Nonlinear Raman Effects

In Eq. (1) it was assumed that the induced dipole varied in a linear fashion with the electric field. However, for electric field intensities above 109 V/m, as are often produced by pulsed lasers, the linear dependence breaks down. New spectroscopic phenomena arise from the nonlinear interaction of a system with intense monochromatic radiation. Each of the four examples considered here involves changes in wavelength of the radiation as a result of interaction with the system and can be considered to be a variant of the Raman effect. The four examples are the hyper-Raman scattering, the surface-enhanced hyper-Raman scattering, stimulated Raman scattering, and coherent anti-Stokes Raman scattering (CARS).

A. Hyper-Raman Spectroscopy

Hyper-Raman scattering arises from illuminating a system with a focused, pulsed laser that has an irradiance just above the threshold for nonlinear interaction. If the incident radiation is of frequency 1v0, the scattered radiation is found to include frequencies of the type 2v0 and 2v0 ± v1, where v1, is a frequency associated with a transition between two levels of the scattering molecules. The hyper-Raman radiation arises from the second-order nonlinear induced dipole. Thus, Eq. (1) can be expanded to

FIGURE 6 Schematic diagram of a confocal Raman microscope.

and the hyper-Raman scattering is controlled by the hyperpolarizability tensor j. The symmetry factors governing the factors of the j tensor are not the same as those for the components of the a tensor. As a consequence, the vibrational selection rules for hyper-Raman scattering are significantly different from those for linear Raman scattering. For example, the torsional vibration in CH2=CH2 is neither IR nor Raman active but is hyper-Raman-active.

B. Surface-Enhanced Hyper-Raman Spectroscopy

Surface-enhanced hyper-Raman scattering (SEHRS) is the analog of hyper-Raman scattering just as SERS is the analog of normal Raman scattering. By adsorbing hyper-Raman-active molecules to a nanoscale roughened Ag, Cu, or Au surface or the corresponding colloids, enhancement factors as large as 1012 have been measured. The main utility of the SEHRS technique lies in the new chemical information available as a consequence of the different selection rules. While low-symmetry molecules will have very similar SER and SEHR spectra, high-symmetry molecules will have spectra that show new vibrational features. The combination of three factors: (1) the 1012 enhancement factor of SEHRS; (2) solid-state, femtosecond lasers with high repetition rate, high peak power, and low average power; and (3) high-efficiency multichannel detectors portends a new era in vibrational spectroscopy using SEHRS to extract new structural information from important chemical and biological systems.

C. Stimulated Raman Effect

When monochromatic radiation from a pulsed laser of sufficiently large irradiance is incident on a scattering system, hyper-Raman scattering is replaced by a different phenomenon: stimulated Raman scattering. In a typical experimental configuration, the laser beam is focused into the sample and the scattering is observed in the forward direction, along the axis of the laser beam direction, and at a small angle to this direction. The forward-scattered radiation is found to consist of the incident frequency v0 and Stokes and anti-Stokes frequencies of the general formula v0 ± nv1, where v1 is usually associated with just one Raman-active vibration of the scattering molecules and n = 1, 2, 3,____For example, if liquid benzene is illuminated, the Stokes and anti-Stokes shifts are all exact multiples of 992 cm-1, which represents the strongest feature in the normal Raman spectrum of benzene.

Stimulated Raman scattering differs from normal Raman scattering in its angular dependence, intensity, and frequency distribution. A major fraction of the incident light is converted to radiation at v0 ± nv1. In benzene,

50% of the incident radiation at v0 may be converted to Stokes radiation at v0 - vi. The high-conversion efficiency of stimulated Raman scattering, taken with the coherent nature of the scattering, gives rise to the possibility that the effect can be used to generate laser-like sources at new frequencies. For example, H2 or D2, gas under pressure is used routinely to shift the incident radiation by 4160 or 2942 cm-1 (the H2 or D2, stretching frequencies, respectively) to gain access to the deep ultraviolet, vacuum ultraviolet, and mid-infrared spectral regions that are not easily accessible in other ways. Stimulated Raman scattering in H2 and D2 has been used extensively in ultraviolet resonance Raman spectroscopic studies of proteins.

