Polarization-sensitive resonance CSRS of deoxy-and oxyhaemoglobin

Polarization-sensitive coherent Stokes Raman scattering (CSRS) measurements of oxy- and deoxyhaemoglobin in aqueous solutions are reported. The excitation wavelengths used were chosen in the region of the Q absorption bands to achieve twofold electronic resonance. The dispersion profiles of all independent susceptibility (3) components and purely anisotropic and anti-symmetric scattering contributions were resolved within the frequency non-degenerate CSRS scheme. Eight bands of oxyhaemoglobin and five bands of deoxyhaemoglobin were observed in the range 1500-1680 cm-1. Simultaneously fitting sets of polarization spectra provided vibrational parameters (positions, bandwidths, amplitudes, phases and CSRS depolarization ratios) for each compound. Major bands were assigned to the non-totally symmetric v10, v11 and v19 modes of the porphyrin macrocycle. The phases calculated exhibited a correlation with the symmetry of the vibrations. On the basis of the spectral fits, the three additional peaks arising in the oxyhaemoglobin spectra could be ascribed to the bands of intermediate deoxyhaemoglobin. The occurrence is due to the partial photolysis of oxyhaemoglobin. Vibrational parameters of these bands were found to be essentially similar to the parameters of the bands observed in the spectra of the stable deoxyhaemoglobin. Despite the asymmetric character predicted, the major bands were all contributed to by a considerable isotropic component. A decrease in the depolarization ratio PR1212 of the anomalously polarized v19 mode from 7.7 in oxyhaemoglobin to 4.3 in deoxyhaemoglobin was observed. Such a decrease in anti-symmetric character of the vibration on release of the ligand supports the occurrence of deformation of the haem ring system

Polarization-xensitive CARS of excited-state rhodamine 6G: induced ansisotropy effects on depolarization ratios

Resonance polarization-sensitive coherent anti-Stokes Raman scattering (PS CARS) spectra of the electronic ground state and excited singlet S1 state of rhodamine 6G in ethanol were obtained with the use of the pump-probe technique with nanosecond time resolution. Variation of the polarization orientation of the pump laser beam showed differences in the excited-state spectra due to optically induced anisotropy. The pure electronic susceptibility of ground-state rhodamine 6G was shown to be small in comparison with nonresonant susceptibility of the solvent, and was neglected in further analyses. The pure electronic susceptibility of excited rhodamine 6G was examined by coherent ellipsometry. The complex third-order susceptibility was analyzed by means of a nonlinear least-squares fit program that provides detailed information on the Raman vibration parameters, including depolarization ratios and phases. In the isotropic case the measured depolarization ratios are close to 1/3, whereas in the anisotropic case, ground-state depolarization ratios are 0.5–0.65 and in the excited state 0.17–0.22. Estimated depolarization ratio changes in ground-state and excited-state rhodamine 6G are in agreement with theoretically predicted values in the case of induced anisotropy under the assumption of parallel dipole moments of the CARS process. The effects of possible changed molecular structure or symmetry and changed enhancement of different electronic transitions cannot be determined without making some assumptions about one of these effects. The obtained phase differences reflect different enhancements and vibronic coupling for ground-state and excited-state vibrations. The ground-state and excited-state hyperpolarizabilities, $\gamma{^E}{s_0}$ and $\gamma{^E}{s_1}$, of rhodamine 6G were estimated to be 3.8·10−35 esu and 27.4·10−35 esu, respectively

In vivo confocal Raman microspectroscopy of the skin: Noninvasive determination of molecular concentration profiles

Confocal Raman spectroscopy is introduced as a noninvasive in vivo optical method to measure molecular concentration profiles in the skin. It is shown how it can be applied to determine the water concentration in the stratum corneum as a function of distance to the skin surface, with a depth resolution of 5 mum. The resulting in vivo concentration profiles are in qualitative and quantitative agreement with published data, obtained by in vitro X-ray microanalysis of skin samples. Semi-quantitative concentration profiles were determined for the major constituents of natural moisturizing factor (serine, glycine, pyrrolidone-5-carboxylic acid, arginine, ornithine, citrulline, alanine, histidine, urocanic acid) and for the sweat constituents lactate and urea. A detailed description is given of the signal analysis methodology that enables the extraction of this information from the skin Raman spectra. No other noninvasive in vivo method exists that enables an analysis of skin molecular composition as a function of distance to the skin surface with similar detail and spatial resolution. Therefore, it may be expected that in vivo confocal Raman spectroscopy will find many applications in basic and applied dermatologic research