Helwan University

Faculty of Engineering
Biomedical Dept.

RAMAN SPECTROSCOPY
Theory, Instrumentation, application and future

Prepared by:
Mohamed Ahmed Mohamed
Abstract

Raman spectroscopy is one of the vibration spectroscopic techniques

used to provide information on molecular vibrations and crystal
structures. This technique uses a laser light source to irradiate a sample,
and generates an infinitesimal amount of Raman scattered light, which
is detected as a Raman spectrum using a CCD camera.
The characteristic fingerprinting pattern in a Raman spectrum makes it
possible to identify substances including polymorphs and evaluate local
crystallinity, orientation and stress.

The improvement of Raman instrumentation and the development of

nanotechnologies in the end of the 1990s dramatically extended the field
of Raman Spectroscopy applications. Nowadays, scientists from a large
range of disciplines including chemistry, physics, material and life
sciences are beginning to use the Raman Spectroscopy technique, it can
be seen in the growing number of publications about Raman
Spectroscopy and its applications as illustrated in Fig.1.
1. Basic Theory

Raman spectroscopy depend on physical phenomena called inelastic

scattering discovered by Smekal in 1923 and then first observational
experiment made by C.V Raman in 1928. So to understand the theory
there must be an introduction about scattering and its types then dive in
the phenomena of inelastic and its mathematical equation.

• Scattering:

Scattering refers to light deflected from the direction of incident-light

propagation.

The interaction of the electric vector of an electromagnetic wave with

the electrons of a compound results in the scattering of the incident
light. Such interactions induce periodic vibrations in the electrons of a
compound, thereby producing oscillating electric moments. Such
oscillating electrons become new sources for emitting radiation, that is,
the scattered light.

There are three basic types of scattering:

1. Elastic: Same frequency (wavelength) as the incident light-

Rayleigh scattering.

2. Inelastic: Lower frequency (longer wavelength) than that of

incident light-Stokes Raman scattering.

3. Inelastic: Higher frequency (shorter wavelength) than that of

incident light-anti-Stokes Raman scattering.

In other words, there are two types of Raman scattering. In one type the
scattered light has lower energy than the incident light, hence it has
lower frequency, and the effect is called Stokes Raman scattering.

In the other, the scattered light has higher energy than the incident
light, hence it has higher frequency than that of the incident light (anti-
Stokes effect of Raman scattering).

Rayleigh scattering (same frequency) does not involve a change in the

energy content of the incident and scattered lights.
 Fig. 2 (a) An electron is excited from the ground level and falls to the
original ground level (Rayleigh scattering). (b) An electron is excited
from the ground level and falls to a vibration level(Stokes Raman
scattering). (c) An electron is excited from a vibration level and falls
to the ground level (anti-Stokes Raman scattering).

As can be seen in (Fig. 2) the incident light interacts with the molecule
and distorts the cloud of electrons to form a “virtual level”.
This level is not stable and the photon is immediately re-radiated as
scattered light. Rayleigh scattering is a process in which an electron in
the ground level is excited and falls to the original ground level. It does
not involve any energy change so Rayleigh scattered light has the same
energy as incident light (which means both lights have the same
wavelength).
Raman scattering can be classified as two types, Stokes Raman
scattering and anti-Stokes Raman scattering. Stokes Raman scattering is
a process in which an electron is excited from the ground level and falls
to a vibrational level. It involves energy absorption by the molecule thus
Stokes Raman scattered light has less energy (longer wavelength) than
incident light.
 By contrast, anti-Stokes Raman scattering is a process in which an
electron is excited from the vibrational level to the ground level.

It involves an energy transfer to the scattered photon thus anti-Stokes

Raman scattered light has more energy (shorter wavelength) than
incident light.

The dominant process is Rayleigh scattering, and Raman scattering is

an extremely weak process in that only one in every (106 – 108 ) photons
scatters.

The ratio of Stokes Raman and anti-Stokes Raman scattering depends on

the population of the various states of the molecule.
At room temperature, the number of molecules in an excited vibrational
level is smaller than that of in the ground level, thus generally the
intensity of Stokes Raman light is higher than anti-Stokes Raman light

From above discussion it can be seen that output scattered light differ
from input incident in wavelength so to make comparison between input
and output we need monochromatic incident light which can be obtained
by using laser with constant wavelength.
Because both the Stokes line and anti-Stokes line involve the same
vibrational-energy difference (Figure 2), the difference between the
incident-light frequency and the scattered frequency in Stokes and anti-
Stokes scattering is identical. That is, the difference in frequency for
Stokes and anti-Stokes scattering is symmetrical.

