FLIM.

Ratiometric method
The ratiometric method measures the change in emission intensities from donor and acceptor fluorophores. During FRET, the amount of emitted photons (emission intensity) from the donor fluorophore decreases and the emission intensity from the acceptor fluorophore increases. The FRET efficiency is basically calculated from the ratio of emission intensities from donor and acceptor before and after FRET occurrence.
To obtain accurate FRET data analyses by the ratiometric method, 7 images are required that have to be taken with appropriate filter cubes. This is because of bleed-through problems: the donor fluorophore will also emit some photons in the wavelength range that is detected as acceptor emission [2], and the acceptor will be excited slightly by the excitation wavelength of the donor [3].

	FLUOROPHORE:	EXCITATION FILTER BP FOR:	EMISSION FILTER BP FOR:
[1]	D	D	D
[2]	D	D	A
[3]	A	D	A
[4]	A	A	A
[5]	D + A	D	D
[6]	D + A	D	A
[7]	D + A	A	A

D = donor fluorophore. A = acceptor fluorophore. BP = band pass.

Disadvantage ratiometric method FRET
Corrections are essential because of bleed-through, so there is a complex data acquisition required (at least 7 images have to be taken). Furthermore, ratiometric measurements are dependent on the intensities, and so on the local concentration of fluorophores, the optical path of the microscope, the local excitation light intensity, and the local fluorescence detection efficiency.