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.