Field-resolved coherent Raman spectroscopy of high frequency vibrational resonances

Electric fields of coherent Raman signals are resolved with sensitivity for high-frequency vibrational resonances utilizing a four-pulse, trapezoidal beam geometry in a diffractive optic-based interferometer. Our experiments show that the heterodyne detected signal phase is stabilized for particular terms in the third-order response function by the cancellation of inter-pulse phases. The C-H stretching modes of cyclohexane and benzene are studied under two polarization conditions. The temporal profiles of signal fields for cyclohexane exhibit a low-frequency recurrence due to the interference between the signals associated with the symmetric and asymmetric C-H stretching modes. In contrast, the electronically nonresonant polarizability response of benzene gives rise to a significant broadband signal component in addition to that associated with its C-H vibrational resonance. Time-frequency shapes of the Raman signal fields are strongly dependent on the properties of the liquid and the polarizations of the laser pulses.     

Solvent structural relaxation dynamics in dipolar solvation studied by resonant pump polarizability response spectroscopy

Resonant pump polarizability response spectroscopy (RP-PORS) was used to study the isotropic and anisotropic solvent structural relaxation in solvation. RP-PORS is the optical heterodyne detected transient grating (OHD-TG) spectroscopy with an additional resonant pump pulse. A resonant pump excites the solute-solvent system and the subsequent relaxation of the solute-solvent system is monitored by the OHD-TG spectroscopy. This experimental method allows measuring the dispersive and absorptive parts of the signal as well as fully controlling the beam polarizations of incident pulses and signal. The experimental details of RP-PORS were described. By performing RP-PORS with Coumarin 153(C153) in CH(3)CN and CHCl(3), we have successfully measured the isotropic and anisotropic solvation polarizability spectra following electronic excitation of C153. The isotropic solvation polarizability responses result from the isotropic solvent structural relaxation of the solvent around the solute whereas the anisotropic solvation polarizability responses come from the anisotropic translational relaxation and orientational relaxation. The solvation polarizability responses were found to be solvent-specific. The intramolecular vibrations of CHCl(3) were also found to be coupled to the electronic excitation of C153.     

Catalytic methanol oxidation over copper: observation of reaction-induced nanoscale restructuring by means of in situ time-resolved X-ray absorption spectroscopy

The catalytically active copper phase for the partial oxidation of methanol is studied by means of time-resolved extended X-ray absorption fine structure (EXAFS) spectroscopy combined with the detection of the catalytic turnover. It is found that the active form of the copper is a strained nanocrystalline form of the metal. The metal is no longer made up from large crystallites but contains a defect structure in which oxygen is already intercalated.     

Quantitative measurement of molecular diffusion coefficients by NMR spectroscopy

An offset-independent adiabatic inversion pulse is used in the diffusion experiment to uniformly excite a sample region that is sufficiently long to ignore the ending effects, yet is short enough to have a homogeneous RF field and to represent the pulsed field gradient with a linear approximation. Under these conditions, the diffusion decay of the peak intensity appears to be Gaussian as a function of the effective gradient field ge as if all the molecules inside the selected region experienced the same ge. Quantitative measurement of molecular diffusion coefficients is therefore made possible.     

The molecular underpinnings of a solute-pump/solvent-probe spectroscopy: the theory of polarizability response spectra and an application to preferential solvation

Recent ultrafast experiments on liquids have made clear that it is possible to go beyond light scattering techniques such as optical Kerr spectroscopy that look at the dynamics of a liquid as a whole. It is now possible to measure something far more conceptually manageable: how that liquid dynamics (and that light scattering) can be modified by electronically exciting a solute. Resonant-pump polarizability-response spectra (RP-PORS) in particular, seem to show that different solvents respond in noticeably distinct ways to such solute perturbations. This paper is a theoretical attempt at understanding the kinds of molecular information that can be revealed by experiments of this sort. After developing the general classical statistical mechanical linear response theory for these spectra, we show that the experimentally interesting limit of long solute-pump/solvent-probe delays corresponds to measuring the differences in 4-wave-mixing spectra between solutions with equilibrated ground- and excited-state solutes-meaning that the spectra are essentially probes of how changing liquid structure affects intermolecular liquid vibrations and librations. We examine the spectra in this limit for the special case of an atomic solute dissolved in an atomic-liquid mixture, a preferential solvation problem, and show that, as with the experimental spectra, different solvents can lead to spectra with different magnitudes and even different signs. Our molecular-level analysis of these results points out that solvents can also differ in how local a portion of the solvent dynamics is accessed by this spectroscopy.     