For instance, a compound illuminated with the blue line of an argon ion
laser at 488 nm (20,492 cm-1 wave number) produces two Raman lines
at 19,992 cm-1 and 20,992 cm-1. Although one cannot see the significant
correlation between these lines merely by inspecting the two values, one
can readily see an interesting result by taking the frequency difference
between the scattered and the incident light (Figure 3).

Normally, Raman scattering is expressed by this wave number difference

(∆wavenumber) rather than the absolute wave number of Raman
scattered light.
A modern Raman spectrometer automatically gives the wave number
shifts, which is what contains the molecular information.
• INDEPENDENCE OF THE INCIDENT LIGHT

The frequency of the Raman-scattered light is independent of the

wavelength of the incident light. One should not confuse this with the
fact that the intensity of Raman scattering depends on the wavelength of
the incident light and, actually, is inversely proportional to 𝜆4 .
Thus whether one excites a molecule with green light (514.5 nm or
19,436 cm-1) or blue light (488.0 nm or 20,492 cm-1) ,one will obtain a
Raman shift line at exactly the same wave number difference.

• Intensity of Strokes Vs Anti-Strokes

Although anti-stroke Raman scattering has higher energy than stroke

scattering and can be easily detected with detector its intensity depend
on temperature of sample so in almost cases it is intensity is small.
• At low temperature most of molecules will be in ground state so
most population of molecules will make stroke scattering rather
than anti-stroke scattering.
• At high temperature population of molecules at high viberational
energy increases relative to that of ground state energy.
This can be modeled using Boltzman distribution low :

Where:
Ni – Number of molecules at high viberational energy Ei
No– Number of molecules at ground state energy Eo
K– Boltzman constant
T – Temperature in the Kelvin scale.
∆E- The energy difference between energy states Ei and Eo

Therefore the ratio of molecules makes anti-Stroke scattering increase

as temperature increases.
• Polarizability and Induced dipole moment

Incident light is electromagnetic wave so when it interact with atom

electrons which are negatively charged will be attracted to positive pole
of electric field while nucleus will be attracted to negative pole as
electromagnetic poles change periodically , electrons in orbital of
matter are vibrate periodically by the incident electric field. The
oscillation of the electrons cloud results in a periodic separation of
charge within the molecules, which is called an induced dipole moment.

The strength of induced dipole moment (P) is proportional to the electric

field (E) of incident light given by this relation:

Where: 𝛼 is poloaizability

Polarizability is how easily electrons can be moved in response to

external field. The polarizability is a material property that depends on
the molecular structure and nature of the bonds.

The electric field is oscillating function depend on the light frequency

Where is incident light frequency

E0 is amplitude of electric field

Substituting in induced dipole equation:

The induced dipole emits or scatters light at the optical frequency of the
incident light wave.

The polarizability α is dependent upon the position of the nuclei in the

molecule. For molecule containing N atoms, there are 3N degrees of
freedom, Of these, 3N - 6 (3N - 5 for a linear molecule) result in
vibrations of the molecule.

Instantaneous positions of the nuclei can therefore be expressed relative

to their equilibrium positions in terms of the normal coordinates Qi'
where i = 1,2, ... ,3N - 6. Considering a diatomic molecule with the
single normal coordinate Qi the dependency of α on Qi is expressed as a
series expansion

Where α0 is equilibrium value of polarizability

The position of the nuclei is time dependent because the molecule is

vibrating with frequency VI' Information on the frequency of vibration
can be obtained from knowledge of the forces between the vibrating
nuclei, and the application of the classical mechanics of small
vibrations. This motion can be expressed as

Now induced dipole can be expressed as:

From this equation we can have the following conclusion:

1. Term α0 Produce scattered light unshifted in frequency (Rayleigh

scattering)
𝜕𝛼
2. If ≠ 0 Raman scattering occurs, and incident light of
𝜕𝑄1
frequency v0 is shifted to scattered light with higher frequency
v0+v1 (anti stokes) and lower frequency v0 - v1 (Stokes).