D. Coherent Anti-Stokes Raman Spectroscopy

The CARS technique involves the use of two tunable dye lasers, a pump laser and a Stokes laser, set at frequencies vP and vS, respectively. If these two light beams cross in the sample at the phase-matching angle 0, coherent anti-Stokes emission at vAS = 2vP - vS is generated through the third-order linear polarization. In fact, /CARS ~ IpIS. The laserlike beam due to vAS is greatly enhanced when the frequency interval, vP - vS = A, is equal to a Raman-active molecular vibrational frequency. Thus, vibrational Raman spectra are obtained by fixing the frequency of vP and varying the frequency of vS. Fluorescence rejection occurs in a CARS experiment because the signal beam, vAS, is spatially and temporally removed from the fluorescence signal.

In previous years, the major application of CARS was in the analysis of gases and gas mixtures, but recent advances have demonstrated that CARS can be applied to nongaseous systems as well. A problem in the application of CARS to molecules in solution is interference from the solvent, which can contribute a background emission resulting from the third-order susceptibility of the solvent. To a certain extent, this problem may be overcome when the CARS experiment is carried out under resonance conditions. As the vP beam frequency approaches an electronic transition of the solute, the solute's CARS signal is resonance enhanced whereas the background emission remains unchanged.


A. Chemical Applications

Five representative applications in which Raman spec-troscopy has proved to be very powerful are cross-sectional imaging of live cells with CARS, investigation of industrial polymers with Fourier Transform (FT) Raman Spectroscopy, the study of self-assembled monolayers (SAMs) with SERS, the assignment of vibrational bands by the combination of SERS and SEHRS, and SPP-enhanced Raman studies of carbon clusters.

1. CARS Gross-Sectional Imaging

Recent CARS experiments have targeted cross-sectional imaging by making use of solid state femtosecond lasers with Xp and Xs in the NIR region. The long wavelength pulses give two major advantages over visible excitation:

(i) the excitation wavelengths are far from electronic transition bands, so the background signals are small, and

(ii) Rayleigh scattering is minimized in heterogeneous samples, allowing deeper penetration into the sample volume. By collinearly coupling the two pulses into an optical microscope and through a high numerical aperture objective, the excitation pulses can be focused tightly onto the sample. Because CARS is a nonlinear optical process, only a small volume of the sample is excited. The advantages of small volume excitation include background signal rejection, decreased photodecomposition of the sample, and the ability to section a three-dimensional object by changing the focal plane of the excitation pulses. The consequence of a long wavelength, small volume excitation is that three-dimensional imaging of live cells has been achieved. Figure 7 shows CARS images of live, unstained bacteria tuned to the Raman shift of 2878 cm-1, an aliphatic C-H stretching band. The lipid bilayer of the bacterial cell membrane is rich in aliphatic C-H bonds. CARS will find more application in coming years because it gives vibrational contrast and high sensitivity, but the power levels are tolerable to living cells.

2. FT-Raman Analysis of Polymers

The spectroscopic advances made in the field of polymer chemistry demonstrate the impact of the FT-Raman

FIGURE 7 CARS image of a live bacterial cell. Imaging was tuned to the Raman shift of 2878-cm-1 band (aliphatic C-H stretch). [Reproduced from Zumbusch et al. (1999). Phys. Rev. Lett. 82(20), 4142, by permission.]

technique. Polymer samples are historically plagued by large fluorescence backgrounds and sample degradation under the heating effects of visible wavelength laser irradiation. In fact, estimates suggest that up to 95% of polymer samples cannot be examined by normal Raman spec-troscopy. Until the advent of FT-Raman, most polymer samples were investigated by infrared (IR) spectroscopy. Often, the strenuous sample preparation required for IR investigations alters the polymer structure, thus sacrificing an accurate spectroscopic view. The high symmetry of most polymer samples also acts as a disadvantage in IR experiments because many symmetric stretching mode vibrations are IR inactive. Often, IR spectra are missing all information regarding the homonuclear polymer backbone.

FT-Raman addresses many of the disadvantages of IR polymer analysis. First, because sample preparation is minimal for FT-Raman analysis, no valuable information is lost. Second, because the Raman selection rule is based on changing bond polarizability rather than on the changing dipole moment (as in IR spectroscopy), the homonu-clear backbone stretches can be seen in Raman spectra. Third, detailed information about the chemical composition, structure, and stereoregularity can be obtained by FT-Raman measurements. Some of the most exciting data gained in FT-Raman investigations of polymers lie in the details of chain conformation due to intermolecular interactions. The chain-packing details help to explain physical characteristics of polymers such as absorbance spectra and transition temperatures.