Analysis of membrane protein cluster densities and sizes in situ by image correlation spectroscopy

Communication between cells invariably involves interactions of a signalling molecule with a receptor at the surface of the cell. Typically, the receptor is imbedded in the membrane and it is hypothesized that the binding of the signalling molecule causes a change in the state of aggregation of the receptor which, in turn, initiates a biochemical signal within the cell. Subsequently, many of the occupied receptors bind to membrane-associated structures, called coated pits, which invaginate and pinch off to form coated vesicles, thereby removing the receptors from the cell surface. The state of aggregation of membrane receptors is obviously in constant flux. Any useful approach to measuring the state of aggregation must, therefore, allow for dynamic measurements in living cells. It is possible to use fluorescently labelled signalling molecules or antibodies directed at the receptor of interest to visualize the receptor on the cell surface with a fluorescence microscope. By employing a laser confocal microscope, high resolution images can be produced in which the fluorescence intensity is quantitatively imaged as a function of position across the surface of the cell. Calculations of autocorrelation functions of these images provide direct and accurate measures of the density of fluorescent particles on the surface. Combined with the average intensity in the image, which reflects the total average number of molecules, it is possible to estimate the degree of aggregation of the receptor molecules. We refer to this analysis as image correlation spectroscopy (ICS). We show how ICS can be used to measure the density of several receptors on a variety of cells and how it can be used to measure the density of coated pits and the number of molecules per coated pit. We also show how the technique can be used to monitor fusion of virus particles to cell membranes. Further, we illustrate that, by calculating cross-correlation functions between pairs of images, we can extend the analysis to measurements of the distributions as a function of time, on the second timescale, as well as to measurements of the movement of the receptor aggregates on the surface. Finally, we illustrate that, by this approach, we can measure the extent of interaction between two different receptors as a function of time. This represents the most quantitative measurement of the extent of co-localization of receptors available and is independent of the spatial resolution of the confocal microscope. The theory of ICS and image cross-correlation spectroscopy (ICCS), focussing on the interpretation of the data in terms of the biological phenomenon being probed, is discussed.     

Complete spectral assignments of methacrylonitrile-styrene-methyl methacrylate terpolymers by 2D NMR spectroscopy

Methacrylonitrile-styrene-methyl methacrylate (N/S/M) terpolymers of different monomer concentrations were prepared by bulk polymerization. The terpolymer compositions were determined by quantitative ¹³C{¹H} NMR spectra and compared with those calculated by Goldfinger's equation using comonomer reactivity ratios: r_NS=0.30, r_SN=0.45; r_NM=0.91, r_MN=0.88; r_SM=0.52, r_MS=0.47. The overlapping and complex ¹³C{¹H} and ¹H NMR spectra of the terpolymers were assigned with the help of distortionless enhancement by polarization transfer and two-dimensional (2D) ¹³C-¹H heteronuclear single quantum coherence experiments. The various vicinal and geminal couplings between the protons in the polymer chains can be seen in the 2D total correlated spectroscopy experiments.     

Narrowing of spectral lines by energy pooling collisions in laser enhanced ionization spectroscopy

A remarkable narrowing of the Doppler profile of the calcium 4s ^{2 1} S ₀–4s4p ³ P ₁ line have been measured by laser enhanced ionization spectroscopy in a thermionic diode. By variation of the laser intensity and the calcium number density, and by analysis of the line profiles, there is evidence that energy pooling in higher states due to the collision of two and also three excited 4s4p ³ P ₁ atoms is responsible for the effect.     

Combustion measurement of thermoacoustic oscillating flames by diode-laser absorption spectroscopy sensors

Diode-laser absorption spectroscopy has been applied to a swirl-stabilized turbulent combustor to detect high frequency combustion oscillation and combustion state related to combustion noise. Two diode-laser absorption spectroscopy techniques of scanned-wavelength method and fixed-wavelength method are adopted. In the scanned-wavelength method, fluctuations of temperature and H₂O mole fraction up to 1 kHz are detected. Two dominant peak frequencies of power spectra of these fluctuations, which are about 125 Hz and 140 Hz, coincide with those of pressure fluctuation in the combustor. In the case of control by secondary fuel injection, the energy at peak frequency of temperature and H₂O mole fraction decreases in accordance with noise reduction. Similar to the combustion noise, temperature fluctuation shows a minimal value at the appropriate frequency of secondary fuel injection. By analysing transmitted signals, the fixed-wavelength method provides power spectra similar to those obtained by the scanned-wavelength method. The advantage of the fixed-wavelength method is capability of detection of high frequency combustion oscillation more than 1 kHz. These results prove that the diode-laser absorption spectroscopy has great applicability as sensors for the combustion measurement of thermoacoustic oscillating flames and active control of turbulent combustion.     

