ABSTRACT
Usually these events can be observed directly by following the fluorescence emission on that time scale. However, steady-state fluorescence measurements integrate the fluorescence signals over this critical timescale, so extra assumptions are required for their interpretation that are not required for the interpretation of lifetime-resolved measurements. Thus, lifetime resolution provides extra parameters that are often critical for correctly interpreting fluorescence measurements, especially in the environment of complex biological systems. Of course, FLIM provides spatial resolution in addition to this temporal information and, just as in single-channel measurements, the lifetime resolution provided by FLIM greatly extends the capabilities of existing fluorescence imaging techniques. Different lifetime contributions can be distinguished, increasing contrast of the image and providing better separation of multiple components and many times better elimination of autofluorescence or other unwanted fluorescent background.
