Vibrational imaging based on stimulated Raman scattering microscopy

A stimulated Raman scattering microscope with near-infrared picosecond laser pulses at high repetition rates (76MHz) and radio-frequency lock-in detection is accomplished. Based on stimulated Raman loss detection, we demonstrate noninvasive point-by-point vibrational mapping of chemical and biological samples with high sensitivity and without the requirement for labeling of the sample with natural or artificial fluorophores. We experimentally demonstrate a major benefit of this technique, which is the capability to respond exclusively to the linear Raman-resonance properties of the sample, thus allowing a direct quantitative interpretation of image contrast in terms of the number density of Raman-active modes. Recent advances in nonlinear coherent microscopy have attracted much interest because it offers three-dimensional visualization of microscopic objects within a complex and heterogeneous biological sample, i.e. a living cell or tissue, without the requirement for labeling the object of interest with external fluorophores or auto-fluorescent proteins. Among the coherent microscopies, those based on coherent Raman scattering (CRS) provide vibrational contrast intrinsic to and characteristic of chemical species, which is similar to confocal spontaneous Raman scattering microscopy. The applicability of the latter, however, is often limited by the inherently weak Raman scattering cross sections. In contrast, because of the coherent nature of the CRS signal, in which the molecular bonds oscillate in phase and the vibrational states are actively pumped, the Raman signal is significantly enhanced. Here, pump and Stokes incident electromagnetic fields with frequencies ωp and ωS (ωp > ωS), respectively, interact 1 Present address: Birla Institute of Technology and Science Pilani, Goa Campus, Goa 403726, India. 2 Present address: Max-Planck-Institut für Metallforschung, Heisenbergstraße 3,70569 Stuttgart, Germany. 3 Author to whom any correspondence should be addressed. New Journal of Physics 11 (2009) 033026 1367-2630/09/033026+09$30.00 © IOP Publishing Ltd and Deutsche Physikalische Gesellschaft