Spectrally resolved fluorescence lifetime and FRET measurements

We present two different approaches that allow multi-wavelength fluorescence lifetime measurements in the time domain in conjunction with a laser scanning microscope and a pulsed excitation source. One technique is based on a streak camera system, the other technique is based on a time-correlated-single-photon-counting (TCSPC) approach. The complete setup consists of a laser scanning microscope (LSM-5 10, Zeiss), a polychromator (250is, Chromex), a streak camera (C5680 with M5677 sweep unit, Hamamatsu Photonics) or a 16-channel TCSPC detector head (PML-16, Becker & Hickl) connected to a TCSPC imaging module (SPC-730/SPC-830, Becker & Hickl). With these techniques it is possible to acquire fluorescence decays in several wavelength regions simultaneously. The fluorescence emitted by the sample can be recorded in a single measurement. No filters have to be used to separate the contributions of different fluorophores to the overall fluorescence signal. When applied to Forster resonance energy transfer (FRET) measurements, the technique allows to separate the decay components of the donor and acceptor fluorescence. In this way, it is possible to reliably determine FRET efficiencies between acceptor and donor fluorophores in given subcellular structures.