2. Instrumentation

Five major component make up Raman spectroscopy consist of the

following :
 1. Excitations source, which is generally a continuous wave (CW)
gas laser
2. Sample illumination and scattered light collection system
3. Sample holder
4. Monochromator or spectrograph
5. Detection system, consisting of a detector, an amplifier and an
output device
Figure 4 illustrates a typical arrangement of these components

1. Excitation sources:
Commonly used excitation sources in Raman spectroscopy are Ar+
(351.1 nm) and Kr+ (337.4 nm) and He-Ne (632.8 nm) also pulsed
 laser such as Nd:YAG, diode.
Laser used in Raman spectroscopy for the following reason:

I. Laser provide beam of light with high power and intensity so

the weak Raman scattering (10-6) can be detected
II. Laser beams are highly monochromatic (band width of
.1 cm-1)
III. Laser beams have small diameters (1-2 mm) which can be
reduced to .1 mm by using simple lens systems. This enable
studies of small samples specially microliquids.
IV. It is possible to produce laser beams in a wide wavelength
range by using dye lasers and other devices.

CW GAS LASERS:

The gas lasers operate mainly in the visible region of the

electromagnetic spectrum. Figure 5 illustrates the basic components
of a noble gas ion laser.

A very high current discharge passes through Ar or Kr gas contained

in the plasma tube. The outside of the tube is water jacketed to cool
the tube. The discharge ionizes the gas and populates the excited state
involved in the lasing.
The ends of the tube are enclosed with Brewster windows that have
an angle defined by tan Ө= n , where n is the index of refraction of
the window material. For fused silica (quartz) in the visible region, È
is 55.6°.
At Brewster's angle, the output beam is almost completely polarized
in a fixed direction.
The resonant cavity, which is defined by the semi-transparent output
mirror and the high reflectance mirror, provides a mechanism for
amplification of photons that are emitted parallel to the cavity axis;
they are

reflected by the two mirrors and interact with other excited ions.
Stimulated emission produces photons of equal energy, phase and
direction, and this process continues until an equilibrium between
excitation and emission is reached. Both mirrors are coated to reflect
the light of wavelength(s) of interest while transmitting all other light.
The output mirror transmits a fraction of the energy that is stored in
the cavity, and the transmitted radiation becomes the output beam of
the laser. The prism is inserted between the two mirrors to force the
laser to lase at a specific wavelength (single-line operation).

2.2 Day Lasers:

Dye lasers are used to extend the wavelength range for Raman
excitation. Basically, three types of dye lasers exist: those that are
pumped by a CW gas laser, those pumped by a pulsed laser, and
those pumped by a flash lamp. Relatively large volumes of organic
dye solutions are required.
A recently developed solid-state laser utilizes a titanium-sapphire
crystal: it is tunable in the 700 to 1,030 nm range and can provide
3W output power when pumped by a 20 W Ar+ laser.

2. Sample Illumination:
Laser beam must be focused onto the sample in order to get
high output intensity of Raman scattering also we need to
collect the scattered light in order to provide high intensity
to detector.
Focusing laser onto the sample can be made because of
small diameter of laser beam (1mm) even small than 1mm
can be achieved by using focus lens .
Collection of scattered light can be accomplished through
use of several optical configuration such as 90o and 180o as
shown in figure 6a and 6b

Collection optics consists of an achromatic lens system

with a collecting lens and a focusing lens.
The light-gathering power of a lens is expressed in terms of
F number, defined by
 Where f is the focal length of the lens and D is its diameter.
The smaller the F, the larger the light-gathering power.

3. The Monochromator

In a single monochromator, extraneous light that bounces

around the spectrometer overlaps the weak Raman
scattered light.
This is caused mainly by undiffracted light scattered from
the face of the grating. Such stray light could be reduced
considerably by arranging two spectrometers in tandem so
that the output of one was purified by the second. Thus, the
construction of double monochromators began. Figure 7 is
a schematic of a double monochromator.
A triple monochromator has even greater stray light
rejection than a double monochromator and allows
observation of Raman bands located very close to the
Rayleigh line. Figure 2-8 depicts schematics of the Spex
Triplemate, in which a double monochromator is coupled
with a spectrograph.
4. Detection:

Since Raman signals are inherently weak, the problems

involved with detection and amplification are severe.
Most of the very early work was done with photographic
detection using long exposure times. Furthermore, the time
to develop plates and examine them with microphotometer
rendered Raman spectroscopy unfit as a routine technique.
This situation has changed considerably since the
development of strong laser sources and sensitive detection
techniques.
Several types of detectors exists we will discuss the
following types:
 I. PHOTON COUNTING:

Scattered light focused and collected on photomultiplier

tube (PM) which convert light into electrical signal , The
PM tube consists of a photocathode that emits electrons
when photons strike it; a series of dynodes, each of which
emits a number of secondary electrons when struck by an
electron; and an anode that collects these electrons as an
output signal figure 10 show PM tube

The background noise is the primary limiting factor in PM

tube performance. This is called the "dark current" and is
primarily caused by the spurious emission of electrons from
the photocathode and dynodes and by dielectric leakage
across the PM tube base pins and resistor chain.

These spurious emissions can be reduced ( a large portion

is of thermonic origin) if the tube is thermoelectrically
 cooled via the Peltier effect so the weak Raman signal may
be easily measured.

5. PHOTODIODE ARRAY DETECTION

In normal Raman measurements, the detection of Raman

signals is made for each frequency, and the Raman
spectrum is obtained through scanning the entire frequency
range. This "single-channel" technique is time consuming,
and it is not suitable when the compound is unstable or
short-lived. Simultaneous detection of Raman signals in the
entire frequency range can be made by using multichannel
detection.

Multichannel photon detectors consist of an array of small

photosensitive devices that can convert an optical image
into a charge pattern that can be read as a Raman
spectrum.
Several types of multichannel detectors are commercially
available.

The silicon vidicon and silicon diode array are based on

the silicon diode detector.

A typical photodiode array detector shown in Figure 11

consists of 1,024 such diodes in an array ~2.5 cm long.
When the dispersed radiation strikes such a multichannel
detector, a charge pattern develops that is related to the
intensity of radiation along the focal plane, and this charge
pattern is detected and ultimately converted into a
spectrum.
Use of an intensifier (multichannel plate) in front of a
photodiode array detector increases the sensitivity of the
order of 103 ~ 104
6. CHARGE-COUPLED DEVICE DETECTION

In recent years the charge-coupled device (CCD) has been

increasingly used in Raman spectroscopy.
The CCD is an optical-array detector based on silicon-
metal-oxide semiconductor research. As shown in Figure
11b the CCD consists of two-dimensional arrays of > 106
pixels (silicon diode), each pixel ranging in size from 6 to
30 ìéôé.

The major advantages of the CCD relative to other

multichannel detectors are the low readout noise, which
makes optical intensification unnecessary, and high
quantum efficiency and sensitivity in a wide wavelength
range (120-1,000 nm).

Thus, the CCD coupled with near IR laser excitation (dye

laser, diode laser) can be used to measure Raman spectra
of fluorescent compounds.

Complete utilization of a large number of detector

elements in the CCD for spectroscopic work is under way.
For example, time-resolved spectra can be obtained by
utilizing a horizontal strip of the CCD and shifting its
position up vertically as a function of time (11). However,
disadvantages of using a CCD for Raman spectroscopy
should also be noted.
7. Application of Raman Spectroscopy in biomedical

Researchers have given much attention to the application

of Raman spectroscopy in biomedical studies, but
progress was slow for a long time until the development
of FT-Raman and NIR-Raman systems.
This is because biological samples generally emit strong
fluorescence that interferes with the Raman measurement.
NIR light has a long enough wavelength to avoid
fluorescence. A human body exhibits low absorption of
light in the spectral region from 700 to 1100nm because it
is between strong absorption bands because of hemoglobin
and water.

When there is no absorption, there is no fluorescence. In

addition, the chromophore must have a large conjugation
system to emit fluorescence in the NIR region, but a
molecule with such a large chromophore is too unstable
to exist in a biological system.
Bergholt et al. reported the in vivo diagnosis of gastric
cancer in human patients. They employed excitation
light at 785nm and an exposure time of 0.5 s. The
measurements were made under an endoscopic guide,
and more than 1000 in vivo spectra were obtained from
the patients. Some of the major Raman bands arising
from proteins, nucleic acids, and lipids were selected
by the ant colony optimization (ACO) method, and
the discrimination model was constructed using linear
discriminant analysis (LDA).
 The sensitivity and specificity of the ACO-LDA analysis
were 94.6% and 94.6%,
respectively, suggesting high reliability of diagnosis based
on Raman spectroscopy. Bergholt et al. also applied
Raman spectroscopy to the in vivo analysis of tissues in
the oral cavity.

