Raman Sprectroscopy

By: Pharma Tips | Views: 14249 | Date: 01-Jul-2010

Since the mid-1990s, surface-enhanced Raman scattering (SERS) has advanced greatly and gained wider application and a renewal of interest. There have been several new and creative developments, e.g. SERS of single molecules, nanostructures and transition metals, tip-enhanced Raman scattering , surface-enhanced hyper-Raman scattering , ultraviolet-excited SERS and surface-enhanced resonance Raman scattering , and their wide applications in biology, medicine, materials science and electrochemistry.

AIM OF WORK :-

            Since the mid-1990s, surface-enhanced Raman scattering (SERS) has advanced greatly and gained wider application and a renewal of interest. There have been several new and creative developments, e.g. SERS of single molecules, nanostructures and transition metals, tip-enhanced Raman scattering , surface-enhanced hyper-Raman scattering , ultraviolet-excited SERS  and surface-enhanced resonance Raman scattering , and their wide applications in biology, medicine, materials science and electrochemistry. 

                It is timely to publish a special issue reporting these initiatives and the progress made in the past 7 years. This issue consists of some invited articles that are roughly divided into three  research themes: theories, methods ,advantages ,disadvantages  and applications. These up-to-date representatives of the research results clearly show that SERS is important not only for Raman spectroscopy and surface science but also for nanoscience.

Raman Spectroscopy

INTRODUCTION :-
          Seventy years ago-The discovery of the RAMAN EFFECT was seen from German physicist Rajinder Singh & Falk riess. Raman spectroscopy is a spectroscopic technique used in condensed matter physics and chemistry to study vibration, rotational, and other low-frequency modes in a system.

           In 1923, SMEKEL predicted theoretically that if a sub stance in the gaseous, liquid or solid state is irradiated with monochromatic light the scattered light should contain radiation with different frequencies than the frequency of incident light.(1)

In 1928, Indian physicist  SIR CHANDRASHEKHAR VENKATA RAMAN (C.V.RAMAN)discovered that  when a beam of  monochromatic light was allowed to pass through a substance in the solid, liquid or gaseous state , the scattered light contain some additional frequencies over & above that of incident frequency. This is known as “RAMAN EFFECT” & is a beautiful confirmation of the SMEKEL’S prediction. He was awarded the 1931, Nobel Prize in physics for this discovery & for his systemic exploration of the phenomenon.(2)
The lines whose wavelength have been modified in Raman effect are called Raman lines .The lines having wavelength greater than that of the incident wavelength  are called Stoke’s lines & those having shorter wavelength are called Anti-stoke’s lines.(3)                                                
                           
          The theory of Raman scattering, which now is well understood & the phenomenon results from the same type of quantized vibrational changes that are associated with I.R. absorption. Thus the difference in wavelength between the incident & scattered visible radiation corresponding to wavelength in the mid I.R. region.
Indeed, the Raman scattering spectra & I.R. absorption spectrum for given species often resemble one another quite closely.
                                                      
DIFFERENCE   BETWEEN  RAMAN SPECTRA & I.R.              SPECTRA:-(1)                          
     RAMAN SPECTRA                                                  
It is due to the scattering light by the vibrating molecules.
Polarizability of the molecule will decide whether the Raman spectra will be observed or not.
It can be recorded only in one exposure.
Water can be used as a solvent.
The method is very accurate but not very sensitive.
Optical systems are made of glass or quartz.
Sometimes photochemical reactions take place in the regions of Raman lines & thus create difficulties.
Substance under investigation must be pure & colourless.
Vibrational frequencies of large molecule can be measured.
As Raman lines are weaker in intensity concentrated solution must be used to increase the intensity of Raman lines.
Homonuclear diatonic molecules are often found to be active.
                      
