What Is Vibrational Raman Spectroscopy: A Vibrational or an Electronic Spectroscopic… DOI: http://dx.doi.org/10.5772/intechopen.86838

similar molecules, i.e., molecules where a number of identical vibrations can be identified in the vibrational signature of the molecules. The method has been applied in combination with polarized resolved fluorescence to study the stability of the Ruthenium-based dye N719 [37] and to study in vitro the stability of N719 and the adsorption and desorption processes of this complex to the TiO<sup>2</sup> substrate in dye-sensitized solar cells [38]. We refer to this paper for details.

Recently the method has also been proposed as a possible noninvasive screening technique for revealing a content of carbon monoxide in fresh tuna fish or meat. Preliminary experimental results were presented at the Raman conference ICORS 2016 in Brazil [39] and are discussed in the following.

The method is based on the presence of dispersive Raman modes combined with a small spectral difference between the visible absorption spectra of similar molecules. The idea is as follows: the resonance condition for a specific molecule in RRS depends, as we have seen above, on the difference in the wave number between the electronic absorption and the excitation laser. Due to the tensor property of resonance Raman scattering, the value of the DPR depends on this difference (polarization dispersion). Thus, a small spectral shift in the absorption will essentially be equivalent to a displacement of the polarization dispersion curve relative to the excitation wave number of the laser, as illustrated in Figure 5. The change of the DPR value at a specific wave number depends on the shape of the dispersion curve, which depends on the nature of the vibration and the Raman tensor. When the molecule has low or no symmetry, most Raman-active vibrations will be dispersive. Although the ideal symmetry of a chromophore is often high, which limits the number of dispersive modes, the real symmetry is frequently lowered due to perturbations of the chromophore, which opens up for dispersion. The heme group with the ideal symmetry D4<sup>h</sup> is an example. This means that in reality the appearance of Raman modes exhibiting dispersion is quite common.

In the modified atmosphere packaging of fresh fish and meat products, the products are frequently exposed to carbon monoxide. Due to the higher binding affinity of CO in comparison with O2, CO replaces O<sup>2</sup> in myoglobin in the muscle tissue with high affinity, which results in the cherry-red carboxy-myoglobin complex MbCO. Due to the red color and high stability of MbCO, the fish or meat products will appear to be more fresh and attractive for a longer time period than the unexposed products. In [40] a quantitative method for the determination of CO bound to myoglobin based on visible absorption spectroscopy has been developed. Although this method has a high accuracy, it requires taking a sample from the product followed by sample preparation before the absorption spectra can be measured. Figure 4 shows a fresh tuna beef sample together with the polarizationresolved RRS spectra measured on the sample without any sample preparation but measured before and after approximately 10 minutes of exposure to CO and exciting the sample with a solid-state 532 nm laser. The experimental DPR values estimated from six Raman modes are collected in Figure 5. Further experimental details including details on the data processing of the polarization-resolved RRS data are obtainable from the author. Figure 5 also shows, as an example, a simulation of the DPR dispersion curves for the mode at 1450cm<sup>1</sup> present in the polarization-resolved RRS spectra and which has been assigned as a b1<sup>g</sup> mode. The spectral shift (color shift) due to the exposure with CO is ≈9nm equivalent to 275cm1, estimated from measuring the diffuse reflectance of the tuna beef sample before and after exposure with CO with a Lambda 900 spectrophotometer equipped with an integrating sphere. The polarization-resolved RRS data were collected by using a fiber-coupled, 180° Raman microscope consisting of a modified OlympusBX60F5, a SpectraPro 2500i spectrograph (Acton) with 1200lines/mm

to give rise to any Raman signal are written. Due to the lowering of the symmetry, there are no longer numerical relations between the tensor components. Considering, e.g., the totally symmetric vibration, we see that a resonating state must have either B3u, B2<sup>u</sup> or B1<sup>u</sup> symmetry and that, e.g., an electronic state with B3<sup>u</sup> symmetry

