**4. Inverse Raman scattering: experiments**

#### **4.1. Experimental setup**

To carry out FTA measurements, a noncollinear geometry in the pump‐probe setup is used. A diode‐pumped Ti:Sapphire femtosecond oscillator generates a ∼100 fs pulse at a repeti‐ tion rate of 78 MHz. The so‐generated pulses are stretched and amplified by a regenerative Ti:Sapphire amplifier, pumped by a Q‐switched Nd3+:YLF laser at 1‐KHz repetition rate, and eventually compressed, leading to 4 mJ, ∼100 fs pulses at 798 nm. A beam splitter sends 90% of the outcoming pulse to an optical parametric amplifier to provide tunability over a broad spectral range (290–2600 nm). This tuneable laser pulse is sent through a depolarizer, an optical chopper, and finally focused on the sample in 1 mm spot, yielding an excitation density of 5 × 1014 photon pulse−1 cm−2. The remaining 10% of the radiation is delayed in time by an optical delay line and focused on a CaF2 crystal to generate a white‐light continu‐ um radiation, spanning between 450 and 800 nm. The WLC radiation is used as probe beam spatially overlapped to the pump pulse on the sample. The light transmitted by the sample is coupled into an optical fiber and sent to a charge‐coupled device (CCD) spectrometer. The temporal resolution (∼200 fs) is determined by the cross‐correlation between the width of pump and probe pulses overlapping on the sample. The chromatic aberrations are removed by chirp correction software.

#### **4.2. The IRS related to FTA experiments**

So far, the nature of the IRS has been described. In this section, the IRS will be treated in relation with femtosecond transient absorption (FTA) experiments. In an FTA experiment, the intensity transmitted by an unexcited medium is given by pr <sup>0</sup> ℏ , when the medium is excited by a pump pulse, a difference in the probe intensity is detected, and can be described as

$$
\Delta I\_{F\Lambda} \left( \hbar \alpha , \pi \right) = I\_{\mu \prime}^{0} \left( \hbar \alpha \right) - I\_{\mu \prime}^{\prime \mu} \left( \hbar \alpha , \pi \right), \tag{17}
$$

where pr pu ℏ, is the probe intensity transmitted by the sample in the presence of the pump, and is the time delay between pump and probe pulses. Conversely, the variation in intensity measured at the Stokes and anti‐Stokes frequencies in the broadband spectrum of the probe pulse will be measured as the difference between the signal with and without the pump pulse: pump−on − pump−off [16]. It results that

The aforementioned variation in intensity IRS will be analyzed for a case of study of a dispersion of eumelanin in dimethyl sulfoxide DMSO‐methanol mixture (1:20 ratio) in the following. In **Figure 4**, the extracted Raman spectrum of the dispersion is depicted. On the red side of the white‐light broadband spectrum (Stokes side), some frequencies resonate with the vibrational modes of the Raman‐active medium. Hence, the signal appears as gain features in the probe pulse at those specific frequencies. At the same time, on the blue side (anti‐Stokes

To carry out FTA measurements, a noncollinear geometry in the pump‐probe setup is used. A diode‐pumped Ti:Sapphire femtosecond oscillator generates a ∼100 fs pulse at a repeti‐ tion rate of 78 MHz. The so‐generated pulses are stretched and amplified by a regenerative Ti:Sapphire amplifier, pumped by a Q‐switched Nd3+:YLF laser at 1‐KHz repetition rate, and eventually compressed, leading to 4 mJ, ∼100 fs pulses at 798 nm. A beam splitter sends 90% of the outcoming pulse to an optical parametric amplifier to provide tunability over a broad spectral range (290–2600 nm). This tuneable laser pulse is sent through a depolarizer, an optical chopper, and finally focused on the sample in 1 mm spot, yielding an excitation density of 5 × 1014 photon pulse−1 cm−2. The remaining 10% of the radiation is delayed in time by an optical delay line and focused on a CaF2 crystal to generate a white‐light continu‐ um radiation, spanning between 450 and 800 nm. The WLC radiation is used as probe beam spatially overlapped to the pump pulse on the sample. The light transmitted by the sample is coupled into an optical fiber and sent to a charge‐coupled device (CCD) spectrometer. The temporal resolution (∼200 fs) is determined by the cross‐correlation between the width of pump and probe pulses overlapping on the sample. The chromatic aberrations are removed

