Multi-dimensional fluorescence lifetime and FRET measurements.
When and where proteins associate with each other in living cells are key questions in many biological research projects. One way to address these questions is to measure the extent of Förster resonance energy transfer (FRET) between proteins that have been labeled with appropriate donor and acceptor fluorophores. When both proteins interact, donor and acceptor fluorophores are brought into close vicinity so that the donor can transmit a part of its excitation energy to the acceptor. As a result, both the intensity and the lifetime of the donor fluorescence decrease, whereas the intensity of the acceptor emission increases. This offers different approaches to determine FRET efficiency: One is to detect changes in the intensity of donor and acceptor emission, the other is to measure changes in the lifetime of the donor molecule. One important advantage of the fluorescence lifetime approach is that it allows to distinguish between free and associated donor molecules. However, like intensity measurements it lacks an intrinsic control ensuring that changes in the measured parameters are only due to FRET and not other quenching processes. Here, we show how this limitation can be overcome by spectrally resolved fluorescence lifetime measurements in the time domain. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting approach. Both approaches allow biologists to record both donor and acceptor fluorescence emitted by the sample in a single measurement.
Biskup
Christoph
C
Institut für Physiologie II, Friedrich-Schiller-Universität, Teichgraben 8, 07740 Jena, Germany. christoph.biskup@mti.uni-jena.de
Zimmer
Thomas
T
Kelbauskas
Laimonas
L
Hoffmann
Birgit
B
Klöcker
Nikolaj
N
Becker
Wolfgang
W
Bergmann
Axel
A
Benndorf
Klaus
K
D002460:Cell Line; D003952:Diagnostic Imaging / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D018613:Microscopy, Confocal; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D011506:Proteins