I.R. SPECTRA
It is the result of absorption of light by vibrating molecule.
The presence of permanent dipole moment in a molecule may be regarded as a criterion of I.R. spectra.
It required at least two separate runs with different prisms to cover the whole region of I.R.
Water cannot be used as solvent because it is opaque to I.R.
The method is accurate & very sensitive.
Optical system made up of         special crystals such as  CaF2 , NaBr.
Photochemical reactions do not take place.
This condition is not rigid.
Vibrational frequencies of large molecule cannot be measured.

Here dilute solutions are preferred.
Homonuclear diatonic molecules are not found to be active.
 
COMPARISION  BETWEEN  RAMAN SPECTRA & I.R. SPECTRA:-

THEORY :-
         Vibrational spectroscopy of molecules can be relatively complicated. Quantum mechanics requires that only certain well-defined frequencies and atomic displacements are allowed. These are known as the normal modes of vibration of the molecule. A linear molecule with N atoms has 3N - 5 normal modes, and a non-linear molecule has 3N - 6 normal modes of vibration. There are several types of motion that contribute to the normal modes. Some examples are: 
stretching motion between two bonded atoms; 
bending motion between three atoms connected by two bonds; 
Out-of-plan deformation modes that change an otherwise planar structure into a non-planar one. 
         Infrared spectroscopy allows one to characterize vibrations in molecules by measuring the absorption of light of certain energies that correspond to the vibrational excitation of the molecule from v = 0  v = 1 (or higher) states. As indicated above, not all of the normal modes of vibration can be excited by infrared radiation. There are selection rules that govern the ability of a molecule to be detected by infrared spectroscopy. 
          The Raman Effect was originally observed in 1928. It is due to the interaction of the electromagnetic field of the incident radiation, Ei, with a molecule. The electric field may induce an electric dipole in the molecule, given by 
p =  Ei                                                                                                           (1) 
where  is referred to as the polarizability of the molecule and p is the induced dipole. The electric field due to the incident radiation is a time-varying quantity of the form 
Ei = Eo cos (2 i t)                                                                                        (2) 
For a vibrating molecule, the polarizability is also a time-varying term that depends on the vibrational frequency of the molecule,  vib 
 =  o +  vib cos(2 vib t)                                                                         (3) 
Multiplication of these two time-varying terms, Ei and , gives rise to a cross product term of the form: 
                                           (4) 
         This cross term in the induced dipole represents light that can be scattered at both higher and lower energy than the Rayleigh (elastic) scattering of the incident radiation. The incremental difference from the frequency of the incident radiation,  i, are by the vibrational frequencies of the molecule,  vib. These lines are referred to as the "anti-Stokes" and "Stokes" lines, respectively. The ratio of the intensity of the Raman anti-Stokes and Stokes lines is predicted to be 
                                           (5) 
         The Boltzmann exponential factor is the dominant term in equation (5), which makes the anti-Stokes features of the spectra much weaker than the corresponding Stokes lines. 
         Infrared spectroscopy and Raman spectroscopy are complementary techniques, because the selection rules are different. For example, homonuclear diatomic molecules do not have an infrared absorption spectrum, because they have no dipole moment, but do have a Raman spectrum, because stretching and contraction of the bond changes the interactions between electrons and nuclei, thereby changing the molecular polarizability. For highly symmetric polyatomic molecules possessing a center of inversion (such as benzene) it is observed that bands that are active in the IR spectrum are not active in the Raman spectrum (and vice-versa). In molecules with little or no symmetry, modes are likely to be active in both infrared and Raman spectroscopy. 


SELECTION RULES :-
Point Groups. Molecules can be classified according to symmetry elements or operations that leave at least one common point unchanged. This classification gives rise to the point group representation for the molecule. Very useful information about the point group is contained in character tables. In this experiment we will study three different molecules: CHCl3, CH2Cl2, and CH3Cl. Both CHCl3 and CH3Cl are represented by the C3v point group and CH2Cl2 is represented by the C2v point group. The character tables describing these two point groups are shown below. 
 