Surface-enhanced Raman scattering (SERS) spectroscopy

is also used for diagnosis. When light is absorbed
by a metal particle of nanometer size, the energy is
transferred into a surface plasmon resonance (SPR). The
electron vibration confined in the nanoparticle raises a
localized SPR on the surface of the particle.
A molecule captured in the localized SPR receives
extremely strong excitation and generates a SERS signal.
Its intensity can be enhanced to 102–1014 times that of a
conventional Raman signal. The features of the SERS
spectrum often differ from those of conventional Raman
spectra. Lin et al. reported using SERS spectroscopy to
analyze blood to detect nasopharyngeal cancer. They
applied SERS measurement to albumin and globulin
purified from blood.

8. LIVE CELL ANALYSIS

It is possible to analyze live cells using Raman
spectroscopy. Oshima et al. succeeded in discriminating
different types of live lung cancer cells in a totally
noninvasive manner.
 This suggests the possibility of long-term monitoring of the
state of live cells without damaging the cells. Hence,
Raman spectroscopy is currently attracting the interest of
many stem cell researchers. Stem cell technologies are
acknowledged to be a key to medicine in the twenty-first
century. It may be possible to cure diseases that have no
known treatment at present, such as Alzheimer’s disease,
Parkinson’s disease, diabetes, and cardiac disease.
However, it is still difficult to induce differentiation to the
target phenotype and maintain the purity of the cells. It is
critically important to develop new technologies to monitor
and control the differentiation of the stem cell. In addition,
it will be necessary to develop a technique to check the
condition of implanted tissues made from stem cells. It is
assumed that Raman spectroscopy can contribute to future
stem cell technologies.

9. 7 FUTURE PROSPECTS

Raman spectroscopy is theoretically complete, but its

applications are growing rapidly.
A high-performance Raman microscope as well as a
portable system with an optical fiber probe is currently
within the reach of biomedical researchers. The nonlinear
Raman spectroscopies, such as CARS systems, will become
commercially available.

Laser performance is improving quickly, and many optical

devices are becoming available.
Much higher performance may be expected in the next
generation of Raman systems.
The range of applications is expanding as the instruments
evolve.

Data analysis methods also contribute to the evolution,

especially in biomedical Raman spectroscopy.
Many applications are appearing in the biomedical Raman
research field.
Active research on cultured cells, such as monitoring the
differentiation of stem cells and the detection of anticancer
drugs, has been reported recently.
It is expected that these studies will be extended to in situ
research in the near future.

Spectral analysis of live tissue is more difficult than that of

live cells because the tissue includes live cells as well as
the extracellular matrix and many other coexisting cells. As
it is difficult to research the human body directly, a period
of animal experiments may be necessary. We already have
Raman instruments for animal study, and new instruments
will be developed.

Raman spectroscopy has another specific potential, which

is the automation of analysis. It is difficult to automate
discrimination analysis of images, although the clinical
diagnosis of cancer depends on histopathology. From this
viewpoint, Raman spectroscopy is well advanced because
many multivariable methods have been developed, and
there is no problem with analyzing Raman spectra
automatically.
This article has described the instrumentation of Raman
systems as well as some new applications.
It has become much easier to make studies using
biomedical Raman spectroscopy within the last two
decades.

However, it is still important to understand the

properties of the instrument and how it works. It is only
one of the available modalities. It is not always necessary
to employ Raman spectroscopy if a better analytical
method is available. Many objects remain uninvestigated
by medical and biological applications.
10. References:

I. Anthony T. TU Raman Spectroscopy in biology

II. John R. Ferraro , Kazuo Nakamoto Introductory

Raman Spectroscopy

III. Hidetoshi Sato, Yasuhiro Maeda, Mika Ishigaki,

Bibin B. Andriana Biomedical Applications of
Raman Spectroscopy

IV. http://www.nanophoton.net/raman/raman-
spectroscopy.html