Make the same lifetime measurement with the acceptor in the absence of the donor, and at the same concentration as in the FRET measurement. Subtract the fluorescence intensity at each time point of the acceptor alone from the donor + acceptor. Then calculate the lifetime from the difference plot. Compare that to the lifetime of the donor alone to get the energy transfer efficiency. (You could also calculate the energy transfer efficiency from the intensity rather than the lifetime, comparing the intensity of donor alone versus [(donor+acceptor)-acceptor alone].)

Adam B Shapiro Should concentration matter in case of lifetime? The reason I want to measure efficiency from lifetime rather than any steady state methods is to avoid the concentration problem in the first place.

So, you're suggesting to make a 1:1 solution of D:A and measure lifetime; then subtract the measured A only lifetime. Repeat the same process with the D-A pair. Then, compare the lifetime of D in these two scenarios?

Should concentration matter in case of lifetime?

A lifetime measurement is a measurement of intensity versus time after excitation, and intensity is concentration-dependent, as well as dependent on quantum yield at the wavelengths of excitation and emission. For a single pure substance, the lifetime is independent of concentration as long as the concentration is low enough to avoid interfering effects. However, my intuition (since I've never tried this) is that if there are 2 fluorophores, deconvoluting the lifetime measurement to extract the individual lifetimes could only be done accurately if the ratio of their intensities were known. This could probably be calculated if the ratio of the concentrations of the two fluorophores and their quantum yields (at the selected wavelengths) were known (maybe someone can supply an equation).

So, you're suggesting to make a 1:1 solution of D:A and measure lifetime; then subtract the measured A only lifetime. Repeat the same process with the D-A pair. Then, compare the lifetime of D in these two scenarios?

No, I'm suggesting using the intensity measurements that go into the lifetime measurement, not the lifetimes. There are 3 measurements needed: one with the D:A pair (the FRET sample), one with D alone, and one with A alone. The concentration of A in the A alone sample must be exactly the same as the concentration of A in the D:A sample. (This is not necessary for the D lifetime measurement.) At each time point in the lifetime curve, subtract the intensity of A from the intensity of D:A. Then calculate the lifetime of the difference curve. That is the lifetime of D in the presence of A, without any contribution from the A fluorescence. Compare this lifetime to the lifetime of the D sample to calculate the energy transfer efficiency.

If this seems too troublesome, then another way to deal with the problem is to change the donor-acceptor pair and the choice of excitation and emission wavelengths so that the acceptor fluorescence intensity is negligible. It is not necessary to use the peak of the donor excitation spectrum, you can shift the donor excitation to shorter wavelengths to reduce excitation of the acceptor. This will have no effect on the energy transfer efficiency. Changing to an acceptor with a longer wavelength of excitation also reduces the acceptor fluorescence, but unfortunately also reduces the energy transfer efficiency.

Quantifying FRET (Förster Resonance Energy Transfer) using fluorescence lifetime measurements can be challenging when the donor and acceptor fluorophores have similar fluorescence lifetimes and overlapping emission spectra. To overcome this issue, one approach is to use fluorescence lifetime unmixing.

Fluorescence lifetime unmixing is a technique that allows the separation of the fluorescence signals from different fluorophores in a sample by analyzing their different fluorescence lifetimes. This can be achieved by using a time-correlated single photon counting (TCSPC) system, which can precisely measure the fluorescence lifetime of each fluorophore.

Here are a few methods for unmixing the fluorescence signals from rsFastLime and EYFP:

It's important to note that the most suitable unmixing method will depend on the specific characteristics of the donor and acceptor and the experimental setup used. It may be necessary totest and optimize the method for the specific system being studied. Additionally, it is also important to consider the potential impact of environmental factors such as pH, temperature, and the presence of other fluorescent compounds on the fluorescence lifetime measurements.

In summary, fluorescence lifetime unmixing is a powerful technique that can be used to separate the fluorescence signals from different fluorophores in a sample, even when their fluorescence lifetimes and emission spectra are similar. By using methods such as two-photon excitation, spectral unmixing, time-resolved anisotropy, or matrix-based unmixing, it is possible to accurately measure FRET without the need for unmixing. However, it's important to use the suitable method and optimize the experimental setup for the specific system being studied to get accurate results.

Time resolved anisotropy measurement is a good idea. When the energy transfer occurs, the anisotropy drops at a rate taht depends on the distance between donor and acceptor. The extent of the anisotropy drop can even give you the stoichiometry of the association.

Article Homo-FRET Microscopy in Living Cells to Measure Monomer-Dime...

This will give you preliminary results until you can label your proteins more properly.

There is a user-friendly program called FRET-Calc, which offers a straightforward interface and is available for free. This program allows users to easily obtain a range of FRET parameters.

https://www.sciencedirect.com/science/article/abs/pii/S0010465523000607