This figure is from Cotton, Chemical Applications of Group Theory, 1963. 
The upper left corner of the character table identifies the point group. The remainder of the first row of the character table identifies the symmetry operations in the point group. The letters in the left column of the character table represent the symmetry species that label the irreducible representations of the group. The numbers in the table are the characters. To the right of the numbers in the table are a set of six symbols, where x, y, and z are placed in the appropriate row of the table to show how the symmetry operations of the point group affect these axes. The Rx, Ry, and Rz are placed in the appropriate row of the table to show how the symmetry operations of the point group affect rotation about these axes. The far right portion of the character table shows how squares and binary products of coordinates are affected by the symmetry operations of the point group. 

Infrared Transitions. For a fundamental transition to occur by absorption of infrared radiation the transition moment integral must be nonzero. The transition moment integrals are of the form: 

where  vo is the wave function for the initial state involved in the transition (the ground state), and  vf is the wave function for the final state involved in the transition (the excited state). The x, y and z involved in the integrals refers to the Cartesian components of the oscillating electric vector of the radiation. If any of these three integrals is nonzero, then the transition moment integral is nonzero and the transition is allowed.

We will use symmetry considerations to determine whether the transition moment integral is zero or nonzero, and hence whether the transitions is allowed or forbidden. The ground state wave function,  vo, belongs to the totally symmetric representation of the point group, A1 for the C2v and C3v point groups shown above. The symmetry representation for the excited state wave function,  vf, depends on the symmetry of the normal mode vibration to be excited. If the product of the three terms in the transition moment integrals above are not the totally symmetric representation, then the integral will be zero. This leads to a very simple rule for the activity of fundamentals in infrared absorption: 
A fundamental transition will be infrared active (that is, give rise to an absorption band) if the normal mode involved belongs to the same symmetry representation as any one or several of the Cartesian coordinates
For the C2v point group, this means that if the symmetry representation of the normal mode is A1, B1 or B2, it will be infrared allowed. Only normal modes with the A2 symmetry representation would be infrared forbidden. For the C3v point group, this means that if the symmetry representation of the normal mode is A1 or E, it will be infrared allowed. Only normal modes with the A2 symmetry representation would be infrared forbidden. 
Raman Transitions. For a fundamental transition to occur by Raman scattering of radiation the transition moment integral must be nonzero. The transition moment integrals are of the form: 
 
where  represents the polarizability of the molecule. The symmetry representations for the polarizability is the same as that of quadratic terms involving the Cartesian coordinates, x2, y2, z2, xy, yz, and xz. The symmetry representations of these terms are presented in the character table. These  's are components of the polarizability tensor and the requirement that the above integrals be nonzero means that there must be a change in polarizability of the molecule when the transition occurs. This leads to a very simple rule for the Raman activity of fundamentals: 

A fundamental transition will be Raman active (that is, give rise to a Raman shift) if the normal mode involved belongs to the same symmetry representation as any one or more of the Cartesian components of the polarizability tensor of the molecule. 

For the C2v point group, this means that all of the symmetry representations of the normal mode: A1, A2, B1 and B2, will be Raman allowed. No normal modes for molecules in the C2v point group will be Raman forbidden. For the C3v point group, this means that if the symmetry representation of the normal mode is A1 or E, it will be Raman allowed. Only normal modes with the A2 symmetry representation would be Raman forbidden. 

Normal Modes of Vibration. The normal modes of vibration for CH3Cl and CHCl3 (both molecules have the same symmetry) are shown in the figure below. 
 

This figure is from Herzberg, Infrared and Raman Spectra of Polyatomic Molecules, 1945. 
This shows that the first three vibrations for these C3v molecules are of A1 symmetry, and that the other three are doubly degenerate E symmetry vibrations.


The normal modes for CH2Cl2 are shown in the figure below. 

