Microscopy Techniques and Culture Surfaces:
Find the Perfect Match
Förster Resonance Energy Transfer (FRET)
FRET determines the precise location and spatial proximity of fluorescently labeled molecules and their interactions in living cells. Using this technique, protein-protein interactions or conformation changes, for example, can be analyzed using a standard widefield or confocal fluorescence microscope. Using specific calcium-sensitive biosensors, FRET can also be applied for the visualization of changes in cellular calcium concentration.
FRET-based visualization of cytoplasmatic calcium concentration. HEK293 cells expressing NK-I on the outer cell membrane and the calcium biosensor Yellow Cameleon 3.6 (YC3.6) in the cytoplasm. Addition of the fluorescently labelled NK-I ligand (SP-TAMRA) results in a red glow of the cell membrane. Upon receptor activation, calcium release induces a change in the YC3.6 fluorescence properties: CFP excitation yields simultaneous CFP and YFP emissions by a FRET phenomenon, appearing as a green glow in the cytoplasm. Provided by M. Roelse, Wageningen, The Netherlands.
A donor fluorophore in its excited state can transfer its excitation energy to an acceptor fluorophore in a non-radiative fashion. Typically, this happens through dipole-dipole coupling in a distance of less than 10 nm. Beyond that distance (Förster radius), the two fluorophores show normal fluorescence behavior.
FRET protein interaction assay. A membrane receptor A and its ligand B are tagged with CFP and YFP, respectively. When the ligand binds to the receptor, the YFP is excited by FRET.
H.C. Ishikawa-Ankerhold, R. Ankerhold, G.P.C. Drummen. Advanced Fluorescence Microscopy Techniques—FRAP, FLIP, FLAP, FRET and FLIM. Molecules, 2012, 10.3390/molecules17044047