So far, the nature of the IRS has been described. In this section, the IRS will be treated in relation with femtosecond transient absorption (FTA) experiments. In an FTA experiment, the intensity

pump pulse, a difference in the probe intensity is detected, and can be described as

( ) () ( ) <sup>0</sup> D =- h hh

 w

, , , *pu*

and is the time delay between pump and probe pulses. Conversely, the variation in intensity

pu ℏ, is the probe intensity transmitted by the sample in the presence of the pump,

wt

pr

 wt

*FTA pr pr I II* (17)

<sup>0</sup> ℏ , when the medium is excited by a

side) a loss in the intensity is achieved at frequencies <sup>=</sup> + .

**4. Inverse Raman scattering: experiments**

**4.1. Experimental setup**

278 Raman Spectroscopy and Applications

by chirp correction software.

where

pr

**4.2. The IRS related to FTA experiments**

transmitted by an unexcited medium is given by

$$
\Delta I\_{\rm IRS} \left( \rho \partial , \tau \right) \propto -\Delta I\_{\rm FTA} \left( \hbar \alpha , \tau \right). \tag{18}
$$

By expressing the transient absorption signal as function of the detected difference in intensity, Eq. (19) can be obtained

$$
\Delta A\left(\hbar o, \tau\right) = -\log\_{10}\left[1 - \frac{\Delta I\_{FT4}\left(\hbar o, \tau\right)}{I\_{\rho\gamma}^{0}\left(\hbar o\right)}\right],\tag{19}
$$

**Figure 5.** Three‐dimensional plot of a femtosecond transient absorption experiment performed in eumelanin dispersed in a DMSO‐methanol mixture. The sample was excited at 2.294 eV and probed over a range spanning from 1.548 to 2.753 eV. From left to right, the Stokes, laser pump, and anti‐Stokes peaks can be identified. The ∆ ℏ, is shown in the first picosecond of time delay between the pump and probe pulses to clearly exhibit the Raman peaks.

and, thus, it is possible to directly relate the IRS effect to the FTA measurements. At Stokes frequencies, the probe‐beam field experiences gain in intensity as already described above. Hence, the argument of the logarithm in Eq. (19) is larger than 1 and the transient absorption ℏ, < 0. In fact, an increase in the photon flux is detected in the transmitted probe beam. Conversely, at anti‐Stokes frequencies, a loss in intensity is experienced in the Raman‐active medium, due to the annihilation of a photon of frequency . Experimentally, the decrease in intensity in the transmitted probe beam can be treated as a real absorption by the sample, being ℏ, > 0. This is precisely illustrated in **Figure 5**. In the middle, the scattered radiation from the incoming pump beam is evident. Symmetrically located with respect to the pump laser, the upside‐down peaks at low energies are the Stokes lines, while the intense peaks at high energies are the anti‐Stokes lines.

Due to the different amount of photons detected in the femtosecond transient absorption experiments caused by the inverse Raman‐scattering effect, it is crucial to recognize the presence of such coherent artifact to avoid misinterpretation in the analysis of the FTA spectra. Hereafter, the relaxation dynamics of a dispersion of eumelanin suspended in a DMSO‐ methanol mixture (1:20 ratio) is investigated by means of FTA. This sample was chosen to demonstrate the influence of the IRS, whose signal arises from the solvent used, in the temporal relaxation of the eumelanin pigments.

#### **4.3. Decoupling IRS features from FTA dynamics**

To investigate the influence of the IRS on the transient absorption dynamics, suitable probing energies have to be chosen accurately. To this end, the temporal evolutions of the Raman features have been analyzed. From **Figure 6**, it is clear that probing the sample dynamics at ℏpr = 1.741 eV allows the investigation of a region free from IRS features (probe frequency lower than the Stokes peaks, ℏpr < ℏ) . Furthermore, this is the energy at which the pigment is showing its maximum absorption, as illustrated in the inset of **Figure 6**. The other two regions where it is worth to investigate the dynamics are ℏpr = 1.823 and ℏpr = 2.460 eV. These two probing energies allow to study regions near the IRS features at energies lower (between the Stokes and the pump frequencies, ℏ < ℏpr < ℏpu) and higher (between the pump and the anti‐Stokes frequencies, ℏpu < ℏpr < ℏ) than the incoming pump pulse, respectively.