For the asymmetric b2<sup>g</sup> vibration, the B3<sup>u</sup> electronic state would result in a tensor where only the component <sup>S</sup>je0¼B3u<sup>i</sup> zx 6¼ 0, whereas one vibrational excitation of this vibration gives a state with the symmetry b2<sup>g</sup>⊗B3<sup>u</sup> ¼ B1u, which is seen to correspond to the transposed state tensor, i.e., only <sup>S</sup>je1¼B1u<sup>i</sup> xz 6¼ 0. From the power expansion of the vibronic state tensor given in Eq. (7), it may be shown that the two tensor elements satisfy the relation: <sup>S</sup>je0<sup>i</sup> zx <sup>¼</sup> <sup>S</sup>je1<sup>i</sup> xz . A closer investigation shows that this relation is valid in general for asymmetric vibrations [11]. The Raman tensor is calculated by inserting the two state tensors in Eq. (3). The DPR is calculated by using Eq. (5) and the relations between the rotational invariants and the Raman tensor derived in [11]. By group theory, it can be shown that an external electronic perturbation with symmetry B1<sup>g</sup> (in D4<sup>h</sup>) would result in the considered shift in the molecular configuration. As shown in Figure 3, the consequence of the symmetrylowering perturbation of the molecular configuration is that the DPR now shows a characteristic symmetric polarization dispersion with maximum half ways between the energy positions of the two states je0i and je1i. The excitation profile is symmetric around the maximum of the DPR curve and has maxima at the positions of these states, i.e., at je0i and je1i. In the point group D4<sup>h</sup>, the state tensor for the a2<sup>g</sup> vibration is purely antisymmetric with a result that the DPR becomes infinity. After the perturbation, where a2<sup>g</sup> ! bg1, the b1<sup>g</sup> state tensor is seen to have the same structure as the tensor for the b2<sup>g</sup> mode, which means that the DPR dispersion curve and excitation profile also become similar. However, the energy of the state je1i will

It follows that through the application of RADIS, it is possible to study small changes of the molecular configuration in excited electronic states and estimate the various molecular parameters influenced by these changes. As shown in numerous RRS papers on biomolecules, these structural changes, which are typically induced by minor changes in the environment of the molecule, can be studied in vivo, which

4.2 Example 2: noninvasive color detection using polarization dispersion

The color of a molecular species is associated with the properties of the electronic excited states of the molecule, and in large biomolecules, it is due the presence of a chromophore being typically a metal complex. The red color of the hemeproteins, which is due to the presence of the Fe-porphyrin complex, is a well-known example. A change in color may be due to a change of the molecular configuration (distortion, aggregation) or be a result of a chemical reaction. By monitoring the color change before and after a chemical reaction, the substance concentration in solutions can be determined from the absorbance measured by a UV/VIS spectrophotometer or in the case of a solid by applying the spectrophotometer with an integrating sphere. In the literature several color detection methods have been developed for the detection of various substances. A book on color detection is in the process of publication by IntechOpen and will be published later in 2019.

In this section a reflection technique with high spectral resolution is discussed. The technique is suitable for the detection of minor color differences between

gives rise to a tensor where only <sup>S</sup>je0¼B3u<sup>i</sup> xx 6¼ 0.

Modern Spectroscopic Techniques and Applications

in general be different for different modes.

is of course a major advantage [31–36].

28

grating, cooled CCD (Princeton Instr/Acton PIXIS), and 532 nm cw laser (VentusLP532) focused with 10x objective with NA = 0.30. Collection optic: Same objective combined with Dichroic mirror and multimode fiber coupling to spectrograph. Av. integration time 30 seconds. No sample preparation.

phenomena in the Raman intensities in the resonance region. Each dispersion curve in Figure 5 is calculated by using only a single adjustable parameter, namely, the energy splitting of the jEui state. The energy splittings, which give the best fit to the experimental values, are as follows: 465cm�<sup>1</sup> and 330cm�<sup>1</sup> for MbO<sup>2</sup> and MbCO, respectively. The energy splittings are small in comparison with the vibrational wave numbers and correspond therefore to a small perturbation of the molecules away from the D4<sup>h</sup> configuration. Since the small energy splittings are also comparable to the estimated bandwidths of the electronic transitions (γjEu<sup>i</sup> � <sup>400</sup>cm�1), they are not resolved in the visible absorption spectra, but as it follows from Figure 5, they give rise to measurable effects in the DPR dispersion. At the excitation wave number 18,797 cm�<sup>1</sup> corresponding to excitation with the 532 nm laser, the change of the DPR value, induced by the exposure with CO, is about 10%. The outcome of the experiment can be improved when the DPR data from all dispersive modes is considered. Another improvement, which would increase the reliability of the results considerably, would be a simultaneous measurement of the parallel and perpendicular polarized spectra, since this opens up for randomization and spatial averaging of the data. Simultaneous measurements of both polarizations can be obtained by modifying the excitation and collection optics in a standard Raman setup with CCD detector, in such a way that the upper and lower halves of the CCD can collect the parallel and perpendicular polarized spectra, respectively. This modification would also permit applications of polarization-resolved