This figure is from Herzberg, Infrared and Raman Spectra of Polyatomic Molecules, 1945. 
This shows that the first four vibrations for this C2v molecule are of A1 symmetry, vibration five is of A2 symmetry, vibrations six and seven are of B1 symmetry and vibrations eight and nine are of B2 symmetry. O
Experimental :-
                  In this experiment you will collect infrared and Raman spectra of liquid samples of the three chloromethanes: methyl chloride, methylene chloride and chloroform. The infrared spectra will be collected using a liquid cell and the FTIR spectrometer. Raman spectra will be collected using the argon ion laser - PTI spectrometer system show in the photograph. Liquid samples are placed in a fluorescence cell and in the sample compartment for the Raman experiments. The excitation of the Raman spectra uses the 488 nm line from the argon ion laser. A computerized spectrometer control and data acquisition is used for this experiment. A photograph is shown of the computerized spectrometer control and data acquisition program. Data can be saved to a data file that can be easily imported into a spreadsheet program for further analysis. The data file consists of a column of wavelengths and a column of signal intensities. These signal intensities are an average of 100 reading of the photomultiplier (PMT) signal. Be sure to record the photomultiplier voltage, and the amplifier and time constant settings (built into the PMT housing). 

 Results :-
              You will need to analyze the Raman data to construct a Raman spectra for the Stokes portion of the spectra. Determine the locations of the fundamental vibrations observed in the Raman spectra. Determine the locations of the fundamental vibrations observed in the infrared spectra. It will be necessary for you to use the literature to help assign the peaks to the particular vibrations in the molecule. Compare the infrared, Raman and literature values of the fundamental frequencies for the transitions for each of the molecules. Are any of the transitions that are infrared or Raman forbidden observed? Discuss the similarities and differences in the data obtained in the infrared and Raman spectra. 

INSTRUMENTATION:-
Basic instrumentation for obtaining Raman spectrum is as follow.        

          Here C is a Raman tube which acts as a container for liquid to be investigated .It is made of glass &is a 1-2 cm in diameter & 10-25 cm long. The one end of the tube is drawn like a horn & blackened outside to provide a suitable background. The other end of the tube is closed with optically plane glass plate .The scattered light emerges through the window W .The Raman tube C is surrounded by water jacket . J in which cold water is circulated in order to prevent the overheating of liquid because of the proximity of the heated arc.

         S is a helium discharge tube which acts as a source of light & is diverted by nickel oxide. Filter F practice mercury arc is used as a source of light because it is difficult to construct & to obtain monochromatic light from helium discharge tube .The mercury arc is placed quite near to the Raman tube & a semi-cylindrical alluminium reflector which enhance the intensity of light .A lens L in front of the spectrograph & the Raman lines are obtained on the photographic plate.

RAMAN SPECTROGRAPH:-
Raman spectrograph consist of

1.Source of light
2.Filter
3.Sample holder
4.Spectrograph.

1] Source of light: - Since the Raman effect is relatively weak. It is essential to have a source of high intensity. The mercury arc is the most common & best source of excitation which is used now days. From mercury arc it is possible to obtain single wave length by the use of  suitable filter .it yields strong lines at 2537,3650,4047,4358,5461,5770 & 5790 A’. Out of this lines corresponding to 2536 A’ (U.V.Region), 4047(violet line), 4358(Blue line) &5461(green line) are most suited for the study of Raman spectra. Again the line  corresponding to  4358 A’ has been found to be  the most commonly used radiation in Raman spectroscopy ,since violet & green lines obtain interfered by other lines & U.V.  Line may often induce decomposition or side reaction.

 TORONTO SOURCE
         Hilger lamp consist of 4 low pressure mercury discharge tubes completely enclosed in hollow jacket whitened internally to act as a reflect or in order to increase the intensity. A tube of water cooled low pressure mercury lamp, referred to as ‘TORONTO’ type developed by welsh &Crawford at the University of Canada.
 
         In recent years lasers are almost universally employed as they produce greater power with a much greater degree of monochromaticity.A laser with ionized argon as its working material gives an output at 488.0 nm & this makes a good Raman source unless it causes fluorescence in the sample.