**Figure 6.** Temporal evolution of the synthetic eumelanin transient absorption spectra acquired in DMSO‐methanol mixture after excitation at 2.254 eV. The spectra have been normalized to the pump‐pulse intensity. The on the right illustrates the delay times between the pump and the probe pulses at which the spectra were acquired. The inset points out the rising of the eumelanin absorption signal, in an enlarged scale.

pump laser, the upside‐down peaks at low energies are the Stokes lines, while the intense peaks

Due to the different amount of photons detected in the femtosecond transient absorption experiments caused by the inverse Raman‐scattering effect, it is crucial to recognize the presence of such coherent artifact to avoid misinterpretation in the analysis of the FTA spectra. Hereafter, the relaxation dynamics of a dispersion of eumelanin suspended in a DMSO‐ methanol mixture (1:20 ratio) is investigated by means of FTA. This sample was chosen to demonstrate the influence of the IRS, whose signal arises from the solvent used, in the temporal

To investigate the influence of the IRS on the transient absorption dynamics, suitable probing energies have to be chosen accurately. To this end, the temporal evolutions of the Raman features have been analyzed. From **Figure 6**, it is clear that probing the sample dynamics at ℏpr = 1.741 eV allows the investigation of a region free from IRS features (probe frequency lower than the Stokes peaks, ℏpr < ℏ) . Furthermore, this is the energy at which the pigment is showing its maximum absorption, as illustrated in the inset of **Figure 6**. The other two regions where it is worth to investigate the dynamics are ℏpr = 1.823 and ℏpr = 2.460 eV. These two probing energies allow to study regions near the IRS features at energies lower (between the Stokes and the pump frequencies, ℏ < ℏpr < ℏpu) and higher (between the pump and the anti‐Stokes frequencies, ℏpu < ℏpr < ℏ) than the incoming pump pulse, respectively.

**Figure 6.** Temporal evolution of the synthetic eumelanin transient absorption spectra acquired in DMSO‐methanol mixture after excitation at 2.254 eV. The spectra have been normalized to the pump‐pulse intensity. The on the right illustrates the delay times between the pump and the probe pulses at which the spectra were acquired. The inset points

out the rising of the eumelanin absorption signal, in an enlarged scale.

at high energies are the anti‐Stokes lines.

280 Raman Spectroscopy and Applications

relaxation of the eumelanin pigments.

**4.3. Decoupling IRS features from FTA dynamics**

**Figure 7.** Transient absorption dynamics acquired in synthetic eumelanin dispersed in DMSO‐methanol mixture. Dis‐ persion has been excited at 2.254 eV and probed at energies ℏpr < ℏ (a), ℏ < ℏpr < ℏpu (b) and ℏpu < ℏpr < ℏ (c), that is, 1.741, 1.823, and 2.460 eV, respectively. The experimental data (full circles) were fitted by a bi‐exponential decay function (red solid line); the blue line shows the instrumental response function (IRF). Figure revised from [17].

The dynamics in the aforementioned regions is shown in **Figure 7** for the eumelanin suspension in DMSO‐methanol mixture [17]. The temporal relaxation of these pigments is well reported [18], and is consistent with the data herein shown. At the same time, it is possible to appreciate a change in the sign of the differential absorption at very short time delays (first hundreds of femtoseconds) upon the probed energy, disclosing the influence of the IRS. When the probe‐ beam frequency is lower than the one at which the Stokes features appears, an IRS‐free FTA dynamics is observed. In fact, as presented in **Figure 7a** (ℏpr = 1.741 eV), the FTA dynamics shows a positive signal which decreases in time in a multi‐exponential way. This signal is indicative of a photo‐induced absorption, where the intensity of the transmitted probe beam in the presence of excitation by the pump pulse is lower than the one collected in the absence of the pump beam. In fact, upon excitation at ℏpu = 2.254 eV, an excited‐state absorption (ESA) process, involving optically allowed transitions to higher‐energy states, occurs in the eumela‐ nin dispersion [19, 20]. When the response of the sample is probed at frequencies between the Stokes features and the incoming pump pulse, a profile like the one shown in **Figure 7b** is observed. Here, the dynamics is made out of a negative signal at ultrashort time delay followed by a sharp rise, ending by a bi‐exponential decay. The observed signal is a convolution of two processes: the absorption of the pigments and the IRS effect arising from the solvent. As observed in the previous case, the eumelanin absorption leads to a positive signal at every time delay between pump and probe pulses.

