RAMAN SPECTROSCOPYOctober 11, 2006 at 1:37 am | Posted in Research, Science & Technology | Leave a comment
Raman Spectroscopy is a spectroscopic
technique used in condensed matter physics
and chemistry to study vibrational, rotational, and other
range. Phonons or other excitations in the system are absorbed
or emitted by the laser light, resulting in the energy of the laser photons being shifted
up or down. The shift in energy gives information about the phonon modes in the system. Infrared spectroscopy yields similar, but complementary information.
Typically, a sample is illuminated with a laser beam. Light from the illuminated spot
is collected with a lens and sent through a monochromator. Wavelengths close to the laser line (due to elastic Rayleigh scattering) are filtered out and those in a certain spectral window away from the laser line are dispersed onto a detector.
Spontaneous Raman scattering is typically very
weak, and as a result the main difficulty of Raman spectroscopy is separating the weak
Raman spectroscopy has a stimulated version, analogous to stimulated emission, called stimulated Raman scattering.
The Raman effect occurs when light impinges upon a molecule
and interacts with the electron cloud of the bonds of that molecule. The amount of
deformation of the electron cloud is the polarizability of the molecule. The amount of the
polarizability of the bond will determine the intensity and frequency of the Raman shift.
The molecule must be symmetric to observe the Raman shift. The photon
(light quantum), excites one of the electrons into a virtual state. When the photon is
released the molecule relaxes back into vibrational energy state. The molecule will
typically relax into the first vibration energy state, and this generates Stokes Raman
scattering. If the molecule was already in an elevated vibrational energy state, the Raman
scattering is then called Anti-Stokes Raman scattering.
Raman spectroscopy is commonly used in chemistry, since vibrational information is specific to the chemical bonds and symmetry of molecules. Therefore, it provides a fingerprint by which the molecule can be identified. For instance, the vibrational frequencies of SiO, Si2O2, and Si3O3 were identified and assigned on the basis of normal coordinate analyses using infrared and Raman spectra. The fingerprint region of organic molecules is in the (wavenumber
) range 500–2000 cm−1. Another way that the technique is used to study changes in chemical bonding, e.g., when a substrate is added to an enzyme.Raman gas analyzers have many practical applications. For instance, they are used in medicine for real-time monitoring of anaesthetic and respiratory gas mixtures during surgery.
In solid state physics, spontaneous Raman spectroscopy is used to, among other things, characterize materials, measure temperature, and find the crystallographic orientation of a sample. As with single molecules, a given solid material has characteristic phonon modes that can help an experimenter identify it. In addition, Raman spectroscopy can be used to observe other low frequency excitations of the solid, such as plasmons, magnons, and superconducting gap excitations. The spontaneous Raman signal gives information on the population of a given phonon mode in the ratio between the Stokes (downshifted) intensity and anti-Stokes (upshifted) intensity.
Raman scattering by an anisotropic crystal gives information on the crystal orientation. The polarization of the Raman scattered light with respect to the crystal and the polarization of the laser light can be used to find the orientation of the crystal, if the crystal structure (to be specific, its point group) is known.
Raman active fibers, such as aramid and carbon, have vibrational modes that show a shift in Raman frequency with applied stress. Polypropylene fibers also exhibit similar shifts. The radial breathing mode is a commonly used technique to evaluate the diameter of carbon nanotubes. In nanotechnology, a Raman microscope can be used to analyze nanowires to better understand the composition of the structures.
Spatially-offset Raman spectroscopy (SORS), which is less sensitive to surface layers than conventional Raman, can be used to discover counterfeit drugs without opening their internal packaging, and for non-invasive monitoring of biological tissue. Raman spectroscopy can be used to investigate the chemical composition of historical documents such as the Book of Kells and contribute to knowledge of the social and economic conditions at the time the documents were produced. This is especially helpful because Raman spectroscopy offers a non-invasive way to determine the best course of preservation or conservation treatment for such materials.
Raman spectroscopy has also been used to confirm the prediction of existence of low-frequency phonons  in proteins and DNA (see, e.g.,     greatly stimulating the studies of low-frequency collective motion in proteins and DNA and their biological functions.
Raman spectroscopy offers several advantages for microscopic analysis. Since it is a scattering technique, specimens do not need to be fixed or sectioned. Raman spectra can be collected from a very small volume (< 1 µm in diameter); these spectra allow the identification of species present in that volume. Water does not generally interfere with Raman spectral analysis. Thus, Raman spectroscopy is suitable for the microscopic examination of minerals, materials such as polymers and ceramics, cells and proteins. A Raman microscope begins with a standard optical microscope, and adds an excitation laser, a monochromator, and a sensitive detector (such as a charge-coupled device (CCD), or photomultiplier tube (PMT)). FT-Raman has also been used with microscopes.