2] FILTERS:- We know that with non monochromatic incident light there will be overlapping of Raman shifts which will give rise to difficulties in the interpretation of the Raman spectrum .It is therefore necessary to have strictly monochromatic radiation. For such a purpose, filter which may be chosen either by trial or by published table son filter for Raman spectrum are employed. They may either be made of glass or may be suitable coloured solutions such as I2 in CCL4  or aqueous solution of ferricyanide.                                                      

3] SAMPLE HOLDER:- For study of the Raman effect, the type of the sample holder depends upon the intensity of the source  & the nature  & viability of the sample. For studying Raman spectra of gases, the sample holder are usually bigger in size than those for liquids. Thus for measuring gases & liquid, the volume of the tube may vary from 50-100 ml. A simple Raman tube, which is easily cleaned & filled, is used in the Raman source unit.

A SAMPLE TUBE & ILLUMINATION UNIT FOR RAMAN     SPECTROSCOPY  

      Solids are usually dissolved before subjecting to Raman spectrograph. Any solvent suitable for U.V. spectra may also be used in the study of Raman spectra. Water has been found to be a very good solvent for inorganic compounds in Raman spectroscopy. For small samples, a four traversal tube is usually employed.

        Since the intensities of Raman lines of gases are very small, very high intensity lights, extremely sensitive detectors & multiple traversal tubes should be used for their measurement.

4] SPECTROGRAPH:- The important feature of a spectrograph, suitable for the study of Raman spectra are :
1)Large height gathering power.
2)Special prisms of high resolving power, &
3)A short focus camera. 
          A lens L in front of the plane window directs the scattered radiation upon the slit of the spectrograph & the Raman lines can be observed on the photographic plate .It should be noted that instrument of high resolving power. e.g.:- gratings are not used in spectrograph, on accounts of the poor luminosity which necessitate long exposures.
          For excitation of Raman effects in solids & gases, certain modification have to be made in the experimental arrangement. The intensity of light scattered from gases is very weak. This difficulty has been removed by using:
1)Very high intensity lights.
2)Spectrograph of great light gathering power.
3)Highly sensitive detectors.
4)Multiple traversal tubes.

5] RECEPTORS OR DETECTORS :-  Raman spectrograph  have been equipped with  either photographic or automatic pen recording device. Photographic emulsion or photomultiplier tube are, therefore are employed in Raman spectroscopy as detectors or receptors. Since Raman lines are usually feeble, photographic plate used should be highly sensitive. In Raman spectroscopy, several hours & even a day is required for exposure. In case of gases time of exposure may be decrease by increasing the pressure in the sample holder.
                         The Carry Model 81 spectrophotometer is the most widely  used at the present time .The cross sectional diagram throught the optical system of the model is shown in figure as follow.
 

ALTERNATIVE TECHNIQUES :-
1.FOURIER TRANSFORM (FT) RAMAN  :-
Unlike dispersive Raman spectroscopy, which obtains a spectrum by diffraction of the different wavelengths, Fourier transform (FT) Raman creates an interference pattern that can be analysed to recover the spectrum. It has the advantage of being faster than the dispersive technique, but is limited in resolution and choice of laser wavelength.
 2.     STIMULATED RAMAN  :-
Stimulated Raman scattering is a non-linear phenomenon that results in a much larger Raman signal than standard scattering (4 to 5 orders of magnitude greater). It can be triggered with a strong laser pulse. Only the strongest Raman active mode is excited at first, but scattering from it can be strong enough to excite the second mode, which in turn can excite the third and so on in a cascade effect.
 
Diagram of Stimulated Raman

3.     RESONANCE RAMAN:-
Another effect that greatly increases the magnitude of scattering is Resonance Raman (RR). This occurs when the energy of the incident radiation is close to that of an electronic excitation energy (i.e. the band gap). Tunable lasers can be used to achieve this. The Raman scattering of vibrational modes around this excited state is then greatly enhanced by resonance effects. 
As shown in figure,Resonance Raman scattering is differ from fluorescence in that relaxation to the ground state is not preceded by perior  relaxation to the the lowest vibrational level of the excited electronic state.The time scale for two phenomenon  are also quite different  with Raman relaxation occurring in less than 10-14 s compaired with the 10-6 to 10-8s for fluorescence emission.
 
