Raman spectroscopy has the ability to generate quantitative and qualitative information even though it has very weak signals which limit its chances to identify very low molecular concentrations (Petry et al., 2003). Improvements have been made especially from 1960’s to enhance Raman signals which can lead to the production of high quality spectra with fine molecular details. Developments in Raman systems are discussed below in this section.
Surface Enhance Raman Spectroscopy (SERS)
In 1977, Jeanmaire and Van Duyne determined the fact that if the sample is placed on a roughened noble metal substrate, it has the ability to significantly increase Raman scattered signals (D.L. Jeanmaire and Duyne, 1977). Silver, gold or copper substrates produce strong electromagnetic fields when excited by visible light. These powerful electromagnetic fields increase the magnitude of dipole as well as intensity of inelastic scattered signals. Special resonators used in SERS allow the magnitude of Raman cross section to be 15 times more than that of basic Raman system which can produce spectra
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even from extremely low concentrations (Kudelski, 2008). This advancement in Raman system is known as Surface Enhanced Raman Spectroscopy (SERS), which promotes the importance of noble metal substrates. SERS is extremely sensitive and can be applied in critical molecular identifications and in recent past it has been excessively used to explore areas of biological and chemical sensing such as trace analysis of pesticides (Weiβenbacher et al., 1997), antigens specific to prostate (Grubisha et al., 2003), nuclear waste (Bao et al., 2006), glucose (Shah et al., 2007) and several others. In this system, there are two ways to amplify Raman signals; 1) chemical enhancement, in which charge transfer takes place between the substrate and adsorbed molecules and 2) electromagnetic enhancement, where an electromagnetic field is created at the metal substrate resulting in amplified signals (Kudelski, 2008, Petry et al., 2003). Selection of substrate is the most important aspect when performing a SERS experiment. Excitation of localized surface Plasmon resonance (LSPR) is directly related to the SERS intensity therefore it is critical to control all factors that may influence LSPR in order to achieve maximum signal strength as well as reproducibility. Sensitive biological samples may get damaged when used with heavy metal surface but this can be resolved by using metal coated glass fibre tip.
SERS compatible lasers include gas, dye and solid-state lasers however Ultra Violet (UV) lasers have certain limitations because LSPR cannot be excited over a specific frequency threshold due to dielectric properties of noble metals (Haynes et al., 2005). A further advancement in SERS is the attachment of a fibre optic probe head with portable Raman system, this is named as tip-enhanced RS (Kudelski, 2008). This can be used for in vivo investigations to measure glucose levels in the body with the help of acquired Raman spectra (Lyandres et al., 2005).
29 Resonance Raman Spectroscopy (RSS)
In a classical Raman system, frequency of light which induces Raman Effect, determines the strength of scattered signals therefore the intensity of a scattered Raman signal varies according to the power of initial frequency. If this initial frequency becomes equal to the electronic transition of an irradiated molecule, Raman active modes are amplified up to 6 orders of magnitude. This phenomenon in Raman systems is known as resonance effect or resonance Raman spectroscopy (RSS). This effect has the ability to measure or target a specific molecule within a complex structure only if it has an absorption transition, which possesses same energy level as of the incoming photons (Robert, 2009). This specific property of RRS allows it to investigate particular molecules in biological tissues such as conformations or chromophores present in proteins can be assessed even though they are embedded in membranes. RRS has been extensively used in biological investigations of specific molecules such as iron-sulfer clusters, hemes and carotenoids (Ruban et al., 2007). Chromophoric biological samples like enzymes have been widely analysed by this technique. Electronic excitation of vibrational mode is generally increased where chromophores are present thus leading to enhanced signals (Kudelski, 2008). In living tissues, haemoglobin oxygen levels have also been measured using resonance effect by evaluating the intensity ratios of oxygenated and de-oxygenated haemoglobin (Ward et al., 2007). Fluorescence contamination can be significantly reduced by combining RRS and SERS, which can generate very sharp and precise spectra and can be extremely helpful in DNA identification (Faulds et al., 2003).
Coherent Anti-Stokes Raman Spectroscopy (CARS)
According to a traditional Raman system, the incident intensity of scattered Raman signals is linear however there is an involvement of two photons. One of these two photons is produced spontaneously. There is a vibrational transition which is obtained
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by virtual excitation and de-excitation of higher energy states (Myers, 1997). The phenomenon of scattering can simply be defined as the energy exchange between incident photon and scattered molecule. Elastic or Rayleigh scattering takes place when there is no energy transfer between the incident photon and the molecule however in inelastic or Raman scattering there is a loss or gain in the energy of scattered photons which means that energy has been transferred (Myers, 1997). In contrast to traditional systems, CARS involve a non-linear Raman technique having two or more coherent monochromatic frequency waves as incident radiation. The overlapping of two monochromatic wave frequencies of the incident radiation is a characteristic feature of CARS. When the first frequency wave is higher than the second, the frequency difference between the two is the frequency of the molecule (Kudelski, 2008). Two special features that this technique offers are high spatial and temporal resolution and high signal generation capacity which allows measuring of the density difference between various levels of vibrational energies (S.A.J. Druet and Taran, 1981). Moreover, signals produced by CARS are generally five orders of magnitude stronger than those of a basic Raman system. According to Druet and Taran (1981), characteristic features of CARS are listed below;
The dispersive mechanism produces enhanced and detailed spectroscopic measurements as compared to spontaneous Raman system.
Excellent spectral and spatial resolution of 0.03 – 1 cm-1
and ˂ 1mm respectively.
Time resolution of approximately 100 fs. Much more luminous (105
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Application of CARS is well documented in the fields of reactive media, plasma, gas laser amplifiers and cells (differentiation between cells) (El-Diasty, 2011, Konorov et al., 2007, Downes et al., 2011).
Confocal Raman Microscopy
The whole sample is uniformly illuminated in a typical Raman spectrometer whereas in confocal Raman system a specific point is illuminated and focused by adding a confocal microscope to the system. The incident light is focused onto the sample and the area of focus is dependent on the magnification of objective used (Petry et al., 2003). The objective which is connected to the spectrometer collects the scattered light from the sample which passes through a pinhole aperture and makes sure that only the reflected light from the focused area passes through and the additional light from any surrounding area is blocked (figure 1.8) (Petry et al., 2003). Fluorescence is difficult to control in these systems as compared to conventional Raman systems because the laser beam is targeted at a very small sample area. The advantage that this system offers is three dimensional measurement with extremely high spatial resolution of >1 μm which is capable of performing single cell investigations (James W. Chan et al., 2006, Zhang et al., 2008).
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Figure 1. 8 Schematic illustration of a basic confocal Raman system