What Is Vibrational Raman Spectroscopy: A Vibrational or an Electronic Spectroscopic…

DOI: http://dx.doi.org/10.5772/intechopen.86838

4.3 Example 3: unpolarized RADIS as a source of three-way multivariate data

In the last decade, chemometrics has become essential in the analysis of small differences of the chemical composition of samples in medical and environmental applications as well as in the food industry. Typically the chemical data are generated by different kinds of molecular spectroscopy: UV/VIS, fluorescence, NIR, IR, Raman, and others. A large number of mathematical methods have been developed and are in most cases part of the software package delivered with the spectrometer. The data from spectroscopy are in most cases two-way data set. Using vibrational Raman spectroscopy as an example, the elements xik in the data matrix are as

application of principal component analysis (PCA). Despite the fact that PCA analysis often gives reliable results in chemical classification problems, the recent analysis of biological samples shows that in order to obtain sufficiently high recognition ratio for secure diagnostics, one has to work with very large data sets. One way is to work with three-way data, which in general has higher information density, instead of two-way data and then apply an appropriate three-way multivariate algorithm. In classification problems involving biological samples, the three-way data has been produced by combining UV/VIS absorption data with fluorescence data in the following way: the UV/VIS absorption is measured at selected wave numbers, and the fluorescence generated at these wave numbers is measured as well. Due to low spectral resolution in both these kinds of spectra, one must produce a data matrix with very high dimension, which has the consequence that a very large number of

In [41] a new application of RADIS has been proposed, where the coherent absorption-emission property of Raman scattering is utilized. When we compare the construction of three-way data by combining UV/VIS and fluorescence data with the RADIS in Figure 2, we see that the unpolarized RADIS data must

Raman <sup>e</sup>νk<sup>Þ</sup> � , i.e., the Raman intensity at the wave number point <sup>e</sup>ν<sup>k</sup> in

th sample. Typically, these data are analyzed by the

Raman imaging.

follows: xik ¼ I

ð Þi

the Raman spectrum for the i

samples must be available.

31

First, it is noticed that the DPR values of five out the six modes indicate that they are dispersive, since all the DPR values are changed due to the exposure to CO. The polarization dispersion shows that the real symmetry of both MbO<sup>2</sup> and MbCO must be lower than D4h, which would give constant values for the DPR. It is also noticed that all the DPR values are smaller than 0.75, which indicate that the polarization dispersion is different from the one considered previously (see Figure 3). The DPR dispersion curve in Figure 5 is calculated for the b1<sup>g</sup> mode at 1450cm�1. It is obtained by adopting the procedure discussed in [11], where instead of considering the exact symmetry of the perturbed molecule (e.g., D2h) it is assumed that the molecule is really a weakly perturbed D4<sup>h</sup> system. In our example it means that the state tensor relation S <sup>j</sup><sup>e</sup>0¼Eu,x<sup>i</sup> xx ¼ �<sup>S</sup> j<sup>e</sup>0¼Eu, <sup>y</sup>i yy is still valid, but the degeneracy of the jEui state is lifted because of the perturbation, so that the two contributions to the Raman tensor now appear at slightly different energies (wave numbers). The splitting of the energy of the jEui state combined with the relation between the state tensors gives rise to constructive and destructive interference

#### Figure 4.

Fluorescence-corrected and normalized polarization-resolved RRS spectra of the tuna beef sample measured before and after 10 minutes. Exposure with CO.

#### Figure 5.

Left: experimental DPR determined from the polarized RRS data in Figure 4 using a 6 mode fit (details available from author). Right: simulation and best fit of the DPR dispersion for the b1<sup>g</sup> mode at 1450cm�<sup>1</sup> applying an "electronic interference model."

What Is Vibrational Raman Spectroscopy: A Vibrational or an Electronic Spectroscopic… DOI: http://dx.doi.org/10.5772/intechopen.86838