In direct imaging, the whole field of view is examined for scattering over a small range of wavenumbers (Raman shifts). For instance, a wavenumber characteristic for cholesterol could be used to record the distribution of cholesterol within a cell culture.
The other approach is hyperspectral imaging or chemical imaging, in which thousands of Raman spectra are acquired from all over the field of view. The data can then be used to generate images showing the location and amount of different components. Taking the cell culture example, a hyperspectral image could show the distribution of cholesterol, as well as proteins, nucleic acids, and fatty acids. Sophisticated signal- and image-processing techniques can be used to ignore the presence of water, culture media, buffers, and other interferents.
Raman microscopy, and in particular confocal microscopy, has very high spatial resolution. For example, the lateral and depth resolutions were 250 nm and 1.7 µm, respectively, using a confocal Raman microspectrometer with the 632.8 nm line from a Helium-Neon laser with a pinhole of 100 µm diameter. Since the objective lenses of microscopes focus the laser beam to several micrometres in diameter, the resulting photon flux is much higher than achieved in conventional Raman setups. This has the added benefit of enhanced fluorescence quenching. However, the high photon flux can also cause sample degradation, and for this reason some setups require a thermally conducting substrate (which acts as a heat sink) in order to mitigate this process.
By using Raman microspectroscopy, in vivo time- and space-resolved Raman spectra of microscopic regions of samples can be measured. As a result, the fluorescence of water, media, and buffers can be removed. Consequently in vivo time- and space-resolved Raman spectroscopy is suitable to examine proteins, cells and organs.
Raman microscopy for biological and medical specimens generally uses near-infrared (NIR) lasers (785 nm diodes and 1064 nm Nd:YAG are especially common). This reduces the risk of damaging the specimen by applying higher energy wavelengths. However, the intensity of NIR Raman is low (owing to the ω4 dependence of Raman scattering intensity), and most detectors required very long collection times. Recently, more sensitive detectors have become available, making the technique better suited to general use. Raman microscopy of inorganic specimens, such as rocks and ceramics and polymers, can use a broader range of excitation wavelengths.
The polarization of the Raman scattered light also contains useful information. This property can be measured using (plane) polarized laser excitation and a polarization analyzer. Spectra acquired with the analyzer set at both perpendicular and parallel to the excitation plane can be used to calculate the depolarization ratio. Study of the technique is useful in teaching the connections between group theory, symmetry, Raman activity, and peaks in the corresponding Raman spectra.
The spectral information arising from this analysis gives insight into molecular orientation and vibrational symmetry. In essence, it allows the user to obtain valuable information relating to the molecular shape, for example in synthetic chemistry or polymorph analysis. It is often used to understand macromolecular orientation in crystal lattices, liquid crystals or polymer samples.
Several variations of Raman spectroscopy have been developed. The usual purpose is to enhance the sensitivity (e.g., surface-enhanced Raman), to improve the spatial resolution (Raman microscopy), or to acquire very specific information (resonance Raman).
- Surface Enhanced Raman Spectroscopy (SERS) – Normally done in a silver or gold colloid or a substrate containing silver or gold. Surface plasmons of silver and gold are excited by the laser, resulting in an increase in the electric fields surrounding the metal. Given that Raman intensities are proportional to the electric field, there is large increase in the measured signal (by up to 1011). This effect was originally observed by Martin Fleischmann but the prevailing explanation was proposed by Van Duyne in 1977. A comprehensive theory of the effect is that given by Lombardi and Birke in 2008 called the a Unified Approach to Surface-Enhanced Raman Spectroscopy.
- Resonance Raman spectroscopy – The excitation wavelength is matched to an electronic transition of the molecule or crystal, so that vibrational modes associated with the excited electronic state are greatly enhanced. This is useful for studying large molecules such as polypeptides, which might show hundreds of bands in “conventional” Raman spectra. It is also useful for associating normal modes with their observed frequency shifts.
- Surface-Enhanced Resonance Raman Spectroscopy (SERRS) – A combination of SERS and resonance Raman spectroscopy that uses proximity to a surface to increase Raman intensity, and excitation wavelength matched to the maximum absorbance of the molecule being analysed.
- Hyper Raman – A non-linear effect in which the vibrational modes interact with the second harmonic of the excitation beam. This requires very high power, but allows the observation of vibrational modes that are normally “silent”. It frequently relies on SERS-type enhancement to boost the sensitivity.
- Spontaneous Raman Spectroscopy (SRS) – Used to study the temperature dependence of the Raman spectra of molecules.
- Optical Tweezers Raman Spectroscopy (OTRS) – Used to study individual particles, and even biochemical processes in single cells trapped by optical tweezers.
