In this study, we present two different approaches that allow multi-wavelength 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 singlephoton-
counting (TCSPC) approach. The setup consists of a confocal laser-scanning microscope (LSM 510, Zeiss) and a
Titanium:Sapphire-laser (Mira 900D, Coherent) that is used for pulsed one- and two-photon excitation. Fluorescence
light emitted by the sample is dispersed by a polychromator (250is, Chromex) and recorded by 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. We
applied these methods to Förster resonance energy transfer (FRET) measurements and discuss the advantages over
fluorescence techniques that are already well established in the field of confocal microscopy, such as spectrally resolved
intensity measurements or single-wavelength fluorescence lifetime measurements.