Raman amplification spectroscopy using pulsed dye lasers

A picosecond laser system consisting of a mode-locked argon-ion laser synchronously pumping two dye lasers is used for studies of Raman amplification spectra. The two dye laser beams, one kept constant in frequency while the other is tunable, coincide in the Raman sample. Recording the gain or the loss in intensity of one of the lasers as a function of frequency difference produces the Raman spectrum. Good signal to noise ratios have been obtained for a variety of liquids and solids. Fluorescing samples can be studied in the Inverse Raman method where the loss on the higher frequency laser is monitored.     

Fast determination of milk fat content using Raman spectroscopy

In our work, we have demonstrated the capability of VIS Raman spectroscopy in combination with partial least square regression (PLS) as a rapid technique for direct milk fat determination. Raman spectra of milk samples revealed contributions from proteins, but mainly from their fat content with different spectral characteristics. Three different methods of sample preparations were applied: (i) liquid milk contained in an open dish, (ii) dried milk droplets on glass plates covered with Al foil, and (iii) liquid milk contained in quartz cuvettes. Methods (i) and (ii) showed a good PLS model for milk fat prediction with low root mean square errors and high correlation coefficients. The main advantage of milk sample contained in the dish lies in its simplicity as well as the fact that the open container maximizes the signal of interest avoiding background contributions. Our results show that Raman spectroscopy is suited for in-line monitoring purposes.     

Ultrafast heme dynamics in ferrous versus ferric cytochrome c studied by time-resolved resonance Raman and transient absorption spectroscopy

Cytochrome c (Cyt c) is a heme protein involved in electron transfer and also in apoptosis. Its heme iron is bisaxially ligated to histidine and methionine side chains and both ferric and ferrous redox states are physiologically relevant, as well as a ligand exchange between internal residue and external diatomic molecule. The photodissociation of internal axial ligand was observed for several ferrous heme proteins including Cyt c, but no time-resolved studies have been reported on ferric Cyt c. To investigate how the oxidation state of the heme influences the primary photoprocesses, we performed a comprehensive comparative study on horse heart Cyt c by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We found that in ferric Cyt c, in contrast to ferrous Cyt c, the photodissociation of an internal ligand does not take place, and relaxation dynamics is dominated by vibrational cooling in the ground electronic state of the heme. The intermolecular vibrational energy transfer was found to proceed in a single phase with a temperature decay of approximately 7 ps in both ferric and ferrous Cyt c. For ferrous Cyt c, the instantaneous photodissociation of the methionine side chain from the heme iron is the dominant event, and its rebinding proceeds in two phases, with time constants of approximately 5 and approximately 16 ps. A mechanism of this process is discussed, and the difference in photoinduced coordination behavior between ferric and ferrous Cyt c is explained by an involvement of the excited electronic state coupled with conformational relaxation of the heme.     

Resonance Raman optical activity and surface enhanced resonance Raman optical activity analysis of cytochrome c

High-resolution resonance Raman (RR) and resonance Raman optical activity (ROA) spectra of cytochrome c were obtained in order to perform full assignment of spectral features of the resonance ROA spectrum. The resonance ROA spectrum of cytochrome c revealed a distinct spectral signature pattern due to resonance enhanced skeletal porphyrin vibrations, more pronounced than any contribution from the protein backbone. Combining the intrinsic resonance properties of cytochrome c with the surface plasmon enhancement achieved with colloidal silver particles, the surface enhanced resonance Raman scattering (SERRS) and surface enhanced resonance ROA (SERROA) spectra of the protein were successfully obtained at concentrations as low as 1 microM. The assignments of spectral features were based on the information obtained from the RR and resonance ROA spectra. Excellent agreement between RR and SERRS spectra is reported, while some disparities were observed between the resonance ROA and SERROA spectra. These differences can be ascribed to perturbations of the physical properties of the protein upon adhesion to the surface of the silver colloids.     

