Andreas Volkmer

We have analyzed the chemical variety obtained by Miller-Urey-type experiments using nuclear magnetic resonance (NMR) spectroscopy and coherent anti-Stokes Raman scattering (CARS) spectroscopy, gas chromatography followed by mass spectrometry (GC/MS) and two-dimensional gas chromatography followed by mass spectrometry (GCxGC/MS). In the course of a running Miller-Urey-type experiment, a hydrophobic organic layer emerged besides the hydrophilic aqueous phase and the gaseous phase that were initially present. The gas phase mainly consisted of aromatic compounds and molecules containing C≡C or C≡N triple bonds. The hydrophilic phase contained at least a few thousands of different molecules, primarily distributed in a range of 50 and 500 Da. The hydrophobic phase is characterized by carbon-rich, oil-like compounds and their amphiphilic derivatives containing oxygen with tensioactive properties. The presence of a wide range of oxidized molecules hints to the availability of oxygen radicals. We suggest that they intervene in the formation of alkylated polyethylene glycol (PEG) in the oil/water interface. CARS spectroscopy revealed distinct vibrational molecular signatures. In particular, characteristic spectral bands for cyanide compounds were observed if the broth was prepared with electric discharges in the gaseous phase. The characteristic spectral bands were absent if discharges were released onto the water surface. NMR spectroscopy on the same set of samples independently confirmed the observation. In addition, NMR spectroscopy revealed overall high chemical variability that suggests strong non-linearities due to interdependent, sequential reaction steps.

We propose an approach, based on wavelet prism decomposition analysis, for correcting experimental artefacts in a coherent anti-Stokes Raman scattering (CARS) spectrum. This method allows estimating and eliminating a slowly varying modulation error function in the measured normalized CARS spectrum and yields a corrected CARS line-shape. The main advantage of the approach is that the spectral phase and amplitude corrections are avoided in the retrieved Raman line-shape spectrum, thus significantly simplifying the quantitative reconstruction of the sample’s Raman response from a normalized CARS spectrum in the presence of experimental artefacts. Moreover, the approach obviates the need for assumptions about the modulation error distribution and the chemical composition of the specimens under study. The method is quantitatively validated on normalized CARS spectra recorded for equimolar aqueous solutions of D-fructose, D-glucose, and their disaccharide combination sucrose.

Coherent Raman Scattering Microscopy. Edited by Ji-Xin Cheng and X. Sunney Xie © 2013 CRC Press/ Taylor & Francis Group, LLC. ISBN: 978-1-4398-6765-5. 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 2.2 Classical Wave Propagation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 2.2.1 Electric Field and Material Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 2.2.2 Wave Equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47 2.2.3 Angular Spectrum Representation of the Electric Field . . . . . . . . . . . . . 48 2.3 Coherent Raman Scattering in the Plane Wave Approximation . . . . . . . . . . . . . 50 2.3.1 CARS in the Plane Wave Approximation . . . . . . . . . . . . . . . . . . . . . . . . . . 53 2.3.2 SRS in the Plane Wave Approximation . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 2.4 Formation of the Focal Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 2.4.1 Transformation by a Lens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 2.4.2 Tightly Focused Field . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 2.5 Coherent Raman Scattering in Tightly Focused Volumes . . . . . . . . . . . . . . . . . . .61 2.5.1 Spatially Dependent Polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61 2.5.2 Far-Field Dipole Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61 2.5.3 CARS Far-Field Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 2.5.4 SRS Far-Field Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 2.6 Imaging of Objects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69 2.6.1 Imaging with CRS Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69 2.6.2 Linear Scattering Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 2.6.3 Nonlinear Interference Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 2.7 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74

At present, tumor diagnostic imaging is commonly based on hematoxylin and eosin or immunohistochemical staining of biopsies, which requires tissue excision, fixation, and staining with exogenous marker molecules. Here, we report on label-free tumor imaging using confocal spontaneous Raman scattering microspectroscopy, which exploits the intrinsic vibrational contrast of endogenous biomolecular species. We present a chemically specific and quantitative approach to monitoring normal human skin cells (keratinocytes and fibroblasts) as well as the human HaCaT in vitro skin carcinogenesis model and the tumor-derived MET in vivo skin cancer progression model. Mapping the amplitudes of two spectrally well isolated Raman bands at 752 and 785 cm^–1 allowed for direct visualization of the distributions representative of tryptophan-rich proteins and nucleic acids, respectively, with subcellular spatial resolution. Using these Raman markers, it was feasible to discriminate between normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (NHDF) and to confine all tumorigenic cells from both the NHEK and NHDF. First evidence for the successful application of the proposed intracellular nucleic acid and tryptophan Raman signatures for skin cancer diagnosis was further demonstrated in an organotypic cutaneous squamous cell carcinomas model, allowing for the identification of tumor cells and their surrounding stroma in the tissue context.

Near bandwidth-limited 7-fs-pulses were delivered at the foci of high-NA objectives by employing broadband mirrors for dispersion compensation. The impact of 7-fs-pulses on the signal generation efficiency and contrast in nonlinear optical imaging was investigated.