V.C. Raman was awarded the Nobel prize two years after discovering the Raman effect in 192855. If a specimen is illuminated by a monochromatic light source, and the scattered light is recorded, the collected spectrum will contain a very strong exciting line at the same frequency as the source. This is due to the elastic scattering of the specimen's molecules to the incident photons. More importantly, there will also be other weaker signals on either end of the line, due to the inelastic scattering of the incident photons (Raman scattering)56. These lines will be very weak relative to the excitation line as the probability of Raman scattering is 1 to 107.
Raman effect takes place when a photon is scattered inelastically from the electric dipole of a molecule. Scattering is explained by quantum mechanics as an excitation to a virtual energy state, lower than a true electronic transition.
Followed by its prompt de-excitation (10-14 seconds or less) with a change in vibrational energy57–59.
Stokes and anti-Stokes scattering modes energy difference originates from the difference between
the starting and final state of the excited molecules60 as shown in Figure 2. In Stokes scattering, the molecule is initially in a ground state and ends up in an excited state. With the increase in the molecule's energy compensated from the scattered photon's energy. In this case, the scattered photon has lower energy than the incident one. On the other hand, the situation is reversed in anti-Stokes scattering. The molecule is initially in an excited state and ends up in the ground state with the excess energy being added to the incident photon, resulting in a higher energy scattered photon.61 At room temperature, the number of excited molecules is significantly lower than those in the ground state. Thus, the anti-Stokes-shifted spectrum is always weaker than the Stokes-shifted spectrum62. Both spectra have the same frequency information.
A change in vibrational, rotational, or electronic energy of a molecule is accompanied by Raman scattering. The resulting vibrational spectrum consists of bands representing the
the states involved in Raman spectra.
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molecule's vibrations. This spectrum depends on the masses of the atoms in the molecule, the strength of their chemical bonds, and their atomic arrangement63. Consequently, different molecules have different vibrational spectra or different “fingerprints”.
Consequently, molecular species can be resolved from their Raman spectrum by analyzing the peak positions.
Further information about the molecules' relative concentration can be derived from relative peak amplitudes64. Conformational information on the other hand can be extracted from peak width, where wider peaks mean a larger variation in conformation between the same molecular species in the sample. Figure 3 shows an example of a high amplitude-low variation peak against a low amplitude–high variation peak.
2.1. Fundamentals of Raman Spectroscopy in Biological Tissues
The application of Raman spectroscopy in biology is rapidly increasing as it provides molecular-level information, allowing investigation of functional groups, bonding types, and molecular conformations. In addition to being a relatively simple, reproducible, non-destructive technique, it requires a small sample size with minimal sample preparation. It also does not suffer from water interference, which is abundant in biological tissues. Raman peaks are relatively narrow, easy to resolve, and sensitive to molecular structure, conformation, and environment, resulting in a high chemical specificity modality65.
Various studies66–71 have mapped Raman peaks in biological tissues to their underlying structures whether it is a particular chemical bond or a functional group. Perhaps the most important for Raman analysis of proteins are the amide bands.
The amide bands originate from the vibrational normal modes of the protein backbone. There are nine modes of vibration labeled A, B, and I-VII in order of decreasing frequency. In the fingerprint range (750 – 1800 cm-1) only amide I, II, and III - shown in Figure 4 - are present,
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of them, amide II is very weak and could only be observed in the absence of resonance excitation. The specific positions of these bands are indicative of the structure/conformation of the proteins at hand. Other important spectral features in protein spectra include aromatic amino acids like proline, hydroxyproline, phenylalanine, tyrosine, and tryptophan shown in Figure 5.
Raman bands from these amino acids provide further information about the environment of the proteins.
2.2. Raman Spectroscopic Characterization of Articular Cartilage.
Figure 6 shows a typical Raman spectrum of bovine articular cartilage. Various studies have made use of the information this spectrum contains to characterize articular cartilage. In 201139, Esmonde-White et al carried out a proof-of-concept study for adapting Raman spectroscopy in arthroscopic
measurements. The study used a custom-designed Raman fiber optic probe to examine the knees of human cadavers and tissue phantoms. In a following study in 201972, they utilized Raman spectroscopy to probe the biochemical composition of the synovial fluid of 40 patients who suffer from OA. The information gained from the spectra allowed joint damage assessment, to determine if the patient is osteoarthritic or not. Another study by Shaikh et al.41 was successful in discriminating between types of cartilage injuries using Raman spectroscopy.
Figure 5: Aromatic amino acids: A) Proline, B) Hydroxyproline, C) Phenylalanine, D)
Tyrosine, E) Tryptophan
Figure 6: Raman spectrum of cartilage
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