 4. SURFACE ENHANCED RAMAN SPECTROSCOPY (SERS) :-
Surface Enhanced Raman Scattering (SERS) is a process that can occur when samples are adsorbed on gold or silver surfaces. It results in a vast increase in the Raman effect, and is therefore useful for spectroscopy. Though the mechanism is not very well understood, it is believed to be a combination of chemical enhancement of polarisability by bonds formed between the sample and the surface, and electromagnetic resonance of small gold or silver particles. This effect can be combined with Resonance Raman for Surface Enhanced Resonance Raman Spectroscopy (SERRS), which results in very strongly enhanced signals, up to 1014 times more intense than standard Raman scattering.

5.COHERENT ANTI-STOKES RAMAN SPECTROSCOPY(CARS):-
          Coherent Anti-Stokes Raman Spectroscopy (CARS) is a technique involving two lasers. One is of a fixed frequency ν1 and the other is tunable to a lower frequency ν2. When combined they result in coherent radiation at frequency
ν' = 2 ν1 - ν2
along with a number of other frequencies.
If there is a Raman active mode with characteristic frequency νm, then when the second laser is tuned such that
ν2 = ν1 - νm
then the coherent emission emerges in a high intensity narrow beam with frequency
ν' = 2 ν1 - ( ν1 - νm) =  ν1 + νm
This is the anti-Stokes frequency, hence the name.
 
Diagram of Coherent Anti-Stokes Raman Spectroscopy

6.   RAMAN OPTICAL ACTIVITY – COMPARES POLARISATIONS:-
Raman optical activity is a technique which compares the different polarisations of Raman scattered light from chiral molecules such as those found in biology, in order to determine more about their structure. This can also be combined with amplifying techniques such as resonance and surface enhancement.

ADVANTAGES & DISADVANTAGES :-
ADVANTAGES
Raman spectroscopy has a number of advantages over other analysis techniques.
Can be used with solids, liquids or gases.
No sample preparation needed. For infrared spectroscopy solids must be ground into KBr pellets or with nujol to form a mull.
Non-destructive
No vacuum needed unlike some techniques, which saves on expensive vacuum equipment.
Short time scale. Raman spectra can be acquired quickly.
Can work with aqueous solutions (infrared spectroscopy has trouble with aqueous solutions because the water interferes strongly with the wavelengths used)
Glass vials can be used (unlike in infrared spectroscopy, where the glass causes interference)
Can use down fibre optic cables for remote sampling.

DISADVANTAGES
Cannot be used for metals or alloys.
The Raman effect is very weak, which leads to low sensitivity, making it difficult to measure low concentrations of a substance. This can be countered by using one of the alternative techniques (e.g. Resonance Raman) which increases the effect.
Can be swamped by fluorescence from some materials.

APPLICATIONS:-
Raman spectroscopy is commonly used in chemistry, since vibrational information is very specific for the chemical bonds in molecules. It therefore provides a fingerprint by which the molecule can be identified. The fingerprint region of organic molecules is in the range 500-2000 cm-1. Another way that the technique is used is 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 (specifically, 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.

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. [4] 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 MICROSPECTROSCOPY:-
            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 interfere very strongly. 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 He-Ne 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 micro spectroscopy, 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 high power. However, the intensity of NIR Raman is low (owing to the ω-4 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. 

POLARIZED RAMAN ANALYSIS:-
             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 pedagogically 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.

REFERENCES :-
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4. J. C. de Paula,  http://www.haverford.edu/chem/302/Raman.pdf. 
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Newyork , Acadamic Press, 1994
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Newyork, Wiley, 1996
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    Newyork, Wiley, 1984
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      Newyork, Wiley, 1991
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