Fast inspection of fruits using nuclear magnetic resonance spectroscopy

High-resolution nuclear magnetic resonance (NMR) spectroscopy is an indispensable technique for obtaining chemical structure information. Its quantitative and noninvasive properties have led to its growing popularity as an analytical tool in many fields, including biology, chemistry, medicine, and food science. During transportation and storage, chemical reactions among the many nutrients lead to a loss of food quality. In these circumstances, portable NMR spectrometers can readily be used for food inspection and quality control. Because of the heterogeneous tissue distribution in food, a high-resolution NMR method is required for detailed food inspection. Therefore, in this study, we demonstrated the feasibility of using an intermolecular double-quantum coherence signal to obtain high-resolution metabolic profiles of several fruits, including grape, cantaloupe, tomato, and watermelon. The resulting high-resolution NMR spectra facilitate the identification of important metabolites, which can be used as biomarkers for food quality control. The method established here may be adapted for food inspection using portable NMR equipment.     

Biogels of cytochrome c : Cytochrome c peroxidase complex studied by electron paramagnetic resonance spectroscopy

Cytochrome c: cytochrome c peroxidase (Cc: CcP in 1: 1 ratio) complex was successfully encapsulated in sol-gel derived glass. The electron paramagnetic resonance (e.p.r.) and optical absorption techniques were used to characterize the coordination number, spin state, charge-transfer activity and structural orientation of Cc: CcP complex and its constituents. The sol-gel encapsulation of metalloproteins allows, for the first time, the detection of e.p.r. signals of biological systems at room temperature. CcP exhibits an e.p.r. spectrum representing the high spin and purely axial symmetry with parameters at g 6 and g 2 and an electronic absorption spectrum with a descent in spectral intensity of shoulder band at 380 nm and a blue-shifted charge-transfer band at 620 nm. Cc shows an e.p.r. spectrum characterizing a mixture of high spin (g 6 and g 2) and low spin (g _x=2.7, g _y=2.2 and g _z=1.8) components. Upon complexation, Cc:CcP pair displays a single and broad e.p.r. spectrum at g 2 and a light absorption spectrum with a red-shifted Soret band at 423 nm, a blue-shifted charge-transfer band at 620 nm and an intensified charge-transfer band at 507 nm. These results suggest that the sol-gel encapsulated Cc:CcP complex has the following chemical and physical characteristics: (a) a hexa-coordination, (b) a high-spin state, (c) an active charge-transfer (or redox) pair, and (d) the direction of the g paramagnetic center of Cc : CcP complex lies nearly parallel to that of the heme normal. The structural coordinations of the sol-gel encapsulated Cc, CcP and Cc : CcP are examined. Moreover, the possible use of biogels at the sol, gelation, and xerogel stages during gel processing to control the structural rigidity and spatial separation/orientation of the encapsulated heme proteins and to study their possible routes of long-range electron transfer reactions are also discussed.     

Direct detection of 8-oxo-deoxyguanosine using UV resonance Raman spectroscopy

8-oxo-deoxyguanosine (8-oxo-dG) is a major oxidative lesion in DNA and is responsible for mutation and cancer. Current techniques for detecting 8-oxo-dG are indirect methods. Thus, development of new methodologies is needed to directly detect such oxidative lesions. In this article, we have used ultraviolet resonance Raman (UVRR) spectroscopy as a novel analytical technique for the detection of 8-oxo-dG. Here, the UVRR spectrum of 8-oxo-dG was acquired and compared to that of deoxyguanosine (dG) and deoxyadenosine (dA). Data analysis shows a distinct UVRR spectrum of 8-oxo-dG with characteristic peaks. Detection of 8-oxo-dG was easily achieved from a mixture with dG. These results reveal that UVRR spectroscopy shows promise as a direct method for detecting 8-oxo-dG.     

Discharge-flow kinetics measurements using intracavity laser absorption spectroscopy

Intracavity laser absorption spectroscopy ("ICLAS") has been demonstrated as a feasible detection method for trace species in a discharge flow tube. This implementation has been used to measure the rate of the reaction between atomic hydrogen and NO to form HNO in helium carrier gas. A reaction rate constant of (4.3 +/- 0.4) x 10(-32) cm(6) molecule(-2) s(-1) at 295 K was measured for the reaction H + NO + M --> HNO + M (M = He). The pressure and concentration range enabled by ICLAS detection has allowed us to limit reactive pathways that would inhibit the formation of HNO. The sensitivity of ICLAS, coupled with the versatility of the discharge flow technique, suggests that intracavity absorption spectroscopy will be a useful technique for kinetics measurements on free radicals and other reactive species.     

