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FRET Microscopy

Further Topics


Data Processing




Spectral FRET


What is FRET?

Förster (Fluorescence) Resonance Energy Transfer (FRET) is a process involving the radiationless transfer of energy from a donor fluorophore in the excited state to an appropriately positioned acceptor fluorophore in the ground state. It is advantageous that the quantum yield (QY) of the fluorophore be higher than or equal to the acceptor fluorophore. There are three important conditions for FRET to occur:

  1. FRET can occur when the emission spectrum of a donor fluorophore significantly overlaps the absorption spectrum of an acceptor (see Figure). There is no FRET if the spectrum is not overlapped by at least 30%.

  2. The efficiency of energy transfer varies inversely with the sixth power of the distance (see Equation) separating the donor and acceptor fluorophores, the distance over which FRET can occur is limited to between 1-10 nm. In practice, we were able to detect about 2-8 nm in a microscopy system (see Chapter).

  3. Molecular dipole-dipole coupling is important for energy transfer from one molecule to another. The emission dipole of the donor and the acceptor absorption dipole must be oriented towards each other and must not be oriented perpendicular to each other (see Figures). The magnitude of the relative orientation of the dipole-dipole coupling value is 1-4.

The main application of FRET is as a "spectroscopic ruler," based on Förster's basic rate equation for a donor and acceptor pair at a distance 'r' from each other (Lakowicz, 2006),

kT = (1/τD) (R0/r)6                                                                     (1)

where kT is the rate, τD is the donor excited state lifetime in the abscence of the acceptor and R0 is the Förster distance, or the distance between the donor and the acceptor at which half of the excitation energy from the donor is tranferred to the acceptor while the other half is dissipated by all other processes, including light emission. The energy transfer efficiency (E) and the distance between donor and acceptor molecule (r) are calculated using the following equations: 

E = R06 / (R06 + r6)                                                                   (2)

E = 1 – (IDA/ ID)                                                                          (3)

r  = R0{ (1/E) - 1}1/6                                                                   (4)

where IDA and ID are the donor intensity in the presence of acceptor and in the absence of acceptor, respectively; r is the distance between D & A; R0 is the Förster distance at which half of the excited-stated energy of the donor is transferred to the acceptor (E = 50%). This characteristic distance (R0) can be estimated based on Equation 5 and expressed in Angstroms. 


where κ2 (dimensionless, ranging from 0 to 4) is a factor that depicts the relative orientation between the dipoles of the donor emission and the acceptor absorption (see Figure 2c); n is the medium refractive index; QYD is the donor quantum yield; J (in units of M-1 × cm-1 × nm4) expresses the degree of the overlap between the donor emission and the acceptor absorption spectra (see Figure 2b). εA (in units of M-1 × cm-1) is the extinction coefficient of the acceptor at its peak absorption wavelength; λ is the wavelength in nanometers; both fD(λ) (donor emission spectrum) and fA(λ) (normalized acceptor absorption spectrum) are dimensionless.  

The Ro value is directly proportional to the dipole moments between donor emission to the acceptor absorption κ2, the quantum yield of the donor QYD, the overlap integral (the spectral overlap) and inversely proportional to the refractive index of the medium. A dipole is an electromagnetic field that exists in a molecule that has two regions with oppositely chared areas, a region with a negative charge and another region with a positive charge. If the spectra are overlapped, the donor's oscillating emission dipole will look for a matching absorption dipole of an acceptor to oscillate in synchrony. 

Why Microscopy?

Fret Main

Within the living cell, interacting proteins are assembled into molecular machines that function to control cellular homeostasis. These in vitro screening methods have the advantage of providing direct access to the genetic information encoding unknown protein partners. These techniques do not allow direct access to interactions of these protein partners in their natural environment inside the living cell, but the FRET microcopy method provides spatial and temporal resolution from single living cells. 

FRET microscopy relies on the ability to capture weak and transient fluorescent signals efficiently and rapidly from the interactions of labeled molecules in single living or fixed cells. The occurrence of FRET signal (sensitized signal) can be verified by acquiring the two-emission signal bands of the double labeled cells excited with donor wavelength. If FRET occurs the donor channel signal will be quenched and the acceptor channel signal will be sensitized or increased. In principle, the measurement of FRET in a microscope can provide the same information that is available from the more common macroscopic solution measurements of FRET; however, FRET microscopy has the additional advantage that the spatial distribution of FRET efficiency can be visualized throughout the image, rather than registering only an average over the entire cell or population. Because energy transfer occurs over distances of 1-10 nm, a FRET signal corresponding to a particular location within a microscope image provides an additional magnification surpassing the optical resolution (~0.25 mm) of the light microscope. Thus, within a voxel of microscopic resolution, FRET resolves average donor-acceptor distances beyond the microscopic limit down to the molecular scale. This is one of the principal and unique benefits of FRET for microscopic imaging: not only colocalization of the donor- and acceptor-labeled probes within ~0.09 mm2 can be seen, but intimate interactions of molecules labeled with donor and acceptor can be demonstrated.

Several FRET techniques exist based on wide-fieldconfocal, spectral and 2p microscopy as well as FRET/FLIM, each with its own advantage and disadvantage. All FRET microscopy systems require neutral density filters or other means to control the excitation light intensity, a stable excitation light source (Hg or Xe or combination arc lamp; UV, Visible or Infrared lasers), a heated stage or a chamber to maintain the cell viability and appropriate filter sets (excitation, emission, and dichroic) for the selected fluorophore pair. It is important to carefully select filter combinations that reduce the spectral bleed through (SBT) to improve the signal-to-noise (S/N) ratio for the FRET signals.


The widely used donor and acceptor fluorophores for FRET studies come from a class of autofluorescent proteins, called Green Fluorescent Proteins (GFPs). The spectroscopic properties that are carefully considered in selecting GFPs as workable FRET pairs include: sufficient separation in excitation spectra for selective stimulation of the donor GFP, an overlap (>30%) between the emission spectrum of the donor and the excitation spectrum of the acceptor to obtain efficient energy transfer and reasonable separation in emission spectra between donor and acceptor GFPs to allow independent measurement of the fluorescence of each fluorophore. GFP-based FRET imaging methods have been instrumental in determining the compartmentalization and functional organization of living cells and for tracing the movement of proteins inside cells.

There are number of combination of FRET pair can be used depending on the biological applications. Selected popular FRET pair fluorophore are - CFP-YFP, CFP-dsRED, BFP-GFP, GFP or YFP-dsRED, Cy3-Cy5, Alexa488-Alexa555, Alexa488-Cy3, FITC- Rhodamine (TRITC), YFP-TRITC or Cy3, etc. You can find the FRET Pair Spctra from here.

Problems with FRET microscopy Imaging

One of the important conditions for FRET to occur is the overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. As a result of spectral overlap, the FRET signal is always contaminated by donor emission into the acceptor channel and by the excitation of acceptor molecules by the donor excitation wavelength (see Figure 1). Both of these signals are termed spectral bleed-through (SBT) signal into the acceptor channel. In principle, the SBT signal is same for 1p- or 2p-FRET microscopy. In addition to SBT, the FRET signals in the acceptor channel also require correction for spectral sensitivity variations in donor and acceptor channels, autofluorescence, and detector and optical noise, which contaminate the FRET signal. How to correct the contaminated signals is explained in data process part.

Figure 1