Sampled single-photon detection

To record a fluorescence waveform, the count rate is first adjusted to reduce the probability of two-photon events to less than 5% as discussed 

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Harris, J. M. and Lytle, F. E. (1977). Measurement of subnanosecond fluorescence decays by sampled single-photon detection. Rev. Sci. Instrum. 48, 1469-76. 

Figure 4.6 shows an apparatus for the fluorescence depolarization measurement. The linearly polarized excitation pulse from a mode-locked Ti-Sapphire laser illuminated a polymer brush sample through a microscope objective. The fluorescence from a specimen was collected by the same objective and input to a polarizing beam splitter to detect 7 and I by photomultipliers (PMTs). The photon signal from the PMT was fed to a time-correlated single photon counting electronics to obtain the time profiles of 7 and I simultaneously. The experimental data of the fluorescence anisotropy was fitted to a double exponential function. 

The lifetime resolution is the smallest variation in lifetime that can be detected. If external noise sources are ignored, the lifetime resolution depends essentially on the photon-economy of the system. For instance, if a 2 ns lifetime is measured with a 4 gate TG single-photon counting FLIM (F = 1.3) and 1000 photons, variations of about 80 ps can be resolved. However, for reasons discussed earlier, in biological samples these values could be higher. 

Figure 13.1—X-ray fluorescence. The sample, when excitated by a primary X-ray source, emits fluorescence that can be detected according to two modes 1) simultaneous detection by a cooled diode that detects the energy of a single photon 2) sequential detection of the emitted wavelengths (using a "6,26 goniometer assembly).

Until recently we were unable to determine k for 1(4) and 1(6) via this method, since this not only requires a time resolution better than 10 ps, but especially since the quenching of the donor fluorescence, that accompanies the electron transfer, makes the measurements extremely sensitive to the presence of minor, fluorescent impurities. After careful recrystallization a sample of 1(6) was now obtained for which the level of impurity fluorescence is sufficiently low to detect the very short lived ( 3-4 ps) residual donor fluorescence. A typical fluorescence decay as observed for this sample in ethylacetate via picosecond time correlated single photon counting (Bebelaar, 1986) is shown in Fig. 3. Via a biexponential reconvolution procedure the lifetime of the short component was determined to be 3.5 0.5 ps, while that of the impurity background is comparable to the lifetime of the isolated donor (-4500 ps) and thus probably stems from one or more species lacking the acceptor chromophore. Similar results were obtained in tetrahydrofuran (3 1 ps) and in acetonitrile (4 lps). 

In the time-correlated single-photon counting (TCSPC) technique, the sample is excited with a pulsed light source. The light source, optics, and detector are adjusted so that, for a given sample, no more than one photon is detected. When the source is pulsed, a timer is started. When a photon reaches the detector, the time is measured. Over the course of the... 

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