Fluorescence lifetime imaging in scanning microscopes: acquisition speed, photon economy and lifetime resolution

In this paper a detailed discussion is presented of the factors that affect the fluorescence lifetime imaging performance of a scanning microscope equipped with a single photon counting based, two‐ to eight‐channel, time‐gated detection system. In particular we discuss the sensitivity, lifetime resolution, acquisition speed, and the shortest lifetimes that can be measured. Detection systems equipped with four to eight time‐gates are significantly more sensitive than the two time‐gate system. Only minor sensitivity differences were found between systems with four or more time‐gates. Experiments confirm that the lifetime resolution is dominated by photon statistics. The time response of the detector determines the shortest lifetimes that can be resolved; about 25 ps for fast MCP‐PMTs and 300–400 ps for other detectors. The maximum count rate of fast MCP‐PMTs, however, is 10–100 times lower than that of fast PMTs. Therefore, the acquisition speed with MCP‐PMT based systems is limited. With a fast PMT operated close to its maximum count rate we were able to record a fluorescence lifetime image of a beating myocyte in less than one second.