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Abstract

In this thesis, the high degree of control offered by the combination of a broadband femtosecond excitation source and a pulse shaper is exploited to achieve high spectral resolution nonlinear optical microscopy. Especially the coherent anti-Stokes Raman scattering (CARS) process is very important for nonlinear microscopy since it provides label-free three-dimensional contrast based on the vibrational atomic motion. The flexibility of the developed setup not only allows to switch between optimal conditions for the generation of CARS and other nonlinear signals but enables their simultaneous measurement. The complementary contrast of signals like second-harmonic generation (SHG), two-photon fluorescence (TPEF) and the CARS process in a multimodal imaging scheme provides additional information to identify structures and the composition of complex biological samples. It is shown how the individual control over the frequencies taking part in the CARS process can be used to achieve resonant vibrational imaging, overcoming the limitations usually associated with the unspecific excitation of using broadband pulses. Within this tailored spectral focusing approach, CARS signals are enhanced by an order of magnitude and SHG and TPEF intensities are boosted even more, as demonstrated by multimodal imaging of Skin tissue. Furthermore, the method can be directly applied to control the difference frequency generation process occurring when focusing the laser on a nonlinear crystal, to form a tunable broadband infrared (IR) light source. Besides the direct characterization of the IR spectrum by this method, absorption spectroscopy becomes possible in a single-beam approach. A whole new frequency region is hereby unlocked, paving the way for retrieving complementary information from Raman as well as IR interactions in the same setup for the first time.