Fluorescence resonance energy transfer in the study of cancer pathways

Abstract

Many different signaling pathways are involved in deregulation of cell proliferation leading to cancer. Although genomic approaches successfully identified a great variety of molecules associated with cancerogenesis, other strategies must be applied to elucidate complex interactions between these molecules. One promising approach is fluorescence resonance energy transfer, a proximity-dependent fluorescence phenomenon. With the development of spectrally different fluorescent proteins and improved technologies for fluorescence measurements, this approach gains an enormous potential for future research. The fluorescence resonance energy transfer principle can be applied for studying all kinds of interactions or conformational changes, and it can also be used for microscopic visualization and subcellular localization of biochemical reactions, thereby promoting the progress of cancer research. Moreover, it can be exploited to develop sensitive and efficient drug screening systems and to design valuable diagnostic tools.

Some Secrets of Fluorescent Proteins: Distinct Bleaching in Various Mounting Fluids and Photoactivation of cyan fluorescent proteins at YFP-Excitation

“…6A). The extent of donor fluorescence intensification was in a range of 5 -15% of the initial fluorescence, which is a value that is quite common for donor recovery FRET microscopy and which would be equivalent to a FRET efficiency of 5 -13% (based on the equation: efficiency E = 1 -F DA /F D, where F DA is donor fluorescence in presence of acceptor (before acceptor bleaching) and F D is donor fluorescence in absence of a functional acceptor (after acceptor bleaching, [3]). When we recorded the time course of this increase of the CFPsignal during illumination at the YFP-excitation for fixed cells mounted in PBS/glycerol using confocal laser scanning microscopy, we observed a single exponential characteristic with a plateau (Fig.6B).…”

“…Contrary to that, false-negative results are likely in samples embedded in commercial mounting fluids due to the fast bleaching of CFP in these media. As a consequence, this common technique of FRET microscopy should generally not be used for the CFP/YFP fluorophore pair but instead either 3-filter methods of FRET microscopy [2,3] should be exploited or alternatives techniques such as fluorescence lifetime imaging (FLIM), which can be used to assess a FRET effect in a robust manner via the decrease of the donor fluorescence lifetime [24,25,26]. Another alternative is recording the kinetics of photobleaching of the donor, which is slowed done in presence of a FRET acceptor.…”

Fluorescence colocalization microscopy analysis can be improved by combining object‐recognition with pixel‐intensity‐correlation

“…FRET images were taken on the Nikon A1 confocal laser‐scanning microscope as described above, using a three‐filter‐configuration based approach 18, 20. DONOR‐fluorescence (ECFP emission at ECFP excitation; ECFP‐channel) was obtained with a 403 nm laser and a 482/35 (465–500 nm) filter cube.…”

“…Based on the assumption that the cFRET signals equals the fluorescence increase of the donor after complete bleaching of the acceptor and the known correlation 20:…”

“…Furthermore, we intended to complement colocalization analysis with FRET microscopy, which gives positive signals just in case two fluorescent molecules are closer than about 10 nm, thereby reporting only real physical interaction rather than random colocalization. This method relies on fluorescence resonance energy transfer from a donor fluorophore to an acceptor fluorophore (with a longer excitation and emission wavelength) via a dipole interaction leading commonly to a decrease in donor emission and an increase in acceptor fluorescence 14, 15, 16, 17, 18, 19, 20. While the physical background of this phenomenon is quite complex, the technical realization is rather simple and can be performed on standard fluorescence microscopes.…”