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Plötz, Evelyn Christine (2011): Spektrale Modulationen in der Femtosekunden-Stimulierten Raman-Mikroskopie. Dissertation, LMU München: Faculty of Physics
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Abstract

Femtosecond stimulated Raman microscopy (FSRM) is an upcoming technique in vibrational microscopy that combines optical imaging with the chemical sensitivity of Raman scattering. It allows for the quantitative, space resolved representation of microscopic samples. In FSRM, chemical contrast relies on femtosecond stimulated Raman scattering (FSRS), a nonlinear Raman process providing signal levels, that are strongly amplified with respect to spontaneous Raman scattering. Therefore, FSRM benefits from shortened exposure times and improved signal/noise-ratios. Even though FSRS is an optically nonlinear effect, its spectral signature resembles the (polarised) spontaneous Raman spectra. Autofluorescence of the sample does not contribute to the FSRS signature. FSRM turns out to be suitable for chemical imaging of biological systems, as cells and tissues. Since their signal strengths are quite low, data with high signal/noise-ratios is essential for the specific analysis of the sample. These requirements can be provided by FSRM. Moreover, the linear dependency of the FSRS signal on the sample concentration facilitates the quantitative analysis of its molecular composition. Given that tissue shows a wide heterogeneity on the molecular level, spectrally broad information, which is indeed provided by FSRM, is indispensable for chemically sensitive imaging. The intention of the present work was to establish FSRM as a tool for biological and medical imaging. FSRM is a scanning technique that relies on the nonlinear interaction of two ultrashort laser pulses with a Raman active medium. One of them is a spectrally narrow and intense picosecond pulse (Raman pump pulse), the other a spectrally broad and weak femtosecond pulse (Raman probe pulse). Both pulses are coupled collinearly into a scanning microscope where stimulated Raman scattering occurs in the sample positioned at the focus. This interaction leads to a spectral modification of the Raman probe pulse, which is recorded by the aid of a multi-channel detector. Its FSRS spectrum can be obtained by referencing the femtosecond pulse in presence of the Raman pump pulse to the one in absence. By raster-scanning the sample space-resolved FSRS spectra are retrieved. In the first FSRM setup a laser/amplifier system served as laser source. Its peak intensities are not compatible with the requirements of biological systems. Therefore a new light source has been developed for FSRM. It is based on a laser oscillator running at high repetition rate, which provides ultrashort femtosecond pulses with a spectral width over more than 3000 cm-1. These serve directly as Raman probe pulses. The Raman pump pulse is generated out of the laser spectrum by means of a ytterbium based fiber amplifier. Stimulated Raman microscopy is considered as a disturbance-free, nonlinear microscopy technique. In the context of this work, a further nonlinear contribution to the FSRS signal is reported for the first time. It shows up as a strong oscillation of the baseline in the spectrum and deteriorates the signal/noise-ratio as well as the spectral selectivity. This background can be identified as a spectral interference between the Raman probe pulse and an additional electric field, which is generated by a four-wave-mixing process between the Raman pump and Raman probe pulse. Properties and methods to suppress the spectral interferences are presented.