The second contribution occurs only in the first hundreds of femtoseconds instead. This is due to the fact that the vibrational coherence needed to achieve the inverse Raman scattering persists as long as pump and probe pulses are temporally overlapped. Since the frequency of the probe pulse resonates with one of the Stokes features, a reinforcement of the vibrational modes of the solvent follows. Due to the resonance at the Stokes frequency, a gain in the intensity of the probe beam is achieved and the IRS appears as an emission of photons as described by the theoretical model presented by Rai et al. [4]. An increase in transmitted intensity is registered as a negative signal in an FTA experiment. Being a stimulated process, the IRS intensity overwhelms the absorption signal carried by the eumelanin pigments until the vibrational coherence lasts. When the delay between pump and probe pulses increases, the IRS artifact disappears, revealing the positive absorption of the eumelanin. Right after, the suspension relaxes back to the ground state following a bi‐exponential decay.

In **Figure 7c**, the FTA dynamics of the eumelanin probed at frequencies between the incom‐ ing pump beam and the anti‐Stokes features is presented. If ℏpu < ℏpr, a depletion of the ground state is achieved through the action of the pump pulse. This process is called ground‐state bleaching and results in a negative signal, since the transmitted intensity of the probe beam is higher when the sample is excited. However, the ground‐state bleaching cannot explain the presence of a sharp and intense positive peak in the first few hundreds of femtoseconds of the dynamics. Again, the IRS effect plays a major role at ultrashort delay times. In fact, the resonance between the incoming fields and the vibrational modes of the solvent occurring this time at the anti‐Stokes frequencies leads to the stimulated Raman process. The loss in intensity experienced by the probe pulse can be accounted for as addi‐ tional absorption, described by a positive signal. When the pump and probe pulse are no more overlapped in the sample, the phase‐matching condition for the IRS is not satisfied, and the eumelanin dynamics is disclosed, recovering the signal in a bi‐exponential fashion.

The decay time obtained from the fit of the eumelanin dynamics is reported in **Table 1** for the DMSO‐methanol suspension. It is worth noting that regardless of the frequency of the probe pulse, the decay times of the samples are comparable. In fact, the IRS does not affect the


relaxation dynamics of the pigment. However, the IRS influences the sign and the amplitude of the FTA measurements.

**Table 1.** Fitting decay time values of eumelanin suspensions, carried out in DMSO‐methanol mixture. The suspension was excited at 2.254 eV. Table adapted from [17].

#### **4.4. Ultrafast Raman loss spectroscopy as diagnostic tool**

dynamics is observed. In fact, as presented in **Figure 7a** (ℏpr = 1.741 eV), the FTA dynamics shows a positive signal which decreases in time in a multi‐exponential way. This signal is indicative of a photo‐induced absorption, where the intensity of the transmitted probe beam in the presence of excitation by the pump pulse is lower than the one collected in the absence of the pump beam. In fact, upon excitation at ℏpu = 2.254 eV, an excited‐state absorption (ESA) process, involving optically allowed transitions to higher‐energy states, occurs in the eumela‐ nin dispersion [19, 20]. When the response of the sample is probed at frequencies between the Stokes features and the incoming pump pulse, a profile like the one shown in **Figure 7b** is observed. Here, the dynamics is made out of a negative signal at ultrashort time delay followed by a sharp rise, ending by a bi‐exponential decay. The observed signal is a convolution of two processes: the absorption of the pigments and the IRS effect arising from the solvent. As observed in the previous case, the eumelanin absorption leads to a positive signal at

The second contribution occurs only in the first hundreds of femtoseconds instead. This is due to the fact that the vibrational coherence needed to achieve the inverse Raman scattering persists as long as pump and probe pulses are temporally overlapped. Since the frequency of the probe pulse resonates with one of the Stokes features, a reinforcement of the vibrational modes of the solvent follows. Due to the resonance at the Stokes frequency, a gain in the intensity of the probe beam is achieved and the IRS appears as an emission of photons as described by the theoretical model presented by Rai et al. [4]. An increase in transmitted intensity is registered as a negative signal in an FTA experiment. Being a stimulated process, the IRS intensity overwhelms the absorption signal carried by the eumelanin pigments until the vibrational coherence lasts. When the delay between pump and probe pulses increases, the IRS artifact disappears, revealing the positive absorption of the eumelanin. Right after, the

