Take the absorption and emission spectra for both of the drugs separately.

Then overlap the absorption spectrum of the one drug with the emission of the another. If you find a reasonable overlap region between absorption of one and emission of another then you can claim them as FRET pair.

And if they do not overlap at all then they cannot act as FRET pair. Higher is the extent of overlap better is the FRET efficiency.

If you want to differentiate static from dynamic quenching you have to examine both the steady state as well as the time resolved fluorescence quenching. In the steady state fluorescnece measurement you will examine static + dynamic quenching, but in the time resolved experiment you will only measure the dynamic quenching.

The above answer from Sengupta established wether FRET is possible - as a resonant process the energies of the states involved need to be the same, which is usually established in the way he describes. Note that the chromophore with higher energy absorption is termed the donor, the other is the acceptor.

To tell if FRET is occurring you need to establish that energy is actually transferred from the donor to the acceptor. This involves observing the depopulation of the donor excited state while the acceptor state is populated. The methods listed below can all be used to accomplish this:

(1) Donor Quenching: While carefully ensuring that the solution concentrations are the same, you could measure the fluorescence of the donor alone as compared to the donor in the presence of the acceptor. The donor fluorescence should be quenched. The energy transfer rate depends strongly on donor-acceptor separation distance and thus on concentration. These measurements require a commercial fluorimeter or a home uilt experiment using spectrometers for selecting both excitation and emission wavelength.

(2) Acceptor Sensitization: Similar to the above, you could excite a solution of acceptor alone with a wavelength of light that the donor (and not the acceptor) absorbs while monitoring the fluorescence. There should be little to none. But in a solution of donor and acceptor you should see acceptor fluorescence.

(3) Time-resolved photoluminescence: Similar to the donor quenching experiment, the donor fluorescence lifetime should decrease in the presence of the acceptor. The new decay should be dominated by the FRET rate. Similarly, the acceptor fluorescence should have a delayed onset with a rate constant roughly the same as the donor decay constant measured in a solution of donor and acceptor. These measurements are done with the donor selectively excited with light of a wavelength not absorbed by the acceptor. A common method of time-resolved PL is time-correlated single photon counting.

(4) Similar to TRPL, pump-probe spectroscopy can be used to accomplish very similar measurements. In this case either ground state bleaching or stimulated emission dynamics are probed.

(5) "Single-particle FRET" measurements are (essentially) more complicated forms of acceptor sensitization measurement involving a confocal microscope, laser, and single-photon detectors. However, instead of using a large ensemble, these measurements are performed on very dilute solutions such that roughly one chromophore enters the laser beam path.

There is a wealth of information in physical chemistry and biophysical journals on the methods I've listed. However, the first two are the easiest and require the least specialized equipment.

I totally agree with Paul but if you use methods (1) or (2) you should correct the emission spectra by the inner filter effect.

Acceptor photobleaching FRET is the most easy way if FRAP facility is available. But a negative FRET experiment doesn't tell you anything.

It depends of your FRET pair. However, if two fluorophores are resonating, the emission from the donor must increase upon "removal" of the acceptor. To functionally remove the acceptor, bleaching the acceptor fluorophore is the easiest way to go. However, you have to be careful. Bleaching by excitation leads to the production of free-radical (look for CALI or FALI for more information) . So, you must be cautious when bleaching the fluorophore. Should you bleach by excitation, start by bleaching your acceptor 10-20% and verify that the emission of the donor increases. If so, keep going another 10-20%. This way, you can also estimate how efficient your FRET is.

Good luck.

simply measure the fluorescence spectra of your sample and use the donor excitation wavelength. then, check if you have acceptor emission or not. If you have acceptor emission upon donor excitation, then you have FRET.

That`s easy: if you have the spectra of both pairs ready you just need to check whether there is a large area of overlap between FRET donor emission (shorter wavelength fluorescence dye) and FRET acceptor (longer wavelength dye). Maximum FRET transfer is given at around 50 angström distance between fluorophores (R(exp6) law). For the example CFP and YFP, which is an excellent FRET pair (Vielreicher M et al., 2007) the spectral overlap is around 70%. If excitation spectrum of donor is well separated from emission spectrum of the acceptor good spectral deconvolutions (separations) are possible enabling good analysis and FRET efficiency calculation. Good luck.

Paul´s overview is exhaustive. However, you mentioned that you aim to detect FRET from freely diffusing molecules. Note that FRET occurs effectively when only when the donor and acceptor are close enough - for freely diffusing molecules, you need very high acceptor concentration, typically in order of 10 mM (depends on R0 of the D-A pair). Then Leonid´s point becomes very important. FRET can be considered also as a special type of quenching, in your case, it will be very difficult to discriminate it from dynamic quenching, which also causes the fluorescence lifetime decrease.