Analytical applications of ultraviolet resonance Raman spectroscopy

UV resonance Raman spectroscopy (UVRR) is a new analytical technique with a unique selectivity which is capable of speciating individual analytes in complex samples. The new instrumentation is discussed as are applications of this technique to studies of polycyclic aromatic hydrocarbons (PAHs) in coal liquids and in tissue. UVRR can also be used to speciate PAHs eluting from high-performance liquid chromatography columns. Other applications to studies of protein structure are also described.     

Photodissociation of heme distal methionine in ferrous cytochrome C revealed by subpicosecond time-resolved resonance Raman spectroscopy

Cytochrome c (cyt c) is an electron-transfer heme protein that also binds nitric oxide (NO). In resting cyt c, two endogenous ligands of the heme iron are histidine-18 (His) and methionine-80 (Met) side chains, and NO binding requires the cleavage of one of the axial bonds. Previous femtosecond transient absorption studies suggested the photolysis of either Fe-His or Fe-Met bonds. We aimed at unequivocally identifying the internal side chain that is photodissociated in ferrous cyt c and at monitoring heme structural dynamics, by means of time-resolved resonance Raman (TR3) spectroscopy with approximately 0.6 ps time resolution. The Fe-His stretching mode at 216 cm-1 has been observed in photoproduct TR3 spectra for the first time for a c-type heme. The same transient mode was observed for a model ferrous cyt c N-fragment (residues 1-56) ligated with two His in the resting state. Our TR3 data reveal that upon ferrous cyt c photoexcitation, (i) distal Met side chain is instantly released, producing a five-coordinated domed heme structure, (ii) proximal His side chain, coupled to the heme, exhibits distortion due to strain exerted by the protein, and (iii) alteration in heme-cysteine coupling takes place along with the relaxation of the protein-induced deformations of the heme macrocycle.     

Fast resonance raman spectroscopy of short-lived radicals

We report the first application of pulsed resonance Raman spectroscopy to the study of short-lived free radicals produced by pulse radiolysis. A single pulse from a flash-lamp pumped tunable dye laser is used to excite the resonance Raman spectrum of the p-terphenyl anion radical with an initial concentration of 4 × 10^?5 moles per liter and a half life of 2 μs. The spectrum is recorded by an optical multichannel system consisting of an image intensifier coupled to a TV-camera.     

The dushinsky effect in resonance Raman spectroscopy

The effect of normal-coordinate rotation on resonance Raman excitation profiles and depolarization dispersion curves is investigated theoretically for two nontotally symmetric modes of the same symmetry.     

Understanding excited-state structure in metal polypyridyl complexes using resonance Raman excitation profiles,time-resolved resonance Raman spectroscopy and density functional theory

This article discusses the use of Raman spectroscopy, in concert with density functional theory, as a strategy for understanding excited-state structure in metal polypyridyl complexes. The first sections of the article discuss how one can use resonance Raman spectra of the ground-state molecule to understand the resonant Franck-Condon excited state. The theories behind these analyses are based on the sum-over-states and time-dependent approaches; a brief introduction to each of these methods is given. The use of density functional theory and its use in the determination of normal modes of vibration and infrared and Raman band intensities are discussed, with reference to a number of recent papers. The application of these methods is illustrated through the analysis of a number of selected examples which exemplify the strategies used to extract data from probing the Franck-Condon region. These data include the displacements of the resonant excited state with respect to the electronic ground state, the reorganisation energies associated with photoexcitation, bond length changes with excitation and other electronic parameters. The use, and limitations, of these methods are discussed. The direct calculation of resonance Raman band intensities is introduced. The direct measurement of excited-state vibrational spectra through time-resolved methods is discussed in the latter section of the article; with particular regard to the use of transient resonance Raman and time-resolved resonance Raman techniques to probe structural changes in metal polypyridyl complexes.     