Morphosynthesis of poly(ether ketone) by reaction-induced crystallization during polymerization

Morphology control of poly(ether ketone) (PEK) was examined by using the crystallization during the nucleophilic aromatic substitution reaction of potassium salt of 4-fluoro-4′-hydroxybenzophenone. Polymerizations were carried out at 290 °C. The PEK was obtained as precipitates and its morphology was highly influenced by the polymerization condition such as the solvent, the concentration and the polymerization time. High crystalline spindle-like crystals were obtained by the polymerization in diphenyl sulfone (DPS) at a concentration of 5.0% for 2 h with the yield of 86%. The average length and width were 1.4 μm and 300 nm respectively, and the maximum thickness was 130 nm. The surface was not smooth and it was hilly. The spindle-like crystal was likely consisted of multilayered lamellae comprised of the microcrystallites. The molecules were oriented perpendicular to the lamella. The polymerization in DPS at a higher concentration of 10.0% afforded the networks of nanofibres, of which the diameter was 100–250 nm. The obtained PEK precipitates possessed excellent thermal properties.     

Determination of relative rate of spectral events by novel modification of two-dimensional correlation spectroscopy

Sign of two-dimensional (2D) correlation peaks provides information on sequence of spectral events. This information is related to molecular mechanism of changes in a given system. Recently, few papers addressing the problems with interpretation of the sign of 2D correlation peaks have been published. To overcome these problems, a modification of the generalized 2D correlation method has been proposed. This method compares variations in the dynamic spectrum with a linear change at a reference point. The rates of spectral responses at individual wavenumbers are proportional to magnitudes of the peaks in the slice of asynchronous spectrum at the reference point. This way, analysis of complex 2D contour plots is replaced by a simple examination of one-dimensional (1D) slice spectrum. In spite of reduced ability of the resolution enhancement, in special cases the proposed method provides information not accessible from the classical 2D correlation analysis. At first, the principles of this method are shown with the synthetic data. Next, the influence of spectral separation, band width and position changes on the slice spectrum is evaluated. Finally, the proposed approach is applied to the experimental spectra of two hydrogen-bonded systems.     

Computer-aided spectral assignment in nuclear magnetic resonance spectroscopy

The proton-carbon correlation spectra, HMBC (heteronuclear multiple bond correlation) and HMQC (heteronuclear multiple quantum correlation), respectively, provide direct and remote connectivity information with high sensitivity. Their combination enables carbon-carbon proximity relationships to be deduced, which are formally identical to those produced by a fictitious INADEQUATE-2D experiment, where correlations would be established exclusively between atoms linked by one or two bonds. The CASA program uses these relationships, as well as DEPT spectra and elementary chemical-shift considerations to assign the ¹³C spectrum of a compound if its structure is known or assumed. If the structure conflicts with the experimental data, no assignment is produced. The CASA program serves as an aid to either spectral assignment or structural elucidation.     

Direct measurement of the transbilayer movement of phospholipids by sum-frequency vibrational spectroscopy

The direct measurement of the transbilayer movement of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in a planar supported lipid bilayer (PSLB) at the fused silica/D2O interface was obtained with sum-frequency generation (SFG) vibrational spectroscopy. The intrinsic sensitivity of SFG to the symmetry of an interface was used to measure the asymmetric distribution of DSPC and perdeuterated DSPC (DSPC-d83) lipids in asymmetrically prepared DSPC/DSPC-d83 PSLBs. Changes in the membrane lipid composition due to exchange between leaflets was monitored by measuring the decay in the CH3 symmetric stretch intensity at 2875 cm-1 with time. The activation energy for transverse motion was determined directly from spectral relaxation measurements at several temperatures and was determined to be 206 +/- 18 kJ/mol. At room temperature (25 degrees C) the half-time of lipid flip-flop was calculated to be approximately 25 days. At 51 degrees C, only 7 degrees C below the main phase-transition temperature of DSPC, the half-time decreases to 25 min. These results have important implications for understanding the transbilayer movement of lipids in biological membranes.     