An additional note: mutual orientation of the dipoles of the 2 fluorophores is also very important for FRET transfer to occur. So if the fluorophores are bound to 2 different interacting proteins you cannot predict how fluorophores will be finally oriented so there could be very good or rather poor FRET efficiencies even though the mutual distance is fine. Same is true in the case of intramolecular FRET in FRET sensors.

How to proof interaction via FRET: a common method is to do acceptor photobleaching. If then donor fluorescence rises FRET was present before bleaching out the acceptor, because FRET transfer is then not possible anymore.

Wow, lots of good answers to this simple question. Hopefully you understand what everyone is saying. If not, as this is basic test. I will spell it out with a simple method.

Find a spectrophotometer.

Prepare the following solutions: 1 Donor only at fixed known concentration. 2 Acceptor only at fixed known concentration. 3. Mixture of Donor and Acceptor at the exact same molar concentrations as when alone. Good to start at mixture of 1:1, you may need to change this.

Then obtain the following spectra for all samples:

1. Excite at optimal donor excitation and collect optimal donor emission.

2. Excite at optimal acceptor excitation and collect optimal acceptor emission.

3. Excite at optimal donor excitation and collect optimal acceptor emission.

Now you have all the data, you can look to see if there is more emission of acceptor in presence of donor (more than alone) and you can see if the emission of donor is less in the presence of acceptor (less than when alone). These are the tell tail signs of FRET. You also have all the numbers at the end to calculate the specifics.

Hope that helps.

As others mentioned it is very hard to detect FRET if the molecules are freely difusing is solution. Do you have any reason believe that thre is stable interction between them? Actually, I would not believe in any quenching, lifetime or intensity measurements in solution as proof of FRET. The dynamic quenching in solution causes changes in all these parameters without any FRET. A simple Stern-Volmer plot will show you if this is dynamic (straight line) or static (not a straight line) quenching, or if there is any excited state phenomena going on. This would be a suggestion of FRET. For FRET, not only distance is important but also orientation of the dipoles of the molecules involved. If they are freely difusing (no binding) it is very unprobable that they will be in position for efficient FRET. Also remember the limitation in distance. A theorectical limit would be 10 nm, but usually it is not possible to observe FRET if the distance is larger than 8 nm.

Best advices are to observe acceptor fluorescence using donor excitation only or bleaching the acceptor and observing donor fluorescence increase. Just be sure that you observe acceptor emission upon donor excitation!

Lifetime of the donor will be decreased in dynamic quenching in solution, so this is not a good option in your case. .

Something not mentioned above is the "red edge effect" on polarization. When there is FRET the energy transfer changes the dipole orientations and the polarization of the donor excitation light is lost, when measuring the polarization of the acceptor emission. Since you are measuring in solution, polarization would also be low. But sometimes the transitions stimulated at the red edge of the excitation spectra are not effcient for FRET and larger acceptor polarization may be observed. This may be a proof of FRET, but again, in solution.....

Yes, I agree with Paul Gunningham and Leonid Patsenker recommendations but can offer an additional argument - linear dependence between quenching ratio I / I0 of donor (0 is index) and 1 / C acceptor (C means concentration of acceptor). Also in the acceptor excited spectra the bandwidth of donor should be appeared. More important to use data collection rather than one.

Hey, before measuring, make sure the emission of the donor overlaps with the excitation spectrum of the acceptor. To find out about their FRET in an experiment, you may order two complementary short single strand DNA, oligonucleotides one labeled with the donor the other with the acceptor so that you can anneal them to become double stranded (double labeled) DNA later. For this assay you know the donor-acceptor distance given by the length of the DNA strand. A negative control could be a double labeled long DNA strand respectively. Now, compare the donor and acceptor fluorescence in absence (single strand) and presence (double strand) of the other, and relate this to the non-FRET case (long strand) to find out the dynamic range of your experiment and the FRET rates you can exspect. You can perform this experiment in solution using a fluorospectrometer or in a single molecule microscopy setup.

see the following:

Elongovan, M., Wallrabe, H., Chen, Y., Day, R. N., Barroso, M., and Periasamy, A. (2003) Methods 29, 58-73

Cunningham answer is the one to use, you can suplement that with reading my 1996 review paper in Chemical Revies

Cool it a bit to increase the viscosity so that the diffusion is slower than ns. TIme-resolve it and see if it fits to Forster function!

David Bateman's answer is ideal.

Prepare the solutions as he indicated and measure the spectra at the donor excitation wavelength.

Once you have donor only, acceptor only (will give you the direct excitation of the acceptor at donor excitation wavelength) and the mixture you will be able to do spectral unmixing (routines are available for that, just look it up).

The spectral unmixing will tell you if there is quenching of the donor and sensitized emission of the acceptor.

