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.
Gerritsen
H C
HC
Debye Institute, Utrecht University, PO Box 80.000, NL 3508 TA, Utrecht, the Netherlands. H.C.Gerritsen@phys.uu.nl
Asselbergs
M A H
MA
Agronskaia
A V
AV
Van Sark
W G J H M
WG
D000818:Animals; D005453:Fluorescence; D008855:Microscopy, Electron, Scanning / Q000295:instrumentation* / Q000379:methods; D032342:Muscle Cells / Q000648:ultrastructure; D017785:Photons; D051381:Rats