Structural analysis of lignin by resonance Raman spectroscopy

Resonance Raman (RR) spectroscopy, combined with Kerr gated fluorescence rejection in the time domain, has recently elucidated lignin structure with unique sensitivity and selectivity. This promises structural studies of fluorescent natural macromolecules, such as lignin, which were previously not possible. Such studies rely on an improved understanding of the RR spectral behavior of lignin, which is today scarcely understood. We explain for the first time this behavior by a semi-empirical theory, and observe its pertinent features for lignin in vascular plants. We have used well-defined oxidative treatments as means of probing lignin structural elements, and show that RR sensitivity and selectivity depend crucially on excitation wavelength. Through the theory we relate these results to basic structural aspects of lignin. Spectra obtained by blue light laser excitation (400 nm) are dominated by low redox potential syringyl lignin groups, whereas lower photon energy (500 nm) decreases the selectivity markedly. RR bands depend on molecular structure but also on molecular environment. Thus charge transfer donor-acceptor interactions within lignin reduce the intensity of bands associated with electron rich moieties. New possibilities for basic and selective structural information on fluorescent natural materials, such as lignin, have thus appeared.     

Ultraviolet resonance Raman microprobe spectroscopy of photosystem II

Photosystem II (PSII) carries out photosynthetic oxygen production and is responsible for the maintenance of aerobic, heterotrophic life. In PSII, protein amino acid residues play an important role in the light-driven electron transfer reactions. Here, we describe an approach to enhancing vibrational signals from PSII proteins through ultraviolet resonance Raman (UVRR) and a microprobe jet flow technique. Our work shows that pump-probe UVRR can be used to monitor intermediates during photosynthetic oxygen evolution.     

Characterization of thalidomide using Raman spectroscopy

Thalidomide is a potent anticancer therapeutic drug whose mechanism of action has not yet been elucidated. In this report, experimental Raman spectroscopy is used to determine and characterize the vibrational frequencies of the drug. These normal modes are then compared to their quantum mechanical counterparts, which have been computed using density functional theory. Upon analysis of the spectra, we found that there was a high level of agreement between the wavenumbers. As such, this spectroscopic technique may be a viable tool for examining the way in which this drug interacts with its target molecules.     

Immunoassay for P38 MAPK using surface enhanced resonance Raman spectroscopy (SERRS)

A micro-bead sandwich assay for P38 mitogen-activated protein kinase using surface enhanced resonance Raman spectroscopy (SERRS) detection is reported. Monoclonal capture antibodies were immobilised on a solid phase of magnetic micro-beads with secondary detection using a rhodamine-labelled antibody. Quantitative SERRS detection of the secondary antibody was possible with a limit of detection of 9.5 x 10(-12) mol dm(-3). The sandwich assay was quantitative and sensitive to 6 ng ml(-1). The mechanism of the SERRS detection in the immunoassay was investigated. The addition of SERRS aggregating agents causes the dissociation of the immuno-complex from the magnetic beads. Scanning electron microscopy images indicate that the colloidal suspension rather than adsorbed silver nanoparticles on the beads provide the SERRS signals, that the aggregate size is partially controlled and that there is some inhomogeneity in the distribution of organic matter on the nanoscale.     

Excited-state structural dynamics of cytosine from resonance Raman spectroscopy

Cytosine, a nucleobase found in both DNA and RNA, is known to form photoproducts upon UV irradiation, damaging the nucleic acids and leading to cancer and other diseases. To determine the molecular mechanism by which these photoproducts occur, we have measured the resonance Raman spectra of cytosine at wavelengths throughout its 267 nm absorption band. Self-consistent analysis of the resulting resonance Raman excitation profiles and absorption spectrum using a time-dependent wave packet formalism yields both the excited-state structural changes and electronic parameters. From this analysis, we have been able to determine that, at most, 31% of the reorganization energy upon excitation is directed along photochemically relevant modes.     

The use of fluorescence quenchers in resonance Raman spectroscopy

We discuss methods of increasing the resonance Raman signal to noise in the presence of a strong fluorescence background. By considering the time dependence of the competing processes, we conclude that for many commonly encountered systems the Raman signal can be enhanced over the fluorescence by introducing a quencher that decreases the lifetime of the radiative electronic state. The efficacy of this approach is demonstrated by the appearance of resonance Raman from fluorescein in water with the progressive addition of KI.