Measurement of the Instrument Response Function

An accurate fluorescence lifetime measurement normally requires measurements of the fluorescence and the instrument response function (IRF). The lifetime or the lifetime components of the decay function are then obtained by deconvolution of the fluorescence curve from the IRF [389]. 

The attempt is often made to use Raman lines to eonvert the excitation wavelength into a wavelength in the spectral region of the fluorescence. However, the problem is the same as for short lifetime fluoreseenee - the Raman effect is so weak that the slightest background fluorescence makes the result useless. 

It should be noted that the IRF recorded in the cuvette is not identical with the laser pulse recorded directly. The horizontal path length of the laser in a 10-mm cuvette adds about 45 ps of transit-time spread to the IRF. If a large beam diameter is used, there are also path length differences for different depth in the cuvette. Moreover, reflection at the cuvette walls and scattering at the sample holder contributes to the IRF. However, these effects are essentially the same for IRF measurement by a dilute scattering solution and fluorescence measurement of a trans- 

The results show that the full resolution of a fast TCSPC system cannot be exploited for measurements in the eommonly used 10 mm cuvettes. The optical transit-time spread can be reduced by recording only from a small spot in the cuvette. Another solution is a thin cuvette under front illumination. 

In special cases it can be useful or even required to excite the fluorescence by two-photon absorption [132, 319, 493, 494, 521]. The excitation wavelength may then be entirely outside the spectral sensitivity range of the detector. Moreover, the pulse profile obtained by detecting the laser pulse directly may not correspond to the effective temporal two-photon excitation profile of the sample. A useful way to obtain an IRF in a two-photon system has turned out to be second harmonic generation from a suspension of gold particles [206, 375]. 

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