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Fluorescence correlation spectroscopy
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SUMMER TRAINING REPORT
on
Study and Application of Spectrometers: Steady State and Fluorescence Correlation Spectrometers
June-July, 2012
Submitted by
Ms. SALONI SHARMA
ISERC,Visva-Bharati UniversityShantiniketan(West Bengal)
Under the Supervision ofDr. Sobhan Sen
Assistant professorSchool of Physical SciencesJawaharlal Nehru University
New Delhi 110067
JAWAHARLAL NEHRU UNIVERSITYSCHOOL OF PHYSICAL SCIENCES
New Delhi - 110067
May 08, 2012
Certificate from the Supervisor
This is to certify that Ms. Saloni Sharma, student of Integrated M.Sc. Seventh semester, Integrated Science Education and Research Center, Visva-Bharati University, West Bengal, has successfully completed semester project entitled “Study and Application of Spectrometers: Steady State and Fluorescence Correlation Spectrometers” under my supervision during the Summer Training, from June to July, 2012 at the Spectroscopy Laboratory, School of Physical Sciences, Jawaharlal Nehru University, New Delhi. I found Ms. Saloni Sharma, intelligent, sincere and very much suitable for sophisticated experiments. I wish her all the success in her future endeavors.
(Sobhan Sen)
Signature of the Supervisor
E-mail: [email protected] [email protected]
Phone: +91-11-26738803 +91-9999191840
Dr. Sobhan SenAssistant Professor
Acknowledgment
Spending last two months in Spectroscopy laboratory (School of Physical Science,
JNU) as a summer project student was an experience of great learning a lot about
molecular spectroscopic and its sophisticated techniques such as FCS ( Single Particle
Detection Technique), Curve Fitting and with a lot of enjoyment.
I am extremely grateful and obliged to Dr. Sobhen Sen who has allowed me to
complete my summer project in Speclab and given his valuable guidance,
suggestions and encouragement .
And I am highly thankful to all my seniors Mr. Mohammad Firoz Khan, mmr. Sachin
Dev Verma, Mr. Kiran Moingarethem, Ms. Nibedita Pal and Ms. Him Shweta for their
continuous help, guidance and support from beginning to the last .
Date:14th July, 2012 SALONI SHARMA
Contents
Chapter 1: Steady State Spectroscopy
1.1 Introduction
1.2 Absorption
1.2.1 Mechanism of Absorption
1.2.2 Absorption Band and Franck-Condon Principle
1.2.3 Lambert-Beer’s Law
1.3 Photophysical Pathways
1.3.1 Internal Conversion
1.3.2 Intersystem Crossing
1.3.3 Emission
1.4 Instrumentation
1.4.1 UV-Visible Spectrophotometer
1.4.2 Measurement of concentration of Rhodamine6G in water
Chapter 2: Fluorescence Correlation Spectroscopy
2.1 Introduction
2.2 Theory
2.3 FCS Set-up
2.4 Application of FCS: Measurement of number of molecules
inside confocal volume.
2.3.1 Preparation of sample
2.3.2 Result and Discussion
Appendix
Chapter 1
Steady State Spectroscopy
Steady State Spectroscopy
1.1 Introduction
Molecular spectroscopy deals with the interaction of light with matter. These
interactions include absorption, emission and scattering etc. It is one of the most
sophisticated techniques used to measure dynamics and characterisation of various
systems. In biochemistry, it has becomes an important tool to determine the structure
and function of proteins, detection of biomolecules like mRNA and study of DNA
dynamics. In fluorescence spectroscopy, there are two types of measurement, one is
steady state measurements in which continuous illumination is performed and the
other one is time resolved in which the sample is irradiated with a pulse of light. In
these processes, fluorescent molecules are used as a probe to study a wide range of
biological processes.
1.2Absorption Spectroscopy
It is concerned with the amount of incident radiation absorbed by any substances.
Molecules selectively absorb certain wavelengths of light than the rest of wavelength
which is intrinsic properties of the molecules. When a photon of suitable energy hit a
molecule in ground state (E0), energy can be absorb and molecule is raised to
electronically excited state (E1). The energy of the photon absorb by the molecule
during the transition is given by
hν = E0 - E1
where h is planck’s constant and ν is frequency of the photon. Since,
c = λ ν
ΔE= hc/λ
1.2.1 Mechanism of absorption: Light is electromagnetic wave which has two
oscillating components i.e, oscillating electric field and magnetic field. Now out of
two fields, the electric field interact with electric charge cloud of the molecule and
created an induce dipole in the molecule. The induce dipole start oscillating with the
electric field. When resonance condition is established between the two interacting
partners (photon and the molecule) a photon is absorbed[1]. The energy of that photon
is use to shift electron of the molecule from its lower energy-state to higher energy-
state. After absorption the time spend by electronically excited molecule in the higher
energy states of an atom or a molecule , if left unperturbed by the environment is
known as the natural radiative lifetime of the molecule.