High-accuracy measurement of specular spectral reflectance and transmittance

The realization of spectrophotometric quantities at the Helsinki University of Technology is based on our reference spectrometer. The reference spectrometer is a high-accuracy instrument developed for measuring spectral specular transmittance and reflectance in a wavelength range extending from ultraviolet to near-infrared. The relative uncertainty estimates for transmittance measurements of neutral-density filters are ca. 0.05%. For spectral reflectance the estimated uncertainties are between 0.14% and 0.34% depending on the sample reflectance and the measurement geometry. We have derived and verified equations that enable both the reflectance and transmittance of various samples to be predicted. Utilizing these equations, the reflectance and transmittance can be accurately calculated for samples with known refractive index. For precise calculations, the characteristics of the measurement beam must be taken into account.     

The outer valance orbital electron densities of cyclopentane by binary (e,2e) spectroscopy

The binding energy spectra and electron distributions in momentum space of the valence orbitals of cyclopentane (C(5)H(10)) are studied by Electron Momentum Spectroscopy (EMS) in a noncoplanar symmetric geometry. The impact energy was 1200 eV plus binding energy and energy resolution of the EMS spectrometer was 1.2 eV. The experimental momentum profiles of the outer valence orbitals are compared with the theoretical momentum distributions calculated using Hartree-Fock and density functional theory (DFT) methods. The shapes of the experimental momentum distributions are generally quite well described by both the Hartree-Fock and DFT calculations when the large and diffuse basis sets are used.     

Rapid measurement of pseudocontact shifts in metalloproteins by proton-detected solid-state NMR spectroscopy

Pseudocontact shifts (PCSs) arise in paramagnetic systems in which the susceptibility tensor is anisotropic. PCSs depend upon the distance from the paramagnetic center and the position relative to the susceptibility tensor, and they can be used as structural restraints in protein structure determination. We show that the use of (1)H-detected solid-state correlations provides facile and rapid detection and assignment of site-specific PCSs, including resolved (1)H PCSs, in a large metalloprotein, Co(2+)-substituted superoxide dismutase (Co(2+)-SOD). With only 3 mg of sample and a small set of experiments, several hundred PCSs were measured and assigned, and these PCSs were subsequently used in combination with (1)H-(1)H distance and dihedral angle restraints to determine the protein backbone geometry with a precision paralleling those of state-of-the-art liquid-state determinations of diamagnetic proteins, including a well-defined active site.     

Methyl torsional potentials of rotational isomers of m-methylanisole studied by spectral hole-burning spectroscopy

Laser-induced fluorescence (LIF) excitation spectra of m-methylanisole in a supersonic jet were measured. Two series of progressions were observed in the spectrum, originating at 36048 and 36115 cm⁻¹, which were successfully assigned to the transitions to the methyl internal rotational vibronic levels of the two isomers, i.e. cis and trans isomers, with the aid of hole-burning spectrum measurements and quantum-chemical calculations. The progression for the trans isomer was observed up to the 6a₁ band, while only the 3a₁ band in addition to the 0a₁ and 1e bands was observed for the cis isomer. This finding can be explained by the conformational change upon the electronic excitation; the 60° rotation of the methyl torsional angle takes place for the trans isomer but not for the cis isomer.     

Photosensitization reaction-induced acute electrophysiological cell response of rat myocardial cells in short loading periods of talaporfin sodium or porfimer sodium

Electrophysiological responses of rat myocardial cells to exogenous photosensitization reactions for a short period of incubation with two photosensitizers, talaporfin sodium or porfimer sodium, were measured in a subsecond time scale. The loading period of the photosensitizer when the photosensitizer might not be taken up by the cells was selected as 15min, which was determined by the fluorescence microscopic observation. We measured the intracellular Ca(2+) concentration ([Ca(2+) ](in) ) by using a fluorescent Ca(2+) indicator, Fluo-4 AM, under a high-speed confocal laser microscope to evaluate the acute electrophysiological cell response to the photosensitization reaction. The measured temporal change in Fluo-4 fluorescence intensity indicated that the response to the photosensitization reaction might be divided into two phases in both photosensitizers. The first phase is acute response: disappearance of Ca(2+) oscillation when irradiation starts, which might be caused by ion channel dysfunction. The second phase is slow response: [Ca(2+) ](in) elevation indicating influx of Ca(2+) due to the concentration gradient. The continuous Ca(2+) influx followed by changes in cell morphology suggested micropore formation on the surface of the cell membrane, resulting in necrotic cell death.