ABSTRACT
Since the rst report on the uorescence phenomenon in 1845,1 interest in uorescence has rapidly increased. During the last few decades, uorescence spectroscopy, and particularly time-resolved uorescence, has become recognized as an important research tool in the biological sciences.2-7 Timeresolved uorescence is of interest due to its higher information content compared to steady-state measurements. However, the information content of the time-resolved uorescence is usually not accessible with classical imaging techniques such as uorescence microscopy. e situation has changed as remarkable developments in the time-resolved uorescence methods have recently facilitated the transfer of time-resolved uorescence from the solution spectroscopy to the eld of lifetime-based sensing and imaging.8-16
Fluorescence emission is a radiative process that occurs on the nanosecond time scale for most uorophores. Normally, the uorescence is a rst-order kinetic process and the intensity decay obeys the exponential law. e uorescence lifetime τ characterizes the average amount of time that a molecule spends in the excited state following absorption of a photon. e importance of the time-resolved uorescence for imaging can be understood from the fact that unlike intensity, the uorescence lifetime is mostly independent of the probe concentration, photobleaching, and lightpath. All these parameters are extremely dicult to control during microscopic cellular experiments. e diculties can be overcome by uorescence ratiometric measurements. However, the number of uorescent ratiometric probes is much smaller than the class of the lifetime sensors.
