FRET Imaging by FLIM


Table of Contents:


Förster Resonance Energy Transfer (FRET) – Exploring Protein-Protein Interaction

Förster resonance energy transfer (FRET) is an interaction of two molecules in which the emission band of one molecule overlaps the absorption band of the other. In this case the energy from the first molecule, the donor, can transfer into the second one, the acceptor. FRET can result in an extremely efficient quenching of the donor fluorescence and, consequently, in a considerable decrease of the donor lifetime. The energy transfer rate from the donor to the acceptor decreases with the sixth power of the distance. Therefore it is noticeable only at distances shorter than 10 nm . FRET is used as a tool to investigate protein-protein interaction. Different proteins are labelled with the donor and the acceptor, and FRET is used as an indicator of the binding between these proteins. FRET is the most frequent FLIM application.

Principle of Förster Resonance Energy Transfer (FRET)

Because of its dependence on the distance FRET has become an important tool of cell biology. Different proteins are labelled with the donor and the acceptor; FRET is then used to verify whether the proteins are physically linked and to determine distances on the nm scale.  

Understanding Protein Interactions with FLIM-FRET: How to Interpret Data and Visualize Results

The interpretation of FLIM-FRET data is simple: In the absence of FRET the lifetime of the donor is unchanged. When FRET is present the donor is loosing its excitation energy into the acceptor, and the lifetime decreases, see figure below.

Protein interaction experiment by FRET. When the proteins are interacting FRET occurs, and the fluorescence lifetime of the donor decreases.

The use of FLIM for FRET has the obvious benefit that the FRET intensity is obtained from a single lifetime image of the donor. When FRET occurs the fluorescence lifetime of the donor decreases, when FRET is absent it remains constant. All that is needed to detect protein interaction is a single lifetime image of the donor. Donor bleedthrough and directly excited acceptor fluorescence therefore have no influence of FLIM-FRET measurements. An example is shown in the figure below.

FRET Image of a cell that has interacting proteins in the cell membrane

Double-Exponential FLIM-FRET Analysis – Accurate Distance Measurements

In all protein-interaction experiments, there is usually a mixture of interacting and non-interacting proteins. Both the fraction of interacting proteins and the distance between the proteins influence the net FRET efficiency derived from the intensities. It therefore cannot be told whether a variation in FRET efficiency is due to a variation in the distance or a variation in the fraction of interacting proteins.

TCSPC FLIM solves the problem of interacting and non-interacting donor by double-exponential lifetime analysis. The resulting donor decay functions can be approximated by a double exponential model, with a fast component from the interacting donor molecules and a slow lifetime component from the non-interacting donor molecules. There are several reasons why a donor does not interact. The protein may just not be linked to each other, an acceptor protein may not be labelled with the acceptor, or the orientation between the donor and the acceptor may be wrong. Orientation is usually considered random, and taken into account by the κ2 factor. If the labelling is complete, as it can be expected if the cell is expressing fusion proteins of the GFP variants, the decay components represent the fractions of interacting and non-interacting donor molecules. Corrected by κ2, the amplitudes, a and b, represent the fractions of interacting and non-interacting protein molecules. The composition of the donor decay function is illustrated in the figure below.

A mixture of interacting and non-interacting proteins delivers a double-exponential donor decay function. Double-exponential decay analysis separates the interacting and non-interacting donor fractions.

Precision measurement of the decay curves and double-exponential FLIM analysis by SPCImage NG directly deliver the classic FRET Efficiency, the FRET Efficiency of the interacting donor fraction, the donor-acceptor distance, and the relative amounts of interacting and non-interacting donor. The technique also has another charm: The lifetime of the non-interacting donor fraction is the reference lifetime for the FRET analysis. This makes the technique calibration-free. This is a significant advantage over classic FRET measurement with single-exponential decay analysis.

Classic FRET Efficiency, FRET Efficiency of interacting donor, donor-acceptor distance, and fraction of interacting donor, derived from double-exponential FLIM-FRET data

For more information and references please see bh TCSPC Handbook, chapter ‘Förster Resonance Energy Transfer (FRET)’.



References for FRET Imaging

For more references on FLIM-FRET please see bh TCSPC Handbook, chapter ‘Förster Resonance Energy Transfer (FRET)’.

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