3. SERS of Self-Assembled Monolayers at the Solid/Liquid Interface

SERS experiments give scientists a window into the complex processes occurring at solid/liquid, solid/gas, solid/UHV, and solid/solid interfaces. When molecules adsorb to a SERS-active substrate, the measured spectra give valuable information concerning the structure and reactivity of the adsorbate molecules. Comparison of SERS spectra to bulk Raman spectra demonstrates any chemical or conformational variance between the free and adsorbed states. This information is important when using SERS to understand complex phenomena such as heterogeneous catalysis and electrochemistry at the molecular level.

As an example, consider the pervasive use of self-assembled monolayers (SAMs) for the surface modification of gold and silver substrates. A recent SERS/SERRS study of the Au film/SAM adsorbate interface demonstrates that thin films of Au(111) on mica substrates, previously thought to be SERS-inactive, is SERS-active without any additional treatment. This discovery is quite significant because it allows SERS experiments to be executed on undisturbed, unroughened Au(111)/SAM adsorbate surfaces. The impact of better understanding SAM adsorption to a Au(111) substrate is significant because this system acts as a model for other substrate/adsorbate systems.

In the aforementioned experiment, the SERS activity of rough Au films is compared with that of single crystal Au(111) films. Figures 8A and B show nanometer-scale AFM images of a rough Au surface and a Au(111) surface before SAM modification. The corresponding SERRS of SAMs on these surfaces are in Figs. 8C and D, respectively. The resonance Raman condition is a result of overlap between the adsorbate molecule's electronic absorption band and the excitation wavelength. Although not shown within this text, SERS spectra were also measured for each sample. With these two spectra, the surface resonance Raman (SRR) enhancement (~102) could be separated from the EM enhancement (~103). This weak EM enhancement, compared to the standard EM contribution of 104-105, is

FIGURE 8 SERS-active surface topography and SERRS spectra for SAM on rough Au and Au(111) substrates. (A) depicts the AFM image and line scan of the rough Au substrate. (B) depicts the AFM image and line scan of the Au(111) substrate. (C) is the SERRS spectrum of SAM adsorbed to the surface shown in (A). (D) is the SERRS spectrum of SAM adosrbed to the surface shown in (B). [Reproduced from Caldwell et al. (1994). Langmuir 10, 4109, by permission.]

FIGURE 8 SERS-active surface topography and SERRS spectra for SAM on rough Au and Au(111) substrates. (A) depicts the AFM image and line scan of the rough Au substrate. (B) depicts the AFM image and line scan of the Au(111) substrate. (C) is the SERRS spectrum of SAM adsorbed to the surface shown in (A). (D) is the SERRS spectrum of SAM adosrbed to the surface shown in (B). [Reproduced from Caldwell et al. (1994). Langmuir 10, 4109, by permission.]

attributed to the ~ 100 nm wide, atomically flat islands that comprised the surface of Au(111)/mica samples shown in Fig. 8B or the roughness features between the terraces of the Au(111) surface. Future experiments will address these hypotheses.

4. SEHRS Assisted Assignment of Vibrational Bands

A particularly salient example of the complementarity of normal Raman spectroscopy with other techniques is expressed in a recent publication of the combined data from infrared, normal Raman, SERS, SEHRS, and theoretical predictions for one molecule. First, ab initio theoretical predictions were made for the vibrational characteristics of trans-1,2-bis(4-pyridyl)ethylene (BPE) at the Hartree-Fock 6-31G* level. When the spectra were collected, comparisons were made between the theoretical and experimental results as well as among the different spectra. Based on the known selection rules for each spectroscopy and the matching of wavenumber shifts to theoretical predictions, all vibrational bands were assigned.

Though this study only gives exact information for BPE, it demonstrates the power of combined theoreti-cal/spectroscopic characterization. The development, in recent years, of ab initio electronic structure calculations of high accuracy has revolutionized theoretical chemistry. The ability to efficiently calculate vibrational properties from first principles has or will completely replace normal coordinate analysis.

Figure 9 shows the SER and SEHR BPE spectra as well as the theoretical prediction for each. Although the hyper-Raman scattering efficiency is eight orders of magnitude smaller than the linear Raman scattering efficiency, the SEHR signals are only three orders of magnitude smaller than the SERS signals. This translates to 1012-fold enhancement over normal hyper-Raman scattering. There has been renewed interest in hyper-Raman spectroscopy due to this immense SEHR enhancement. Vibrational modes that are active in only hyper Raman spectroscopy can be measured reliably for the first time.