1.2.2 Lambert Beer’s Law: Quantity of light absorbed or rate of absorption by
any substance is based on Lambert Beer’s Law. It is combination of two laws which
are-
Lambert’s law – Absorbance of incident radiation is independent of intensity of
incident radiation and equal amount of incident radiation is absorbed by each small
fraction of layer of the medium.
Beer’s law- Absorbance is directly proportional to the concentration of molecules of
absorbing sample and the thickness of the layer of sample. Combining these two laws
we get,
-dI= ICdl
-dI/I= Cdl
Where I=intensity of incident radiation
c=concentration of molecules in
sample
L=thickness of the medium/sample
=constant of proportionality
Figure1 shows physical view of Lambert Beers law. Integrating both sides from limit
I0 to I, we get
ln(I0/I)= CL or
log(I0/I)=( /2.303) CL or
Optical Density (OD) = Absorbance = ε(λ)CL
Where log (I0/I) is called as absorption density or OD and ε(λ) is the molar extinction
coefficient which is λ dependent.
Fig.1: Derivation of Lambert-Beers’ Law
1.2.3 Franck-Condon principle : At room temperature most of the molecules
reside in the zero vibrational level of the ground state potential function. The time
taken for absorption is about 10-15s and for vibration it is around 10-13s[1]. Due to this
difference in the time-scale of electronic transition and vibration, the electronic and
nuclear motion can be separated and the total energy can be written as,
Etotal=Erotation+Evibration+Eelectronic
According to Born Oppenheimer Approximation “electronic transitions are so fast in
comparison to the nuclear motion that immediately after the transition, the nuclei have
a nearly the same relative position and momentum.” This fact that the vibrational
motion takes place in a time period of 100 times slower than the time period of
absorption and born oppenheimer approximation
forms the basis of Franck-Condon principle which
is “ the most probable transitions are those for
which position and momentum do not change very
much.” Therefore, the transitions can be represented
by a vertical line arising from lower energy level
curve to higher energy level curve parallel to
potential energy axis as there is no change in
internuclear distance after electronic transition
which is shown in figure2. A few more transitions
are also possible from other positions of =0 level
giving the width of absorption band. The intensity
of the transition is given by the square of the Franck-Condon overlap integral.
Fig.2: Franck-Condon energy
diagram
I0IC
L
dl
I0IC
1.3 Photophysical pathways: After absorption the molecule has two options
either it gets involved in a photochemical reaction and losses its identity or reverses
transition with emission. There are different pathways available to the excited
molecules for dissipation of excitation energy is grouped under “photophysical
pathways”. All these photophysical process occurs in a time period less then the
natural radiative lifetime of the molecule. To represent the various processes taking
place between absorption and emission (photophysical processes) we use Jablonski
diagram[2].
A Jablonski diagram is shown ain figure3 which illustrate the various photophysical
processes effectively i.e, internal conversion, intersystem crossing, fluorescence and
phosphorescence.
1.3.1 Internal conversion: When a molecule from higher vibration level of
excited state comes down to lower energy level within the excited state levels by
radiating heat is called as internal conversion as the non radiative loss of energy
occurs between the same spin manifold. The time scale for these processes is 10-13 to
10-12 s.
1.3.2 Intersystem crossing: When a molecule cross over to a lower energy triplet
state from higher energy singlet state by radiating energy in the form of heat and vice
versa is called Intersystem crossing. The time-scale for this process is 10 -6 s to 10-3s.
1.3.3Emission: In the case of molecules at very low pressure and temperature
where
collisional perturbation are absent, the excited species may return to ground state
directly by emitting the same frequency as it has absorbed. For polyatomic and
molecules at condensed state the excess vibrational energy obtained in vibronically
coupled electronic transition is quickly lost to surrounding in a time period of 10 -13s. If
the radiative transition to the ground state is allowed then the fluorescence emission is
observed. Again the fluorescence emission takes place according to Frank-Condon
principle and the most probable transition will depend on the internuclear geometries.
