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

Abstract

SummaryIn 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 timegate 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.

Fluorescence lifetime imaging in an optically sectioning programmable array microscope (PAM)

“…1). Although the treatment presented here applies to the frequency domain, a related set of issues, including photon statistics and optimal selection of experimental parameters (31), arises in time domain measurements, particularly those based on the collection of a small number of time windows (9,10). Lifetime determinations of specimens with only a single type of unperturbed fluorophore (a condition we will refer to as fluorophore homogeneity) or thin specimens with multiple fluorophores that are spatially separated but lie within the diffraction limit will not be improved by optical sectioning in general.…”

“…Image reconstruction procedures applied to widefield and structured illumination data have also been used to generate sectioned fluorescence lifetime images. Different optimized lifetime imaging systems for the time domain (9,10) and frequency domain (11,12) have been reported; see Suhling et al (1) and Bunt and Wouters (13) for recent reviews.…”

Fluorescence lifetime imaging in biosciences: technologies and applications

“…Considering the fact that many endogenous chromophores (quenched donors in FRET, NADH, NADPH) are characterised by fluorescence lifetimes in ps-range, the time-accuracy provided by FD FLIM is often insufficient. In contrast, TD FLIM techniques reach time-resolutions in the low ps-range or even fs-range [8][9][10][11]40,[45][46][47] .…”

“…FLIM in time-domain is primarily more advantageous than FLIM in frequency-domain because it provides a direct measurement of the fluorescence decay and, thus, a direct access to the parameter/parameters of interest 9,10,12,[40][41][42][43][44][45] . Considering the fact that many endogenous chromophores (quenched donors in FRET, NADH, NADPH) are characterised by fluorescence lifetimes in ps-range, the time-accuracy provided by FD FLIM is often insufficient.…”

“…In order to obtain feasible fluorescence decays, measurements of the sample and of a calibration reference, i.e. usually scattered excitation radiation, are necessary 8,26,45 . Since the IRF of most PMT strongly depends on the wavelength, this calibration is rather difficult especially in the two-photon microscopy because the PMTs are often not able to detect radiation at both the excitation and the emission wavelengths.…”