phenomena in the Raman intensities in the resonance region. Each dispersion curve in Figure 5 is calculated by using only a single adjustable parameter, namely, the energy splitting of the jEui state. The energy splittings, which give the best fit to the experimental values, are as follows: 465cm�<sup>1</sup> and 330cm�<sup>1</sup> for MbO<sup>2</sup> and MbCO, respectively. The energy splittings are small in comparison with the vibrational wave numbers and correspond therefore to a small perturbation of the molecules away from the D4<sup>h</sup> configuration. Since the small energy splittings are also comparable to the estimated bandwidths of the electronic transitions (γjEu<sup>i</sup> � <sup>400</sup>cm�1), they are not resolved in the visible absorption spectra, but as it follows from Figure 5, they give rise to measurable effects in the DPR dispersion. At the excitation wave number 18,797 cm�<sup>1</sup> corresponding to excitation with the 532 nm laser, the change of the DPR value, induced by the exposure with CO, is about 10%. The outcome of the experiment can be improved when the DPR data from all dispersive modes is considered. Another improvement, which would increase the reliability of the results considerably, would be a simultaneous measurement of the parallel and perpendicular polarized spectra, since this opens up for randomization and spatial averaging of the data. Simultaneous measurements of both polarizations can be obtained by modifying the excitation and collection optics in a standard Raman setup with CCD detector, in such a way that the upper and lower halves of the CCD can collect the parallel and perpendicular polarized spectra, respectively. This modification would also permit applications of polarization-resolved Raman imaging.

#### 4.3 Example 3: unpolarized RADIS as a source of three-way multivariate data

In the last decade, chemometrics has become essential in the analysis of small differences of the chemical composition of samples in medical and environmental applications as well as in the food industry. Typically the chemical data are generated by different kinds of molecular spectroscopy: UV/VIS, fluorescence, NIR, IR, Raman, and others. A large number of mathematical methods have been developed and are in most cases part of the software package delivered with the spectrometer. The data from spectroscopy are in most cases two-way data set. Using vibrational Raman spectroscopy as an example, the elements xik in the data matrix are as follows: xik ¼ I ð Þi Raman <sup>e</sup>νk<sup>Þ</sup> � , i.e., the Raman intensity at the wave number point <sup>e</sup>ν<sup>k</sup> in the Raman spectrum for the i th sample. Typically, these data are analyzed by the application of principal component analysis (PCA). Despite the fact that PCA analysis often gives reliable results in chemical classification problems, the recent analysis of biological samples shows that in order to obtain sufficiently high recognition ratio for secure diagnostics, one has to work with very large data sets. One way is to work with three-way data, which in general has higher information density, instead of two-way data and then apply an appropriate three-way multivariate algorithm. In classification problems involving biological samples, the three-way data has been produced by combining UV/VIS absorption data with fluorescence data in the following way: the UV/VIS absorption is measured at selected wave numbers, and the fluorescence generated at these wave numbers is measured as well. Due to low spectral resolution in both these kinds of spectra, one must produce a data matrix with very high dimension, which has the consequence that a very large number of samples must be available.

In [41] a new application of RADIS has been proposed, where the coherent absorption-emission property of Raman scattering is utilized. When we compare the construction of three-way data by combining UV/VIS and fluorescence data with the RADIS in Figure 2, we see that the unpolarized RADIS data must

grating, cooled CCD (Princeton Instr/Acton PIXIS), and 532 nm cw laser

graph. Av. integration time 30 seconds. No sample preparation.

it means that the state tensor relation S

Modern Spectroscopic Techniques and Applications

before and after 10 minutes. Exposure with CO.

applying an "electronic interference model."

Figure 4.

Figure 5.

30

(VentusLP532) focused with 10x objective with NA = 0.30. Collection optic: Same objective combined with Dichroic mirror and multimode fiber coupling to spectro-

First, it is noticed that the DPR values of five out the six modes indicate that they are dispersive, since all the DPR values are changed due to the exposure to CO. The polarization dispersion shows that the real symmetry of both MbO<sup>2</sup> and MbCO must be lower than D4h, which would give constant values for the DPR. It is also noticed that all the DPR values are smaller than 0.75, which indicate that the polarization dispersion is different from the one considered previously (see Figure 3). The DPR dispersion curve in Figure 5 is calculated for the b1<sup>g</sup> mode at 1450cm�1. It is obtained by adopting the procedure discussed in [11], where instead of considering the exact symmetry of the perturbed molecule (e.g., D2h) it is assumed that the molecule is really a weakly perturbed D4<sup>h</sup> system. In our example

<sup>j</sup><sup>e</sup>0¼Eu,x<sup>i</sup> xx ¼ �<sup>S</sup>

degeneracy of the jEui state is lifted because of the perturbation, so that the two contributions to the Raman tensor now appear at slightly different energies (wave numbers). The splitting of the energy of the jEui state combined with the relation between the state tensors gives rise to constructive and destructive interference

Fluorescence-corrected and normalized polarization-resolved RRS spectra of the tuna beef sample measured