5. SPP-Enhanced Raman Spectroscopy of Carbon Clusters

SERS is generated from molecules adsorbed to a roughened metal surface, but not all molecules can maintain their structure and function in direct contact with a metal surface. Both biomolecules and atomic clusters fall into this category. One solution to this limitation is to put a spacer molecule (such as the SAM suggested in the previous section) between the metal surface and the molecule

FIGURE 9 Theoretical SER (A) and SEHR (C) and experimental SER (B) and SEHR (D) spectra of BPE on a roughened silver surface. [Reproduced from Yang et al. (1996). J. Chem. Phys. 104(11), 4313, by permission.]

of interest. In cluster science, a common solution to the problem of high reactivity is to embed the clusters inside solid matrix of condensed inert gas. SPP-enhanced Raman spectroscopy allows Raman spectra to be measured from smooth surfaces.

A recent SPP-resonance Raman experiment explored the structural conformations of several carbon clusters. The Ci6, Ci8, and C20 clusters were created by laser ablation of a graphite rod and then deposited into a N2 matrix on the silvered SPP prism surface. After finding the SPP resonance condition (as described in Section VI.B), Raman spectra were collected with six different excitation wavelengths. SPP-Raman enhancement is operative in all six spectra. The strong dependence of the Raman spectra for the C20 cluster on Xex shown in Fig. 10 indicates that enhancement due to RRS is simultaneously operative. It is important to emphasize that these signals shown in this figure are obtained from ca. 1010 clusters in the laser focal spot. Upon comparing the Raman peak frequencies for all three carbon clusters to theoretical predictions, the researchers were able to hypothesize that all three carbon clusters adopt either a linear chain or poly-

acetylene ring conformation, but not a fullerene or bowl structure.

B. Biochemical Applications

A major advantage of Raman spectroscopy for the analysis of biomolecules stems from the fact that water has a weak Raman spectrum. Spectra can be recorded for aqueous solutes at 10-1 -10-2 M with little interference from the solvent. For a chromophore under the RR condition the accessible concentration range becomes 10-e-10-6 M. Moreover, the intensity enhancement associated with the RR effect confers the important advantage of selectivity, allowing one to observe selectively the vibra-tional spectrum of a chromophore that is just one component of an extremely complex biological system. Because many biomolecules have chromophores with an ultraviolet (UV) resonance condition, one may also selectively excite a chromophore by irradiating these molecule with UV light. This technique is known as Ultraviolet Resonance Raman Spectroscopy (UVRRS). In recent years, Raman difference spectroscopy (RDS) has been developed in

FIGURE 10 SPP-Raman spectra of matrix-isolated C20 clusters over the energy region 100-2000 cm-1 for excitation wavelengths of (A) 457.9, (B) 488, (C) 514.5, (D) 635, (E) 647, and (F) 670 nm. [Reproduced from Ott, et al. (1998). J. Chem. Phys. 109(22), 9653, by permission.]

order to deconvolute the complicated spectra of biological macromolecules.

Three major classes of biomacromolecules have been studied by normal (nonresonance) Raman spectroscopy: proteins, nucleic acids, and lipids and membranes. The type of information obtained for each class can be summarized as follows.

1. Proteins. Quantitation of polypeptide conformation, a-helix, ¡i-sheet, ¡i-turns, etc.; characterization of cysteine-SH side chains; conformation of disulfide -S-S-linkages; strength of hydrogen bonds to tyrosine-OH; exposure to hydrophobic/hydrophilic environments of tryp-tophan side chains

2. DNA and RNA. Quantitation of the (deoxy)ribose-phosphate backbone conformation and base composition; observation of base pairing and base stacking (often different classes of bases can be monitored separately); H-D exchange in bases

3. Lipids and membranes. Interchain interactions and melting behavior of the lipid aliphatic chains; effects of chemical perturbants (e.g., cholesterol) and proteins on melting behavior; lipid head-group conformation

RR spectra have been obtained from chromophores in live tissue and from bacterial cells under physiological conditions. However, the bulk of the studies in the past 20 years has been on purified materials: the type of information gained is summarized here.

