Raman Spectroscopy and its Clinical Applications
When matter is illuminated with a source of light, several phenomena can happen, one of these can be dispersion of the light. If the light continues to maintain the same frequency as the incident light, it is said that a Rayleigh scattering is observed, if the scattered light undergoes a change in its frequency, it is said to have undergone a Raman scattering.
Raman spectroscopy can be an important clinical tool for real-time diagnosis of various diseases, through in-situ and in-vivo evaluation, which can provide information on the composition of the sample at molecular level.
One application of Raman spectroscopy is the analysis of biological samples such as glucose, blood, urine, among many other analytes, offering a qualitative and quantitative diagnosis. In Raman spectroscopy, incident light is often referred to as the excitation light, and may be of the UV, Visible and Infrared type.
Each molecular species contains its unique package of normal vibrations, so the Raman spectrum of some species in particular will consist of a series of bands or characteristic peaks of the sample, which give meaning of the vibrational frequency of the molecule.
In general the Raman spectrum of a sample, is composed of peaks between 10 to 2o cm^-1, which exhibit the presence of many biochemical compounds. This is the basis for obtaining information on the qualitative and quantitative nature of Raman spectroscopy.
Raman spectroscopy is widely used as an analytical technique complementary to infrared spectroscopy, so it is possible to obtain vibrational spectra of substances in aqueous solution.
Raman scattering or Raman effect
The phenomenon of relaxation is the change of light in its direction, frequency or both, when light strikes a medium.
Raman scattering is a type of inelastic scattering.
In inelastic scattering, the outgoing radiation differs in frequency from the incident, and can be both Raman scattering and fluorescence.
The light’s inelastic scattering can be called Raman scattering, of which there are two types: Raman — Stokes, where scattered light has less energy than incident light, and Raman — anti-Stokes scattering.
Raman scattering is important for anyone interested in the vibrational and rotational states of molecules. The intensity of scattered light depends on the following factors: The size of the illuminated particle or molecule, the observation position, the intensity scattered as a function of the angle with respect to the incident beam, and the frequency of the incident light.
In scattering there is an exchange of energy between light and matter (look the image above).
When light strikes a substance and is scattered, a small fraction of the radiation (approximately 1 in 10⁷ photons) will affect the vibrational energy of the substance’s molecules by interacting with the electric dipole moment of the molecule. In classical terms, the interaction can be seen as a disturbance of the electric field of the molecule. From the quantum point of view, the Raman scattering process can be seen as a transition from a molecule of its base state to an excited vibrational state accompanied by the simultaneous absorption of an incident photon and the emission of a scattered Raman photon.
The photon scattered, will have a lower energy (a wavelength greater) than the excited photon. The scattered light has generally lower optical frequencies than that of the photon incident, this is known as Stokes shift.
The results of the Raman dispersion change the vibrational state of the molecule. This vibrational energy is dissipated as heat, and due to the low intensity of Raman scattering, the dissipated heat does not cause an increase in measurable temperature in the material.
The spreading event occurs in a time of 10¹⁴ seconds or less. The scattered Raman light can be collected by a spectrometer, to obtain a Raman spectrum, where its intensity is shown as a function of the change in the frequency of light or shifts in frequencies.
The intensity of the Raman scatter is proportional to the fourth power of the frequency of the induced oscillating dipole. So the frequency along with the power of a laser, and fluorescence, should be taken into account.
Among the two types of detectors used in Raman are single-channel detectors and photomultiplier tubes (PMT) that have a single solid-state detection element.
PMTs are used in dispersive Raman systems, where they are interested in studying the region of low frequencies, with a good resolution, and close to the Rayleigh lines. Solid state detectors, such as Germanium (Ge) or Gallium Indium Arsenide (InGaAs), are preferred for example when laser line excitation corresponds to an Nd: YAG laser, with wavelength equal to 1064 nm.
Biomedical applications of Raman spectroscopy.
Different research groups use this technique for glucose detection and monitoring, biopsy discrimination of cancerous or atherosclerosis tissue, monitoring proteins, among other analytes in human tissues and fluids.
Detection of blood analytes
At the George R. Harrison Laser Biomedical Research Center at the Massachusetts Institute of Technology, the Michael S. Feld, et al., group, use Raman spectroscopy and multivariate methods for detection of blood analytes (glucose, urea, proteins, triglycerides And hemoglobin) [1]. Https://www.researchgate.net/publication/5814933_Blood_analysis_by_Raman_spectroscopy
Methodology used in this research shows the potential of Raman spectroscopy and the use of multivariate methods in clinical applications.
It is known that approximately 600 million cholesterol tests are performed annually around the world to determine possible risk, and severity of cardiovascular disease. There is a great deal of interest in optical measurements, which would allow the simultaneous analysis of multiple components in blood, without need for conventional processes such as centrifugation, and addition of reagents. Raman spectroscopy by generating a different spectrum for each multiple component, can solve the individual components of this complex mixture.
Biopsy Analysis
Detection of breast cancer is carried out using techniques such as mammography, ultrasound, magnetic resonance, positron emission imaging, among others. However, only 10 to 25% of suspicious lesions are detected using mammograms, this error rate causes unnecessary and costly surgeries [2]. While mammography and ultrasound can show only anatomical changes, they are not sensitive to sub-morphological and biochemical changes that distinguish benign and malignant lesions from the breast. Raman spectroscopy is ideally substitutable in the exploitation of these factors, providing spectral characteristics of the chemical, and morphological composition of the tissue. Recent advances in the technology make it possible to perform Raman spectroscopy in vivo via optical fibers through a biopsy via the use of needles, endoscopes, and other conventional medical devices.
This allowed the creation of a Raman spectra library of individual morphological structures in benign and malignant breast cancer tissue.
We can also use a micro spectroscopy imaging system to obtain Raman spectra, used to develop the chemical combination and the morphological model of macroscopic spectra of the tissue. The model explains the spectral characteristics of normal and diseased tissues of the breast and relates the Raman spectra to the morphological parameters used by pathologists to diagnose breast diseases.
References
[1]. https://www.researchgate.net/publication/5814933_Blood_analysis_by_Raman_spectroscopy
[2]. M. Fitzmaurice, J. L Myles, J.P. Crowe, A.S. Haka. K.E. Shafer-peltier, J.T. Motz, R.R. Dasari, M.S. Feld, G.R. Harrison., Raman spectroscopy and breast cancer: looking beyond mammography for the early detection of breast cancer., (2012)
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