Left: experimental DPR determined from the polarized RRS data in Figure 4 using a 6 mode fit (details available from author). Right: simulation and best fit of the DPR dispersion for the b1<sup>g</sup> mode at 1450cm�<sup>1</sup>

j<sup>e</sup>0¼Eu, <sup>y</sup>i

yy is still valid, but the

automatically be born as three-way data and more importantly the spectral resolution is very high. Consequently, only few data points along the Raman shift axis <sup>e</sup>ν<sup>k</sup> and along the excitation axis <sup>e</sup>ν<sup>j</sup> (eν<sup>j</sup> is an excitation wave number) for small number of samples are really needed. Thus, the elements of the RADIS data matrix are given as xijk ¼ I ð Þi Raman <sup>e</sup>ν<sup>j</sup> ;eνk<sup>Þ</sup> � . In [41] the RADIS data matrix has been analyzed by the application of a Tucker3 multivariate model, and various classification problems have been simulated and studied with the result that only few samples (<10) and few Raman lines (3– 4) and few excitation wave numbers (2–3) are needed to obtain reliable results. We refer to [41] for details.

from the signature obtained from RS, and it depends on the specific wave number of the laser. Due to appearance of overtones and combination bands in the VRRS, the anharmonic corrections to the vibrational potential function in the electronic ground state can be estimated. It follows that VRRS is a vibrational spectroscopic technique, where the properties of the resonating states and the state tensors asso-

What Is Vibrational Raman Spectroscopy: A Vibrational or an Electronic Spectroscopic…

Since the VRRS technique can be applied also as time-resolved spectroscopy, it is an attractive tool for the investigation of both the structure and dynamics of biomolecules. The main advantage of VRRS is the ability to investigate different parts of a large protein molecule by tuning the excitation wave number into the absorption band of the chromophore of interest. In a recent paper [42], the application of VRRS in the study of the structure and dynamics of various proteins is discussed. [42] gives an excellent review of this field covering both visible and UV resonance

The polarization properties of the resonance Raman signal are more important in resonance than in non-resonance. For example, as discussed in [43], the uniqueness

ciated with these have an important influence on the spectral distribution.

of the polarization-resolved VRRS spectra combined with standard PCA

chemometrics enables one to discriminate between closely related biomolecules with almost identical unpolarized VRRS spectra. The key point is that structural molecular change manifests itself through a change of the polarization of the Raman signal (DPR). The DPR defined in Eqs. (4) and (5) is an absolute quantity, which in combination with standard PCA renders the multivariate analysis insensitive to

The discussion in Section 3.3 and the examples presented in Section 4 demon-

The examples discussed in Section 4.2 show that it is possible to detect a small change in color of a molecular sample by determining the change of the DPR of a dispersive Raman mode, applying only a single excitation wave number in the absorption spectrum. To be detected the color change must be due to a modification

strate that RADIS is closer to UV/visible absorption spectroscopy than it is to vibrational spectroscopy. Besides the spectral resolution is much higher enabling the vibrational fine structure of the absorption spectra to be resolved. In resonance, the interference between the state tensors, which is the origin of the sensitivity of the Raman signal with respect to changes of the molecular parameters, is restricted to those with energy denominators closest to the laser wave number. It was also demonstrated that the polarization properties of the Raman signal, expressed through the DPR, play a more important role than in non-resonance. As said already, the DPR is defined as the ratio between two Raman signals with different polarization. The interference, which can be both constructive and destructive, will in general be different in the two Raman signals depending on the wave number of the laser and the structure of the state tensors, which again is determined by the molecular symmetry and physical properties of the molecule. Section 4.1 and Figure 3 illustrate a simple example, where the molecular configuration in an electronically excited state is distorted, which, as seen, creates a significant polarization dispersion. To fully exploit the sensitivity of the DPR to changes in the molecular parameters, one must determine the polarization dispersion, i.e., one must monitor two resonance Raman spectra (the parallel and perpendicular polarized) at each laser wave number available. Traditionally the Raman spectra with different polarizations are measured in sequence. However, with CCD technology it is possible to measure the two Raman signals simultaneously, which will improve the accuracy of the DPR considerably. This requires a modification of the entrance and collection optics of a standard Raman spectrometer, so that the upper and lower halves of the CCD monitor the parallel and perpendicular polarized Raman signals,

Raman as well as cw and time-resolved versions of VRRS.

sample and experimental variations.

DOI: http://dx.doi.org/10.5772/intechopen.86838

respectively [44, 45].

33
