I have a system with two fluorophores. when I measure the fluorescence I observe an increase in relation to the light absorved. This suggests the existence of an energy transfer that could be FRET, but, how can I determine exactly if it is (or not) FRET?
Here is one suggestion. FRET is strongly distance-dependent. It will not occur between molecules that are free in solution. So you can tell if FRET is occurring by comparing the fluorescence intensities of the system when both molecules are in the complex versus when one or both of them is present at the same concentration but free in solution (assuming no difference in quantum yield between bound and free fluorophore).
You can also measure the FRET efficiency by comparing the sum of the fluorescence intensities of the system with each one of the fluorophores omitted to that of the whole system, keeping the excitation and emission wavelengths the same in every case.
You can look at the absorption spectra. If the emission spectrum of one system has overlap with the absorption spectrum, FRET is likely be there. However for confirming FRET you need to take decay data. You can get details in the book by Lackowicz (Principles of Fluorescence spectroscopy). Intensity changes are also likely to occur in case of radiative transfer if there is overlap between the emission of one system and absorption of other. In such case the emitted photons of one system are directly absorbed by other.
Note that there are many situations in which fluorescence of one of the dyes changes, but not necessarily due to FRET (e.g. quenching of different sorts, photochemical and photophysical properties). True, you can measure intensities. If you do so, look for an anti-correlated change in fluorescence, whereby if the donor fluorescence intensity decreased, the acceptor's increased (and vice versa). Still, in some cases (intermolecular FRET of binding for instance), sometimes this anti-correlated change is not indicative (e.g. when you measure FRET between two interacting proteins, and when they bind, the close proximity of the proteins affect the photophysical properties of one of the dyes).
Another way to measure FRET is to compare the donor fluorescence from two samples, one with the presence of an acceptor (DA; hence susceptible for FRET) and one in the absence of an acceptor (DO) and then calculate:
E = 1 - F_DA/F_DO
where F_DA and F_DO are the donor fluorescence intensities, in the presence or absence of an acceptor, respectively.
BUT, this approach is also not advisable, since fluorescence intensity of an ensemble of molecules strongly depends on their concentration,which may have sample to sample variations. Fluorescence lifetimes, however, report on fluorescence independently of concentration. Performing donor fluorescence decay measurements (using e.g. TCSPC) in the absence (DO) and in the presence (DA) of an acceptor will yield:
E = 1 - tau_DA/tau_DO
where, the tau's are the (mean) fluorescence lifetimes derived from the decays.
Still, this requires the measurement of two unsynchronized measurements. There are many ways to retrieve FRET from one measurement. One way involved measuring the fluorescence intensity of both the donor and the acceptor (and anticipate an anti-correlated trend between the two) focusing at single molecules, using single molecule FRET (smFRET). In these experiments you are able to retrieve not only FRET but also, how many different types of FRET sub-populations your system has, whether other photophysical processes are involved in fluorescence change, as well as get donor-only information, following the photobleaching of the acceptor.
Another simple method is to measure the excitation spectrum of the "acceptor" in the presence and absence of the "donor". If there really is FRET then the excitation spectrum of the "acceptor" will change. For this type of analysis even uncorrected excitation spectra will suffice.
A number of phenomena can cause in the quenching of the donor fluorescence. If you are using a time resolved setup, the check the fluorescence lifetime of the acceptor molecule by exciting the donor molecule selectively. You will find a rise component in fluorescence lifetime profile if the process is FRET. It is a convenient way and you will find plenty of reports.
If your fluorophores are reported as FRET pairs, they will undergo FRET if they reside close to each other (not more that 100 Angstorm). If you are not sure of your dyes as FRET pairs, still you can test it. To act as FRET pair, donor emission spectrum and acceptor absorption spectrum must overlap. If you are sure that your dye pairs may act as FRET pairs, you can do the following-
Take emission spectra with only donor and donor+acceptor (in both cases concentration of donor should be same). If FRET occurs, donor intensity should decrease with concomitant increase in acceptor intensity. Hope this helps.
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