In **Figure 7c**, the FTA dynamics of the eumelanin probed at frequencies between the incom‐ ing pump beam and the anti‐Stokes features is presented. If ℏpu < ℏpr, a depletion of the ground state is achieved through the action of the pump pulse. This process is called ground‐state bleaching and results in a negative signal, since the transmitted intensity of the probe beam is higher when the sample is excited. However, the ground‐state bleaching cannot explain the presence of a sharp and intense positive peak in the first few hundreds of femtoseconds of the dynamics. Again, the IRS effect plays a major role at ultrashort delay times. In fact, the resonance between the incoming fields and the vibrational modes of the solvent occurring this time at the anti‐Stokes frequencies leads to the stimulated Raman process. The loss in intensity experienced by the probe pulse can be accounted for as addi‐ tional absorption, described by a positive signal. When the pump and probe pulse are no more overlapped in the sample, the phase‐matching condition for the IRS is not satisfied, and the eumelanin dynamics is disclosed, recovering the signal in a bi‐exponential fashion.

The decay time obtained from the fit of the eumelanin dynamics is reported in **Table 1** for the DMSO‐methanol suspension. It is worth noting that regardless of the frequency of the probe pulse, the decay times of the samples are comparable. In fact, the IRS does not affect the

suspension relaxes back to the ground state following a bi‐exponential decay.

every time delay between pump and probe pulses.

282 Raman Spectroscopy and Applications

The very need of high spatial and temporal resolution to investigate molecular reaction pathways has pushed toward the development of femtosecond stimulated Raman scattering. The aim is to be able to follow structural changes in molecules during a reaction occurring on short timescales, spanning from femtoseconds to picoseconds. The ability of femtosecond stimulated Raman spectroscopy lies in the high temporal resolution with which molecular vibrations can be collected, giving deep insights into reaction dynamics. Charge‐transfer processes have been intensely investigated by FSRS; for example, long‐debated studies on 4‐ (dimethylamino)benzonitrile, due to the discrepancy between the structural simplicity of this push‐pull molecule and the complexity of the excited electronic levels, have been recently come to an end. In fact, the crucial role played by intramolecular and solvent reorganizations has been at the forefront of a systematic investigation, regarding three different dynamics on various timescales: the ππ\* relaxation, the internal conversion, and the vibrational relaxation [21, 22]. By investigating the excited‐state proton transfer by FSRS, Fang et al. attributed to the skeletal motions the origin of the fluorescent form of a green fluorescent protein from *Aequorea victoria*, which is famous for its efficient bioluminescence [23]. Indeed, by looking separately at the low vibrational frequencies of specific modes, it was possible to identify an out‐of‐phase motion of the phenoxil ring in the chromophore, and thus to optimize the chemical structure of the chromophore for improving the excited‐state proton transfer. Another important role in which FSRS is actively utilized is to help reveal the role that molecular symmetry plays in vibrational coherence activity in photosynthetic systems (as carotenoids) and in photochem‐ istry. In particular, internal conversion processes and coupling between electronic states are ruled out [24, 25]. Finally, the vast majority of chemical reactions studied by FSRS concerns the isomerization, because of its key function in chromophores of high significance in biology. For example, Kuramochi et al. presented the first information pertaining to the vibrations in early instants of the photodynamics observed in the chromophore of the photoactive yellow protein. This study provided more insights on how to trigger the photoreceptive functions of the chromophore when embedded in the protein [26]. Kukura et al., instead, explored the spectral evolution of specific vibrational modes explaining how the activation of rhodopsin, a light receptor, is driven by geometric changes in the retinal backbone [27]. These are just few of the large number of examples that can be recalled to demonstrate the power of FSRS to unravel reaction coordinates, chemical configurations, and nuclear dynamics.

