Analysis of Pulses Bandwidth and Spectral Resolution in Femtosecond Stimulated Raman Scattering Microscopy

Featured Application Stimulated Raman microscopy, based on two femtosecond pulsed lasers and with a spectral resolution of about 56 cm(-1), is demonstrated to be sufficient in order to distinguish protein and lipid bands in the C-H region. In the last decade, stimulated Raman scattering (SRS) imaging has been demonstrated to be a powerful method for label-free, non-invasive mapping of individual species distributions in a multicomponent system. This is due to the chemical selectivity of SRS techniques and the linear dependence of SRS signals on the individual species concentrations. However, even if significant efforts have been made to improve spectroscopic coherent Raman imaging technology, what is the best way to resolve overlapped Raman bands in biological samples is still an open question. In this framework, spectral resolution, i.e., the ability to distinguish closely lying resonances, is the crucial point. Therefore, in this paper, the interplay among pump and Stokes bandwidths, the degree of chirp-matching and the spectral resolution of femtosecond stimulated Raman scattering microscopy are experimentally investigated and the separation of protein and lipid bands in the C-H region, which are of great interest in biochemical studies, is, in principle, demonstrated.