Imaging protein interactions by FRET microscopy: FRET measurements by sensitized emission.

This protocol describes a method for measuring fluorescence resonance energy transfer (FRET) by the detection of acceptor-sensitized emission. This approach is useful in situations where donor intensities are low and/or there is contamination with high background (auto) fluorescence in the donor channel. However, absorption spectra characteristically exhibit long tails in the higher-energy, shorter-wavelength (blue) region, which may result in the direct excitation of the acceptor molecule in addition to that of the donor, thus resulting in mixing of direct and sensitized emission. Conversely, fluorescence emission tends to tail into the red part of the spectrum, causing donor fluorescence bleed-through into the acceptor detection channel. Corrections for these effects involve the acquisition of fluorescence images of samples containing the donor, the acceptor, and both of these for three different filter settings. The result is an estimation of the sensitized emission, i.e., the emission induced by FRET from the donor to the acceptor alone. Excitation of a donor fluorophore in a FRET pair leads to quenching of the donor fluorescence and increased emission from the acceptor (sensitized emission). This can be normalized using the acceptor emission, measured after specific excitation of the acceptor, to define apparent energy transfer efficiency in each pixel of the image. It is also proportional to the fraction of acceptor molecules that is bound to a donor-tagged molecule. Alternatively, an apparent energy transfer efficiency can also be defined that is proportional to the bound fraction of donor-tagged molecules.

Imaging protein interactions by FRET microscopy: FRET measurements by acceptor photobleaching.

This protocol describes the detection of fluorescence resonance energy transfer (FRET) by measuring the quenching of donor emission alone. As opposed to sensitized emission measurements, photobleaching can be performed with high selectivity of the acceptor because absorption spectra are steep at their red edge, allowing the acceptor to be bleached without excitation of the donor. When using acceptor photobleaching FRET measurements, care should be taken that the photochemical product of the bleached acceptor does not have residual absorption at the donor emission and, more importantly, that it does not fluoresce in the donor spectral region. Because of mass movement of protein during the extended time required for photobleaching (typically 1-20 min), it is preferable to perform this type of FRET determination on fixed cell samples. Live-cell FRET measurements based only on donor fluorescence are more feasible using fluorescence lifetime imaging (FLIM), because lifetimes are independent of probe concentration and light path length. The former is not easy to determine in cells, and the latter means that cell shape is not a factor.

Imaging protein interactions by FRET microscopy: FLIM measurements.

This image acquisition protocol is a basic plan for taking a fluorescence lifetime imaging (FLIM) series. FLIM makes live-cell FRET measurements based only on donor fluorescence more feasible, because lifetimes are independent of probe concentration and light path length. The former is not easy to determine in cells, and the latter means that cell shape is not a factor.

Imaging protein interactions by FRET microscopy: labeling proteins with fluorescent dyes.

This protocol provides a method for labeling proteins, such as antibodies and purified recombinant proteins, with succinimide esters of sulfoindocyanine (Cy) dyes. Cy dyes covalently bind to free amino groups (the α-amino-terminal or ε-amino groups on lysine side chains).

Imaging protein interactions by FRET microscopy: cell preparation for FRET analysis.

The following protocol describes the preparation of cells for FRET analysis on live and fixed cells. The reagents used have been optimized to minimize the quenching of GFP mutants and fluorescent dyes.