Spektrale Modulationen in der Femtosekunden-Stimulierten Raman-Mikroskopie

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.