- Stimulated Raman Spectroscopy – A spatially coincident, two color pulse (with polarization either parallel or perpendicular) transfers the population from ground to a rovibrationally excited state, if the difference in energy corresponds to an allowed Raman transition, and if neither frequency corresponds to an electronic resonance. Two photon UV ionization, applied after the population transfer but before relaxation, allows the intra-molecular or inter-molecular Raman spectrum of a gas or molecular cluster (indeed, a given conformation of molecular cluster) to be collected. This is a useful molecular dynamics technique.
- Spatially Offset Raman Spectroscopy (SORS) – The Raman scatter is collected from regions laterally offset away from the excitation laser spot, leading to significantly lower contributions from the surface layer than with traditional Raman spectroscopy.
- Coherent anti-Stokes Raman spectroscopy (CARS) – Two laser beams are used to generate a coherent anti-Stokes frequency beam, which can be enhanced by resonance.
- Raman optical activity (ROA) – Measures vibrational optical activity by means of a small difference in the intensity of Raman scattering from chiral molecules in right- and left-circularly polarized incident light or, equivalently, a small circularly polarized component in the scattered light.
- Transmission Raman – Allows probing of a significant bulk of a turbid material, such as powders, capsules, living tissue, etc. It was largely ignored following investigations in the late 1960s but was rediscovered in 2006 as a means of rapid assay of pharmaceutical dosage forms. There are also medical diagnostic applications.
- Inverse Raman spectroscopy.
- Tip-Enhanced Raman Spectroscopy (TERS) – Uses a metallic (usually silver-/gold-coated AFM or STM) tip to enhance the Raman signals of molecules situated in its vicinity. The spatial resolution is approximately the size of the tip apex (20-30 nm). TERS has been shown to have sensitivity down to the single molecule level.
6. Ben Vogel (29 August 2008). “Raman spectroscopy portends well for standoff explosives detection”. Jane’s. http://www.janes.com/news/transport/business/jar/jar080829_1_n.shtml. Retrieved 2008-08-29.
14. Ellis DI, Goodacre R (August 2006). “Metabolic fingerprinting in disease diagnosis: biomedical applications of infrared and Raman spectroscopy”. Analyst 131 (8): 875–85. doi:10.1039/b602376m. PMID 17028718.
16. Jeanmaire DL, van Duyne RP (1977). “Surface Raman Electrochemistry Part I. Heterocyclic, Aromatic and Aliphatic Amines Adsorbed on the Anodized Silver Electrode”. Journal of Electroanalytical Chemistry (Elsevier Sequouia S.A.) 84: 1–20. doi:10.1016/S0022-0728(77)80224-6.
20. Matousek P, Clark IP, Draper ERC, et al. (2005). “Subsurface Probing in Diffusely Scattering Media using Spatially Offset Raman Spectroscopy”. Applied Spectroscopy 59 (12): 393. doi:10.1366/000370205775142548. PMID 16390587.
24. P. Matousek, N. Stone (2007). “Prospects for the diagnosis of breast cancer by noninvasive probing of calcifications using transmission Raman spectroscopy”. Journal of Biomedical Optics 12 (2): 024008. doi:10.1117/1.2718934. PMID 17477723.
- An introduction on Raman Scattering, d3technologies.co.uk
- Raman Spectroscopy Applications, renishaw.com
- Raman Data Search and Storage – The free application with a wonderful database of Raman data (vibrations, assignment) with storage function and Raman spectra (discussions) with search function. ramandata.sourceforge.net
- Romanian Database of Raman Spectroscopy – This database contains mineral species (natural and synthetic) with description of crystal structure, sample image, number of sample, origin, Raman spectrum and vibrations, Raman discussion and references. Also, this site contains artefacts sample with sample image and pigment spectrum; black, red, white or blue pigment. rdrs.uaic.ro
- Chemical Imaging Without Dyeing, witec.de
- DoITPoMS Teaching and Learning Package – Raman Spectroscopy – an introduction, aimed at undergraduate level. doitpoms.ac.uk
- Raman Spectroscopy Tutorial – A detailed explanation of Raman Spectroscopy including Resonance-Enhanced Raman Scattering and Surface-Enhanced Raman Scattering. 22.214.171.124
- The Science Show, ABC Radio National – Interview with Scientist on NASA funded project to build Raman Spectrometer for the 2009 Mars mission: a cellular phone size device to detect almost any substance known, with commercial <USD$5000 commercial spin-off, prototyped by June 2006. abc.net.au/rn
- Raman spectroscopy for medical diagnosis from the June 1, 2007 issue of Analytical Chemistry, pubs.acs.org
- Spontaneous Raman Scattering (SRS), lavision.de
- Painless laser device could spot early signs of disease, BBC News, 2010-09-26