One of the specific methods enrolled by the femtosecond‐stimulated Raman spectroscopy relies on the IRS effect and is called femtosecond inverse Raman scattering (FIRS) [2], or ultrafast Raman loss spectroscopy (URLS) [4]. In URLS, the decrease in intensity of the probe beam, as described in Section 4.2, is completely described by the IRS effect and is used as fingerprint to follow in time the reaction pathways. Moreover, this spectroscopic tool shows some beneficial features missing in the general FSRS. The intensity of the Raman peaks at the anti‐Stokes frequencies results higher than what is measured at the Stokes frequencies (Raman gain), leading to a better signal‐to‐noise ratio [7, 9, 10]. Second, looking at the blue side of the pump pulse to identify the spectral features of the sample helps to reject the fluorescence signal, which appears on the red side [2]. Finally, the detector dynamic range has higher efficiencies on the anti‐Stokes than on the Stokes side, minimizing the noise levels and thus allowing for clearer imaging (FIRS microscopy) [28–32], for example, in tissues [33] and drug‐delivery processes [34–36].

In the previous paragraph, it was shown that investigating the temporal evolution of the signal at specific probing energies can be used to determine the influence of the IRS coherent artifact on the dynamics of the eumelanin pigments. Focusing now on the IRS features, the dynamics of specific bonds, induced by photoexcitation processes, can be probed [37, 38].

First, it is of crucial importance to identify the spectral features encountered in the FTA measurements, and ascribe them to specific vibrational modes. To this end, the Raman spectrum of the solvent mixture (DMSO‐methanol, 1:20 in ratio) was collected. As can be seen in **Figure 8a**, the Raman spectrum is dominated by three narrow peaks and a broad band. These features are recognized as follows: CO stretching and SO stretching in methanol and in DMSO, overlapping at 0.125 eV (peak I); CH2 bending in methanol at 0.177 eV (peak II); CH stretching in methanol at 0.352 and 0.365 eV (symmetric and antisymmetric vibrational mode), and in DMSO at 0.361 eV (peak III); OH stretching in methanol at 0.414 eV (peak IV) [39–41]. A direct correspondence of the Raman peaks shown in **Figure 8a** is found in the FTA meas‐ urements depicted in **Figure 8b**. In fact, the spectral evolution at ultrashort time delays shows specifically the same Raman features occurring symmetrically to the pump pulse at Stokes and anti‐Stokes frequencies.

Tuning the pump pulse to lower energies, the spectral features follow the energy shift, maintaining constant the energy difference between each of them and the pump pulse (spectra from red to blue in **Figure 8b**). Computing = ℏpu <sup>−</sup> ℏ , that is, the difference in energy between the pump pulse and the spectral features, this equals the Raman shift values displayed on the ‐axes for each Raman peak reported in **Figure 8a**. By this analysis, the authors proved that the observed coherent artifact in the FTA measurements at ultrashort time delay is a feature originating from the stimulated Raman‐scattering process [17].

receptor, is driven by geometric changes in the retinal backbone [27]. These are just few of the large number of examples that can be recalled to demonstrate the power of FSRS to unravel

One of the specific methods enrolled by the femtosecond‐stimulated Raman spectroscopy relies on the IRS effect and is called femtosecond inverse Raman scattering (FIRS) [2], or ultrafast Raman loss spectroscopy (URLS) [4]. In URLS, the decrease in intensity of the probe beam, as described in Section 4.2, is completely described by the IRS effect and is used as fingerprint to follow in time the reaction pathways. Moreover, this spectroscopic tool shows some beneficial features missing in the general FSRS. The intensity of the Raman peaks at the anti‐Stokes frequencies results higher than what is measured at the Stokes frequencies (Raman gain), leading to a better signal‐to‐noise ratio [7, 9, 10]. Second, looking at the blue side of the pump pulse to identify the spectral features of the sample helps to reject the fluorescence signal, which appears on the red side [2]. Finally, the detector dynamic range has higher efficiencies on the anti‐Stokes than on the Stokes side, minimizing the noise levels and thus allowing for clearer imaging (FIRS microscopy) [28–32], for example, in tissues [33] and drug‐delivery

In the previous paragraph, it was shown that investigating the temporal evolution of the signal at specific probing energies can be used to determine the influence of the IRS coherent artifact on the dynamics of the eumelanin pigments. Focusing now on the IRS features, the dynamics of specific bonds, induced by photoexcitation processes, can be probed [37, 38].

