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.

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In this paper a versatile, high sensitivity spectrograph is presented for use in uorescence m icroscopy. The high sensitivity is achieved by using a prism for the dispersion in combination with a state-ofthe-art back illuminated charge-coupled device (CCD) camera. The spectrograph, including the CCD camera, has a detection ef ciency of 0.77 6 0.05 at 633 nm. Full emission spectra with a 1-5 nm spectral resolution can be recorded at a maximum rate of 800 spectra per second. Two applications are shown, in which the spectrograph is ber-coupled to a commercia l confocal laser scanning microscope. In the rst example, Fö rster resonance energy transfer imaging experiments were carried out on double-labeled actin laments in the in vitro motility assay. A 160 3 160 point im age was recorded in 1.5 m in at 3 ms dwell time per image point. In the second application, a time-resolved study of single quantum dots is presented at 5.2 ms time resolution.

Endothelial cell cultures, established from bovine umbilical cord arteries and subsequently infected with Cowdria ruminantium, were used as antigen in the indirect fluorescent antibody test. Bovine sera (374) and caprine sera (388) collected in 6 provinces of Mozambique were tested. Overall, 30.4% of goat sera had antibodies to Cowdria, and 43% of sera collected from cattle. North of the River Save, where the tick Amblyomma variegatum is highly prevalent, overall percentages of positive sera were low, 10% in goats and 20% in cattle. However, south of the river where the tick Amblyomma hebraeum is abundant percentages were much higher, 63.5% in goats and 59.4% in cattle. These results are discussed in relation to field observations that clinical disease is rare or absent in the north with enzootic instability in goats and Friesian calves in the south.