We have already stated that PP signals are more complicated than time-resolved fluorescence signals because they contain several different contributions. Let us discuss what these contributions are. For that, let us look at the energy level scheme of a hypothetical molecule shown in Fig. 27A. When the pump pulse excites the sample, some molecules leave the ground state and are transferred to the excited state (solid green arrow). This means that the concentration of ground-state molecules decreases and part of ground state absorption signal disappears. Therefore, at the wavelengths of ground state absorption, the absorption difference becomes negative. The spectral shape of this negative contribution is identical to the ground state absorption spectrum measured by a spectrophotometer (this is what is missing, because some molecules are now in the excited state). This contribution to the difference absorption signal is called ground state bleaching (GSB) and is shown in Fig. 27B by green area curve. Over time, this signal remains until all the excited molecules return to the original ground state, from which they were excited.
Another contribution to the PP signal is related with stimulated emission (SE). It arises, when the probe pulse finds some of the molecules in the excited state, and the photons of the probe pulse stimulate the emission of the sample molecules (red dashed arrow in Fig. 27A). This the phenomenon underlying the principle of lasers. The photons radiated by the molecules have exactly the same polarization, direction and wavelength as the photons that ‘dropped’ the molecules from the excited to the ground state. It is easy to see that this signal, similarly to the GSB signal, will be negative: as equation suggests, when the pump pulse is blocked, the number of photons reaching the detector (intensity ), will be equal to the number of photons impinging on the sample (assuming no absorption). When the pump pulse is unblocked, the detector will also receive all these photons plus the photons emited by the sample. We will then have and signal DA in will be negative. It sounds like a paradox: how can you have a decrease in absorption in the spectral range, where sample does not absorb? But it isn’t, really. The sample emits light and that is perceived as decreased absorption. Within the limits of Einstein coefficients, the relationship between the stimulated and spontaneous emission (fluorescence) spectra is:
Fig. 1) A. Energy levels of a hypothetical molecule and some quantum transitions influencing the difference absorption spectrum. B: The corresponding difference absorption spectrum and with separated contributions of different transtions.
In practice this means that in molecular solutions, SE and fluorescence have nearly identical spectral shapes. Similarly to fluorescence, SE exhibits Stokes shift (red shift compared to the absorption spectrum). In Fig. 27B, SE is depicted by the red area curve. The reason for the Stokes shift is the relaxation of the molecule and its immediate environment in the excited state (discussed in detail below).
The third contribution to the PP signal is induced absorption (IA) resulting from the fact that the molecules in the excited state can absorb another photon and go to a higher excited state (dotted blue line in Fig. 27A). This process can only occur in the excited molecules, therefore, after the excitation, additional absorption appears and the related contribution to DA signal is always positive (blue area curve in Fig. 27B). Note, however, that induced absorption can be caused not just by singlet excited states of molecules. If, for example, the excited molecule has udergone intersystem crossing to the triplet excited state, triplet state absorption will be observed, corresponding to transitions T1 ® Tn. Even if the molecule is back in the ground state, but this state is slightly different from the initial one (for example, the molecule has isomerized, given away or bound a proton, the environment of the molecule is hotter than before, and so on), we will observe induced absorption of the new ground state. This is the advantage of PP spectroscopy compared to time-resolved fluorescence: when the excited state is gone, so is the fluorescence signal. Difference absorption remains, though, and can provide information about the further results of photon absorption in the molecule.