1. Heme proteins. Oxidation and spin state of the heme iron using porphyrin marker bands; detailed chemistry and hemoglobin-CO photolysis on the nano- and picosecond timescales; quaternary structures; chemistry of Fe-ligand bonds

2. Visualpigmentsandbacteriorhodopsin.Retinalcon-formation in the photocycle intermediates; state of protonation of the retinal-protein Schiff base

3. Metalloproteins. Chemistry of the ligands around the chromophoric metal center

Most of the other naturally occurring biological chro-mophores, such as flavins and carotenoids, have now been studied. UVRRS is a technique that complements visible and infrared Raman spectroscopies by providing sensitive, selective information about a small number of vibra-tional bands. In order to make maximal use of the UV Raman technique, one must carefully choose the species in solution so that the Raman cross sections and bands of the solution do not compete with those of the analyte being studied. With a well-chosen system, this technique can give information about the structure and dynamics of small molecules and functionalities and chromophores on larger molecules. UV Raman is most useful because many molecules have absorption bands deep in the UV region, and fluorescent interference is greatly decreased because fluorescence does not usually occur in species with an excitation band below 260 nm.

In classic UV Raman systems, an excimer laser was used to pump a frequency-doubled dye laser which was then directed at the liquid sample as it was jetted along a guided path. Recently, UV Raman spectroscopy has become more feasible due to the advent of a frequency-doubled Ar+ laser that has five excitation wavelengths with substantial power in the UV region. This laser yields better signal-to-noise ratios and allows for UV Raman investigation of solid samples. The most recent UV Raman studies examine protein-nucleic acid interactions, protein-protein interactions, and the structure and behavior of proteins, DNA, and neurotransmitters in aqueous solutions.

However, many biological sites of importance do not contain a suitable chromophore, and for these, the RR labeling technique was developed. An RR label, usually mimicking a natural component, is introduced into the system as a reporter group, and there has been success in using chromophoric ligands to study antibody-haptcn, enzyme-inhibitor (and drug), DNA-drug, and cell-dye interactions. Moreover, the labeling technique can aid the understanding of the molecular details of enzymolysis. The hydrolysis of thionoesters (of the type RC(=O)NHCH2C(=S)OCH3) by the enzyme papain occurs via the formation of a transient dithioester RC(=O)NHCH2C(=S)-S-papain (the thiol sulfur belongs to a cysteine side chain in the active site). The dithioester absorbs at 315 nm, and thus, the label is generated at the time and location of catalysis. The 324-nm excited RR spectrum in Fig. 11 shows how different spectral features monitor conformations in different parts of the enzyme-substrate complex. By this means it is possible to monitor the critical events in the bonds undergoing catalytic transformation in a complex of molecular weight 24,000.

Raman analysis of large macromolecules, such as proteins, often yields complicated, spectrally crowded results.

Resonance Raman labeling is used in order to highlight the spectrum of small portions of the large molecule. This technique has limited applications to protein molecules because the chromophore dominates the observed spectrum and not all proteins will accept chromophore addition. A more generally applicable method known as RDS has become technically feasible in recent years to study events such as protein-ligand binding, enzymatic catalysis, and protein assembly.

In RDS, spectra of both the normal and the modified version of the macromolecule are collected. By subtracting the normal spectrum from the tagged molecules' spectrum, information about the tagged portions of the molecule is obtained. Two methods used in the tagging process are ligand binding and isotopic editing. By subtracting a bare protein's spectrum from the spectrum of the protein bound to a ligand, the RDS spectrum of the bound ligand is obtained. In isotopic editing, the substitution of an atom in the bond of interest with another stable isotope will cause shifts in the Raman bands for motions involving the edited nucleus. By subtracting the normal spectrum from the isotopically edited spectrum, Raman peaks will be seen only for the modes of vibration involving the isotopically edited atoms.

FIGURE 11 Monitoring the group (and its neighbors) undergoing transformation in an enzyme's active site. The RR spectrum shown is of the enzyme-substrate transient PhC(=O)NHCH2(=S)S-papain; 324-nm excitation. [Reproduced from Carey, P. R. (1992). "Raman Spectroscopy," In "Encyclopedia of Physical Science and Technology," 2nd ed., Academic Press, New York, by permission.]

FIGURE 11 Monitoring the group (and its neighbors) undergoing transformation in an enzyme's active site. The RR spectrum shown is of the enzyme-substrate transient PhC(=O)NHCH2(=S)S-papain; 324-nm excitation. [Reproduced from Carey, P. R. (1992). "Raman Spectroscopy," In "Encyclopedia of Physical Science and Technology," 2nd ed., Academic Press, New York, by permission.]

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