Gerald W. Lucassen

Waveguide CARS Spectroscopy: A New Method for Background Suppression, Using Dielectric Layers as a Model:

Waveguide Coherent Anti-Stokes Raman Spectroscopy (CARS) can be used to measure Raman-active vibrations in thin-layer dielectric waveguides. In waveguide CARS experiments, background-free spectra can be obtained when asymmetric mode combinations are applied. The degree of suppression depends on the waveguide parameters and the wavelengths used. A new method using scanning pump and Stokes beams in waveguide CARS experiments is presented, which allows the possibility of maintaining full background suppression conditions over large spectral intervals. A small controlled change in the tuning conditions results in a heterodyning of the signal with a small amount of background, thereby enhancing the small resonant signals. Several simulations for dielectric waveguides are given.

Compensating pulse-to-pulse fluctuations and increasing spectral reproducibility of phase-matched CARS measurements.

The compensation of pulse-to-pulse fluctuations and the improvement of spectral reproducibility of scanned coherent anti-Stokes Raman scattering (CARS) spectra of dispersing media are discussed. A simple reference CARS setup is presented that needs a minimum number of optical components as a result of using the same optical path for both signals. Variations in spectral profile were found to be caused by mechanical play in the translation stage, which is used to adjust the matching angle. Using retroreflection, the matching angle adjustment is made insensitive to these mechanical imperfections. The multiple interference of probe and signal beam that may occur in thin cuvette walls and its effect on the detected CARS signals are shown, and possible solutions are discussed.

Nonresonant background suppression in CARS spectra of dispersive media using phase mismatching

A nonresonant background suppression technique using coherent cancellation through phase mismatching is discussed and applied for a noncollinear beam configuration. A cuvette structure consisting of a glass, a sample, and a glass layer is regarded. Phase mismatching is shown to be a useful method to suppress nonresonant contributions from cuvette glass walls as well as those originating from the sample. A numerical calculation reveals a limit for the background suppression which can be achieved with this technique. Measurements using ethanol as a sample show the possibility to compensate the nonresonant background originating from the cuvette walls and to effectively suppress the nonresonant contribution in the spectrum of the sample by a factor of 10-50, yielding Lorentzian bands biased by a constant background. Direct measurement of depolarization ratios without interfering nonresonant background is demonstrated for ethanol and shows that this technique can readily be combined with the polarization sensitive CARS technique.

Polarization-Sensitive CARS of Excited-State Rhodamine 6G: Induced Anisotropy 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, , of rhodamine 6G were estimated to be 3.8·10−35 esu and 27.4·10−35 esu, respectively.

Possibilities and limitations of off-resonance polarization sensitive cars of short chain proteins

Polarization sensitive CARS in the absence of resonance enhancement is applied to a short chain protein. The minimum concentration to record polarization sensitive CARS spectra of protein solutions is estimated to be 10 mg/ml. The effects limiting the protein concentration are discussed and regarded from an experimental point of view. Signal strength and line parameters of polarization sensitive CARS spectra of the short chain protein Lysyl-Tryptophyl-Lysine are compared with those of a normal Raman spectrum.

Nonresonant background suppression in coherent anti-Stokes Raman scattering spectra of dissolved molecules

The Phase Mismatching CARS technique uses the nonresonant signal of the glass cuvette walls to coherently compensate the nonresonant signal of the sample yielding a method of measuring background free CARS spectra. The technique is especially suitable under (pre) resonant nditions where many molecular Raman vibrationsNend to have depolarization ratios p close to the nonresonant depolarization ratio p In comparison with the Polarization Sensitive CARS technique the Phase Mismatching technique has a higher net suppression ratio because of the lower loss of vibration resonant signal. The combination of the Phase mismatching and Polazation Sensitive CARS technique yields the possibility of direct measurements of XIjkl components and resolving overlapping bands with different depolarization ratios without an interfering nonresonant background. 1.

Polarization properties of v10, v11, and v19 modes of (de)oxyheme of hemoglobin as obtained from three-color CSRS

We report polarization sensitive coherent Stokes Raman scattering (CSRS) measurements of oxy- and deoxyhemoglobin in aqueous solutions that were carried out under electronic resonance with the Q absorption bands. All independent susceptibility (Chi) (3) components as well as anisotropic and anti-symmetric scattering contributions were resolved within frequency nondegenerate CSRS scheme. Eight bands of oxy- and five of deoxyhemoglobin were observed in the range 1500 - 1680 cm-1. Each set of dispersion profiles was simultaneously fitted with a set of parameters including band positions, widths, amplitudes, phases and, importantly, all the CSRS depolarization ratios. On this basis the major bands were assigned to non-totally symmetric (nu) 10, (nu) 11, and (nu) 19 modes of the porphyrin macrocycle. These displayed a clear correlation of vibrational 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 (nu) 10 and (nu) 11 modes were found to exhibit anti-symmetry as well. The decrease in depolarization ratio (rho) 1212R of anomalously polarized (nu) 19 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.

Polarization-sensitive coherent Raman spectroscopy of proteins in solutions

Polarization sensitive coherent Raman spectroscopy (PCARS/PCSRS) was applied to study proteins in solutions in both two and three color configurations. In off-electron resonant PCARS the main problem is the nonresonant background, arising from the solvent. The advantage of PCARS is the resolving power as is demonstrated in the amide-I region of some proteins. Three color PCSRS was used to measure independently the (chi) (3) components of hemoglobin resonantly excited in the (alpha) and (beta) absorption bands. From a simultaneous fit on different PCSRS spectra vibrational parameters were determined including phases and depolarization ratios. Besides the polarization and phase characterization of hemoglobin bands, we decomposed the vibrational amplitudes and phases into electronic contributions. Together with amplitudes, the phases contain information on electronic enhancement, vibronic coupling and symmetry of the molecular vibrations.