## 3 Results and Discussion  
Autocorrelation characterizations of the pulsed laser beams were examined using the aforementioned autocorrelator. The Ti:Sa and OPO laser emission wavelengths were fixed at 811 nm and at 1074 nm, respectively. The collected autocorrelation function of the Ti:Sa and OPO is reported in Figure 4a,b, respectively. As can be seen in these figures, the pulses emitted by the lasers show a Gaussian-like distribution, as expected. Therefore, with the aim to evaluate the pulse width value, both the autocorrelation traces were Gaussian-fitted as displayed in Figure 4a,b, and the corresponding FWHM of the Gaussian curves was calculated to be about 341 fs and 357 fs for Ti:Sa and OPO, respectively. Since the pulse follows a Gaussian line shape and considering Equation (1), the factor of 1.414 was applied to our evaluation, leading to an estimation of the pulses' width duration at the input of the microscope of about \(\tau=241\) fs for Ti:Sa and \(\tau=253\) fs for OPO, respectively. Thus, a small broadening of OPO was observed, while a major one was retrieved for Ti:Sa. These broadenings are related to the dispersion arising during propagation through several optical elements along the beams' path; in particular, the higher widening observed for Ti:Sa can be explained considering that in its optical path a Pockels cell is employed. In Table 1, the features of the two lasers and results of their autocorrelation measurements are summarized. Figure 4: Autocorrelation trace of the pulsed laser sources (**a**) Ti:Sa and (**b**) OPO, respectively. Results of the Gaussian fit are also reported. The second order group velocity dispersion can be evaluated for each pulsed source by applying Equations (2) and (3). The obtained values are GDD = 9905 fs2 and GDD = 11,177 fs2 for Ti:Sa and OPO, respectively. In our case, pulses were not equally chirped. To complete the single beam characterization, the laser spectra were acquired by an optical spectrum analyzer and results are shown in Figure 5, when Ti:Sa is tuned to 811 nm and OPO to 1074 nm, respectively. The resultant bandwidths are approximately the same for both pulsed laser sources and were of about 6.1 nm. The measured FWHM of Ti:Sa and OPO cross-correlation was 371 fs; since the convolution of two Gaussian functions is another a Gaussian function [31], Equation (1) can also be applied when cross-correlation measurements are performed, leading to a pulse duration of 262 fs (see Figure 6). Thus, the obtained experimental spectral bandwidth, which is given by the FWHM of cross-correlation in the frequency domain, was of 56 cm\({}^{-1}\) \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Laser Source**} & **Wavelength Range [nm]** & **Pulse Duration** & **Repetition Rate** & **Widened Pulse Duration [fs]** \\ \hline Ti:Sa & 740–880 & 140 & 80 & 241 \\ OPO & 1000–1600 & 200 & 80 & 253 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Pulsed laser sources properties. Figure 5: Optical spectra of the pulsed laser sources (**a**) Ti:Sa and (**b**) OPO, respectively. Results of the Gaussian fit are also reported. Figure 6: Cross-correlation measure and its Gaussian fit. The retrieved spectral resolution is very useful in biological sample imaging when both lipid and protein bands need to be investigated by SRS microscopy by simply regulating the frequency either of the pump or the Stokes beams in successive scans. The corresponding two stretching signals are CH\({}_{2}\) (2845 cm\({}^{-1}\)) and CH\({}_{3}\) (2940 cm\({}^{-1}\)), respectively; thus, these bands can be collected by choosing one Raman shift at a time, allowing the imaging of the map distributions of the lipid and protein on the same field of the sample.