First, it is of crucial importance to identify the spectral features encountered in the FTA measurements, and ascribe them to specific vibrational modes. To this end, the Raman spectrum of the solvent mixture (DMSO‐methanol, 1:20 in ratio) was collected. As can be seen in **Figure 8a**, the Raman spectrum is dominated by three narrow peaks and a broad band. These features are recognized as follows: CO stretching and SO stretching in methanol and in DMSO, overlapping at 0.125 eV (peak I); CH2 bending in methanol at 0.177 eV (peak II); CH stretching in methanol at 0.352 and 0.365 eV (symmetric and antisymmetric vibrational mode), and in DMSO at 0.361 eV (peak III); OH stretching in methanol at 0.414 eV (peak IV) [39–41]. A direct correspondence of the Raman peaks shown in **Figure 8a** is found in the FTA meas‐ urements depicted in **Figure 8b**. In fact, the spectral evolution at ultrashort time delays shows specifically the same Raman features occurring symmetrically to the pump pulse at Stokes and

Tuning the pump pulse to lower energies, the spectral features follow the energy shift, maintaining constant the energy difference between each of them and the pump pulse (spectra

between the pump pulse and the spectral features, this equals the Raman shift values displayed on the ‐axes for each Raman peak reported in **Figure 8a**. By this analysis, the authors proved that the observed coherent artifact in the FTA measurements at ultrashort time delay is a feature

, that is, the difference in energy

from red to blue in **Figure 8b**). Computing = ℏpu <sup>−</sup> ℏ

originating from the stimulated Raman‐scattering process [17].

reaction coordinates, chemical configurations, and nuclear dynamics.

processes [34–36].

284 Raman Spectroscopy and Applications

anti‐Stokes frequencies.

**Figure 8.** (a) Raman spectrum of DMSO‐methanol mixture (*λ*ex = 488 nm, *P*incident = 6 mW, acquisition time: 30 s) and (b) Spectral evolution of the transient absorption signal detected at ∼ 0 in the DMSO‐methanol mixture for differ‐ ent pump‐pulse energies. The curves are vertically shifted for clarity. Figure taken from [17].

Once identified the Raman vibrational modes, it is possible by URLS to investigate them specifically and, in particular, to address their spectral evolution in time. This can give insights on the transient structure of the molecules and on the dynamics of the specific vibrational modes. Here, we report on a very preliminary analysis ran in such direction on the aforemen‐ tioned sample; in **Figure 9** the C─H Raman vibrational mode located at 2.641 eV, upon pumping the sample at ℏpu = 2.525 eV, is examined in terms of peak shift and intensity over time. The spectra shown in **Figure 9a** have been normalized to the intensity of the incoming pump pulse. The peak position experiences a shift toward the blue region of the spectrum as the temporal decoupling of pump and probe pulses occurs. This can be clearly seen in **Figure 9b** within the first picoseconds. At the same time, its intensity decreases at an exponential rate (**Figure 9c**).

This example should visually explain the potential and the strength of the URLS as spectro‐ scopic tool. In fact, the results here collected, and the many more presented in literature [37, 38, 42], unambiguously demonstrate the ability of the technique to select specific bonds and study their dynamics upon photoexcitation, at ultrafast timescales. However, further investi‐ gations are required to relate the experimental observations to the ultimate structure of the solvent molecules.

 **Figure 9.** (a) ∆ signal in the blue region of the pump pulse. The peak taken into account is the anti‐Stokes feature related to the stretching of the C─H bond in methanol and DMSO, (b) evolution of the Raman peak position as func‐ tion of delay time between the pump and the probe pulses, and (c) temporal evolution of the intensity of the Raman peak.

#### **5. Conclusions**

 In this chapter, the authors presented a complete description of the inverse Raman scattering effect, one of the four‐wave mixing processes contributing to the stimulated Raman scattering process. Feynman dual‐time line diagrams and energy level diagrams were used to explain the theory behind the IRS effect. Once addressed the nature of the IRS effect, its close relation with the transient absorption pump‐probe experiment was described, as well as the influence on the temporal evolution of the sample dynamics. To this end, the dynamics of eumelanin dispersions carried out at different exciting energies were shown, pointing out the crucial role of the IRS in the relaxation dynamics of the sample. Finally, the implementation of the IRS effect as diagnostic tool in determining the structures and interactions among molecules was presented. In fact, the high resolution achieved in the time and spectral domains showed by ultrafast loss Raman spectroscopy enables to follow specifically the electronic structure of molecules while undergoing chemical reactions, even on ultrafast timescales.
