Time-Gated Fluorescence Detection

Often the absorption spectra of several pollutants overlap. It is therefore not possible to determine the specific concentrations of different pollutants from a single absorption measurement at a given wavelength X. Either several well-selected excitation wavelengths A, have to be chosen (which is time consuming for in situ measurements) or time-resolved fluorescence excitation specfioscopy can be used. If the excited states of the different components have sufficiently different effective lifetimes, time-gated fluorescence detection at two or three time delays Ar, after the excitation pulse allows a clear distinction between different components. 

Time-gated fluorescence detection is used to monitor the fluorescence of a sample as a function of time after excitation by a flash or pulse of light. 

This time-resolved fluorescence technique allows a measure of the time dependence of fluorescence intensity after a short excitation pulse. It consists of obtaining a spectrum measured within a narrow time window during the decay of the fluorescence of interest. The usefulness of this technique is now well proven for biochemical assays and immunoassays. Lanthanide chelates have luminescence decay times over 600 ps, which allows time-gated fluorecence detection, with a complete rejection of other fluorecence signals. For these quantitative applications, the primary source is generally a quartz lamp associated with a splitter. 

In TG methods, the fluorescence emission is detected in two or more time-gates each delayed by a different time relative to the excitation pulse (see Fig. 3.3). In the case of a detection scheme equipped with two time-gates, the ratio of the signals acquired in the two time-gates is a measure of the fluorescence lifetime. For a decay that exhibits only a single exponent, the fluorescence lifetime is given by ... 

Fig. 3.3. Principle of time gating (TG). After exciting the specimen with a short light pulse, the fluorescence is detected in a number of time gates that open after a specific delay with respect to the excitation pulse.

In Fig. 3.5A a comparison between time-gated detection and TCSPC is shown. The time-gated detection system was based on four 2 ns wide gates. The first gate opened about 0.5 ns after the peak of the excitation pulse from a pulsed diode laser. The TCSPC trace was recorded using 1024 channels of 34.5 ps width. The specimen consisted of a piece of fluorescent plastic with a lifetime of about 3.8 ns. In order to compare the results, approximately 1700-1800 counts were recorded in both experiments. The lifetimes obtained with TG and TCSPC amounted to 3.85 0.2 ns and 3.80 0.2 ns respectively, see Fig. 3.5B. Both techniques yield comparable lifetime estimations and statistical errors. 

Fig. 3.9. Principle of a wide field FLIM system with simultaneous detection of two time gates. The fluorescence image is split into two images and one of the images is optically delayed with respect to the other. Both images are detected simultaneously with the same time-gated detector.

Fig. 2 Data acquisition for time-domain FLIM. FI fluorescence intensity, h gated image no 1,12 gated image no 2. Left Excitation pulse of the light source and synchronized timegated detection with a CCD camera. Right Lifetime determination by two subsequent time-gates according to Eq. 2...

Fluorescence can be detected at various delay times with adjustable time gates Af in consecutive acquisition cycles (multigate detection). At best, lifetime r can be calculated from four experimental data (for the case of monoexponential decay) ... 

The other intermediates in the Krebs cycle (isocitrate, a-ketoglutarate (KG), succinate, fumarate, L-malate and oxaloacetate) also cause large differences in fluorescence intensity on addition to [Eu(Tc)], which can be imaged with the help of a microwell plate (Fig. 16a). As KG and succinate cannot effectively coordinate with Eu3+, no significant fluorescence enhancement was expected, nor indeed observed. By choosing different lag times for time-gated detections, different intermediates can be seen in different time windows. 

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