Fluorescence Resonance Energy Transfer for Single-Molecule Imaging
Identifying protein molecules that are in close physical proximity is important in tracing metabolic pathways. Scientists at Pacific Northwest National Laboratory (PNNL) use fluorescence resonance energy transfer (FRET) to obtain that information. For this technique, two types of proteins are stained with fluorescent dyes. The emission frequency of the donor dye must overlap the absorption frequency of the acceptor dye. When the molecules are close together, typically 10 to 100 Å, the excitation can pass from donor to acceptor without the emission of a photon. In practice, the sample is stained with both dyes. A laser tuned to the absorption frequency of the donor illuminates the sample. The acceptor emission, as a result of the indirect excitation, is detected. The FRET effect is inversely proportional to the sixth power of the distance between molecules; only molecules in close proximity will be detected.
In the image below, FRET was detected at the single-molecule level between EGFR-13A9-Alexa647 and EGF-TMR, using simultaneous acquisition of donor and acceptor emissions, while toggling between donor and acceptor excitations. In the top panel, the specimen is illuminated at the donor excitation frequency. The donor molecules' emission is shown in the right pane. The acceptor emission, left, is caused by FRET from the nearby donor molecules. In the bottom panel, the specimen is illuminated with the acceptor excitation frequency. The right pane shows that none of the donor molecules were activated; the left pane shows the total distribution of acceptor molecules. Comparing the panes shows which acceptor molecules are associated with donor molecules.
