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

Published: June 15, 2015, 11 a.m.

b'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.\\nIn 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).\\nTherefore, 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. \\t\\nAs 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. \\nThis 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). \\nIn 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.'