A system for time-encoded non-linear spectroscopy and microscopy

Raman scattering can be applied to biological imaging to identify
molecules in a sample without the need for adding labels. Raman
microscopy can be used to visualize functional areas at the
cellular level by means of a molecular contrast and is thus a
highly desired imaging tool to identify diseases in biomedical
imaging. The underlying Raman scattering effect is an optical
inelastic scattering effect, where energy is transferred to
molecular excitations. Molecules can be identified by monitoring
this energy loss of the pump light, which corresponds to a
vibrational or rotational energy of the scattering molecule. With
Raman scattering, the molecules can be identified by their specific
vibrational energies and even quantified due to the signal height.
This technique has been known for almost a century and finds vast
applications from biology to medicine and from chemistry to
homeland security. A problem is the weak effect, where usually only
one in a billion photons are scattered. Non-linear enhancement
techniques can improve the signal by many orders of magnitude. This
can be especially important for fast biomedical imaging of highly
scattering media and for high resolution spectroscopy, surpassing
the resolution of usual spectrometers. In this thesis a new system
for stimulated Raman spectroscopy (SRS) and hyperspectral Raman
microscopy with a rapidly wavelength swept laser is presented. A
time-encoded (TICO) technique was developed that enables direct
encoding of the Raman transition energy in time and direct
detection of the intensity change on the Stokes laser by employing
fast analogue-to-digital converter (ADC) cards (1.8 Gigasamples/s).
Therefore, a homebuilt pump laser was developed based on a
fiber-based master oscillator power amplifier (MOPA) at 1064 nm and
extended by a Raman shifter that can shift the output wavelength to
1122 nm or 1186 nm. This is achieved by seeding the Raman
amplification in the fiber with a narrowband 1122 nm laser diode.
Surprisingly, this also leads to narrowband (0.4 cm-1) cascaded
Raman shifts at 1186 nm and 1257 nm, which is in contrast to the
usually broadband spontaneous Raman transition in fused silica. The
underlying effect was examined and therefore concluded that it is
most probably due to a combined four-wave-mixing and cascaded Raman
scattering mechanism. Experimentally, the narrowband cascaded Raman
line was used to record a high-resolution TICO-Raman spectrum of
benzene. As Raman Stokes laser, a rapidly wavelength swept Fourier
domain mode-locked (FDML) laser was employed which provides many
advantages for SRS. The most important advantages of this fiber
based laser are that it enables coverage of the whole range of
relevant Raman energies from 250 cm-1 up to 3150 cm-1, while being
a continuous wave (CW) laser, which at the same time allows high
resolution (0.5 cm-1) spectroscopy. Further, it enables a new dual
stage balanced detection which permits shot noise limited SRS
measurements and, due to the well-defined wavelength sweep, the
TICO-Raman technique directly provides high-quality Raman spectra
with accurate Raman transition energy calibration. This setup was
used for different applications, including Raman spectroscopy and
non-linear microscopy. As results, broadband Raman spectra are
presented and compared to a state-of-the-art spontaneous Raman
spectrum. Furthermore, several spectroscopic features are explored.
For first imaging results, samples were raster scanned with a
translational stage and at each pixel a TICO-Raman spectrum
acquired. This led to a hyperspectral Raman image which was
transformed into a color-coded image with molecular contrast.
Biological imaging of a plant stem is presented. The setup further
allowed performing multi-photon absorption imaging by two-photon
excited fluorescence (TPEF). In summary, this thesis presents the
design, development and preliminary testing of a new and promising
platform for spectroscopy and non-linear imaging. This setup holds
the capability of biological multi-modal imaging, including
modalities like optical coherence tomography (OCT), absorption
spectroscopy, SRS, TPEF, second harmonic generation (SHG),
third-harmonic generation (THG) and fluorescence lifetime imaging
(FLIM). Amongst the most promising characteristics of this setup is
the fiber-based design, paving the way for an endoscopic imaging
setup. Already now, this makes it a robust, alignment-free,
reliable and easy-to-use system.