Alternatively you can use lifetime.

By measuring the donor lifetime in presence and absence of the donor you should be able to see the donor quenching (keep concentration low maybe uM range depending if the dissociation constant allows you to still see interaction, good TCSPC systems will allow you to measure the lifetime even at very low concentration).

If you're afraid of quenching and want the ultimate proof. You can measure the acceptor sensitized emission fluorescence decay. If you extract a rise time component (negative pre-exponential factor) then there is FRET (as opposed to only direct excitation).

I hope this helps.

For the procedure here you will need to do absorption spectra, tunable (e.g. titanium sapphire laser) with automatic sweep and the equipment registering the signal at one wavelength only and the normal sweep for the emission spectra.

If you are exciting any kind of molecule that you suspect being energy donor to other accepting molecule, the procedure to make sure if there is a transfer between them would be always the same:

1) Assuring you will be able to observe the signal:

For energy transfer to occur, 2 conditions have to be satisfied: a) they have to have overlaps between emission lines of one and absorption line of the other b) molecules have to be close so you need the high concentration. But, if you have high concentration there is a possibility of quenching where the light emitted from the acceptor is absorbed by other acceptors around. You can resolve this by directing the excitation beam parallel and at the very edge of the cuvette and you are collecting your signal in the right angle to your incident beam so that you can recollect directly the light emitted from the sample exiting the glass wall. You need a solvent that is not absorbing at wavelengths of your excitation source, not absorbing at wavelengths where you expect energy transfer to occur nor where you expect the acceptor to emit.

2) Finding energy candidate overlap:

2a. absorption spectra of your acceptor only (without other molecules that would influence your absorption)

2b. emission spectra of your donor only. Graph these two spectra on top of each other and look for overlaps. You have to find cases where you have absorption lines at more or less same wavelength as the emission. If the levels are not exactly resonant, you have to have at least a good overlap of the ‘tails’ of the emission and absorption line, being emission on higher energies than absorption so that the spare energy can be released in the form of heat.

3) Finding the observed wavelength:

Let us suppose that you excited the donor and it transferred to the acceptor. Now your acceptor will emit it either from the manifold where you transferred the energy or other lower energy (larger wavelength) manifolds. So you identify the longest wavelength where your energy overlap from above ends and go to just a bit longer wavelength. This means that your donor cannot emit at the wavelength you are at, but if the energy is transferred to your acceptor, it can. You can check it by doing the excitation spectra. Place your observing monochromator at this wavelength behind a narrow slit. Using the tunable laser (Ti-sapphire) you sweep through the whole range of excitations always collecting the emission at your same wavelength. If you do that experiment with the sample of only your donor, it should not emit when excited at the wavelength you will use for your excitation but in the same experiment the sample of your acceptor only, it does emit.

4) Finding the excitation wavelength:

The key fact is that your excitation wavelength has to be absorbed well by your donor and not absorbed at all by the sample of your acceptor alone.

5) The experiment:

5a. Place the sample with your acceptor only in the configuration described in 1), excite at wavelength from the step 4) and register the spectra. You should not have the emission at the wavelength determined from the step 3).

5a. Place the sample containing both donor and acceptor, excite it at the wavelength from the step 4) and register the spectra. You should detect the emission at the wavelength determined in the step 3)

There is a trick. You do not want to be wrong, not detecting the emission that is there because your experiment is not well focused optically. All cuvettes that you use have to be equal. You can use a stand with a placement to fit the cuvette in and screws for fine tuning of the stand. You can use a pattern sample to tune optically your setup. For pattern sample you use a material with a strong and sharp emission (for eg. Er-doped nanoparticles). At very narrow slit of your collecting monochromator you optimize the setup to collect the light at the right angle from the point your exciting beam enters the cuvette. Than you can carefully replace the pattern sample in the holder with your samples to perform the experiment.

This was what I was asking sometime ago.However, I have some ideas to add to the many well written answers.

First, find out the emission of your donor and acceptor in your D-A pair.

Since changes in concentration affect the PL intensity in steady state spectrophometer, try Time-resolved Fluorescence spectrocopy which is independent of concentration.

So you can easily tell if you have D-A pair by comparing changes in lifetime of D-A pair with reference to the lifetime of acceptor only.

It will increase in the case of FRET.

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. The efficiency of FRET is dependent on the inverse sixth power of the intermolecular separation, making it useful over distances comparable to the dimensions of biological macromolecules. Thus, FRET is an important technique for investigating a variety of biological phenomena that produce changes in molecular proximity. I agree with David Bateman's suggestion to use spectrophotometer and his exeprimental technique.

Suppose if there are two metal oxides acting as D-A pair and both have binding affinity for an enzyme, then how can we analyse FRET after adding analyte which is substrate for  the enzyme?