Polarization-sensitive, multiplex coherent anti-Stokes Raman scattering (ps-MCARS) has been used to detect the vibrational bands of the highly luminescent zinc(II)-octaethylporphyrin (Zn-OEP). We show here that ps-MCARS can be used to measure the vibrational bands under resonant conditions. Polarization-sensitive CARS further helps to resolve composite and overlapping Raman bands on the basis of variations in vibrational symmetry. This paper also exemplifies the advantages of multiplex CARS (MCARS) spectroscopy carried out under tight focusing conditions as proposed earlier. We demonstrate that a 'microscopic' CARS focusing and collection system in combination with the multiplex technique allows a reduction in the amount of sample and a shortening of the measurement time. We analyse the relationship between the coherent vibrational phases and the symmetry of the different vibrational band of Zn-OEP skeletal modes. The values of the coherent vibrational phases are similar for vibrational bands with the same symmetry.

Polarization sensitive multiplex spectroscopy of resonance coherent anti-Stokes Raman scattering (CARS) of copper(II)-tetraphenylporphyrin in solution (CH2Cl2) is reported. The measurements were performed in the Q band range of the porphyrin electronic absorption. Four polarized CARS spectra were resolved in 1300–1650 cm−1 Raman range and were simultaneously fitted with a single set of vibrational parameters (band positions, bandwidths, amplitudes, depolarization ratios, and phases). The obtained coherent vibrational phases of A1g, A2g, and B1g skeletal modes of the porphyrin macrocycle appeared to correlate strongly with the mode vibrational symmetry. The origin of such correlation is analyzed within a model of the third-order nonlinear electric susceptibility χ(3). The proposed model is based on multidimensional displaced harmonic oscillator in the Herzberg–Teller expansion of Raman polarizability. The coherent vibrational phases of modes of different symmetry classes are directly affected by the symmetry dependent vibronic couplings between the pairs of Qx, Qy and Bx, By electronic transitions of the porphyrin. The phase contrast between the modes of various symmetries is most pronounced near the Q00 resonance.

The first application of polarization-sensitive multiplex coherent antiStokes Raman spectroscopy (MCARS) in the absence of resonance enhancement to the resolution of the secondary structure of a protein in solution is reported. Polarization MCARS spectra of bovine albumin in D20 were obtained in the range 1370 to 1730 cm -I with the aid of the background suppression technique. The spectra were fitted simultaneously with a single set of parameters (band positions, bandwidths, amplitudes, and depolarization ratios). Polarized Raman spectra simulated with these parameters revealed a good correspondence with the spontaneous Raman spectra measured. The broad amide I' band was decomposed assuming the three major secondary conformations of protein, of which the contribution of B-sheet structure was found to be negligible. Relative weights of a-helix and random coil conformations agree well with the estimates obtained with Raman and circular dichroism (CD) spectroscopies.

Polarization-semitive 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 x(3) components and purely anisotropic and anti-symmetric scattering contributions were resolved within the frequency non-degenerate CSRS scheme. Eight bands of oxyhaemoglobm and five bands of deoxyhaemoglobin were observed in the range 1500-1680 cm-'. Simultaneously fitting sets of polarization spectra provided vibrational parameters (positions, bandwidths, amplitudes, pbases and CSRS depolarization ratios) for each compound. Major bands were assigned to the non-totally symmetric vl,, , vI1 and ~1 9 modes of the porphyrin macrocycle. The phases cakulated 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 p:212 of the anomalously polarized vI9 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.

We report polarisation sensitive Coherent Stokes Raman Scattering (CSRS) measurements of oxy-and deoxyhemoglobm in aqueous solutions that were carried out under electronic resonance with the Q absorption bands. All independent susceptibility Xt'3' components as well as anisotropic and anti-symmetric scattering contributions were resolved within frequency non degenerate CSRS scheme. Eight bands of oxy-and five of deoxyhemoglobin were observed in the range 1500-1680 cm1. Each set of dispersion profiles was simultaneously fitted with a set ofparameters including band positions, widths, amplitudes, phases and, importantly, all the CSRS depolansation ratios. On this basis the major bands were assigned to non-totally symmetric v10, v11 and v19 modes ofthe porphyrin macrocycle. These displayed a clear correlation ofvibrational phase with the mode symmetry. Of eight bands resolved in oxyHb spectra three were attributed to features of intermediate deoxyHb, caused by a partial photolysis of oxyhemes. On a nanosecond time scale they were found essentially similar to those found for stable deoxyHb. A detectable isotropy was observed for all non-totally symmetric modes of both oxy-and deoxyhemes. The v10 and v11 modes were found to exhibit anti-symmetry as well. The decrease in depolansation ratio p212 of anomalously polarised v19 mode from 7.7 (oxyheme) to 4.3 (deoxyheme) was detected. The latter evidenced heme deformation related with a further doming that occurs upon release of oxygen. and Qoi absorption bands. Our aim was to analyse these modes for both oxy-and deoxyHb on the basis of their CRS depolarisation ratios that should reveal the symmetry of scattering tensor, and therefore expose distortions of O-8194-1715-7/95/$6.O0