Due to large energy gap between S1 and S0 the transition does not occur by non-
radiative pathway. This transition takes place as fluorescence.
Fluorescence: Emission of a photon from lower vibrational level of first excited state
to higher vibrational level of ground state is called fluorescence emission. The time
scale for this emission is about 10-9 s. In most of the case, fluorescence spectrum is
observed on the red side of the spectrum as the wavelength of the emitted wave is
larger than the absorbed wave due to non- radiative loss of excess excitation energy
and this is called as Stock’s Shift as shown in the figure (3)
Fig.3: Jablonski diagram
Phosphorescence: It is the emission of photon from lowest vibration level of first
triplet excited state to highest vibrational level of ground state. The time period for
phosphorescence is 10-6s to 102s. Therefore phosphorescence is a delayed emission,
observed even after duration of hours. This is because of spin restriction i.e, a
transition from triplet state to singlet state.
1.4 Instrumentation
1.4.1UV-Visible Spectrophotometer: It is used to obtain the absorption
spectrum of any molecule. It has two source for radiation i.e, halogen
tungsten(200nm-350nm) and deuterium lamps(350nm-400nm), a monochromator to
select wavelength, beam splitter to split the beam into two equal half , then it has two
arm (in case of double beam spectrophotometer as we have used), one for placing the
sample and other for placing the reference solvent. The beam after absorption by the
sample is collected at detector as ‘I’ and the beam coming from reference is collected
as ‘I0’ then the value of log(I0/I) is calculated according to Lambert Beer’s law to get
absorption spectrum. Detailed ray diagram of the spectrophotometer is shown in
figure (4).
Fig.3: Fluorescence Stock Shift
Fig.4: Ray Diagram of UV-Visible Spectrophotometer
1.4.2 Application: Measurement of the concentration of Rhodamine 6G in
Water
Sample Preparation: We add a very small amount of solute Rh6G in HPCL water to
make a solution of Rh6g whose concentration is unknown.
Fig 5: Steady State Absorption Spectra of Rh6G in
water
Beam Splitter
Chopper
Slit
Light Source
Mirror
Mirror
Sample Cell
ReferenceCell
Grating
Detector
Computer
Procedure: First of all baseline correction is done by putting the solvent without
solute in both arm of spectrometer. After this, we replace solvent of sample arm with
the solution. The radiation obtained after absorption is collected and it goes to
detector and we get an absorption spectrum as shown in the figure (5).
Calculation: From this absorption spectrum we get different OD. Having known OD,
we can calculate the concentration of the Rh6G using Lambert-Beer’s Law as follows,
Here, C = Unknown
530= 10.5 M-1Cm-1, L is 3mm = 0.3cm
OD530= 2.13
Putting these values in the above equation we get the value of concentration(C) which
comes out to be 67.8 M.
Chapter 2
Fluorescence Correlation Spectroscopy
Fluorescence correlation spectroscopy: Theory and
Technique
2.1 Introduction
Fluorescence correlation spectroscopy is used to study dynamics of a very low
concentrated solution at single molecule level. It is useful for determination of
diffusion, concentration and dynamics of molecular interaction.
2.2Theory: In this spectroscopic technique we observe the change in fluorescence
intensity. A beam of laser light is focused by an objective to form a very small
confocal volume of dimension in femtolitre. The intensity of fluorescence fluctuate
due to fluctuation in various physical quantities during diffusion of molecules in the
confocal volume. This fluctuation in fluorescence emission is autocorrelated to get a
temporal progression of a system around its equilibrium state. The autocorrelation is
obtained by comparing the fluorescence signal obtained at a time t to the signal
obtained at a time delay i.e, at ( t+ ). A very low concentration sample is used as the
relative effect of a particular molecule on total measured fluorescence decreases with
increase in number of molecules[3].
The normalised autocorrelation function is given as,
(1)
Where f(T) is the difference between the fluorescence intensity at time t and its
average[4]. If chemical kinetics is neglected and only the single species is observed
then the autocorrelation function for a small confocal volume is given as,
(2)
Where,
N=average number of particle in the confocal volume
=time taken by the molecule to cross the confocal volume
The diffusion constant can be calculated with the help of following equation
= (3)
Then from Stock Einstein’s equation given as,
(4)
Where,
is hydrodynamic radius, =viscosity of the medium, K= boltzman’s constant,
T=temperature
2.3FCS Set Up:
Fluorescence correlation spectrometer consists of following components:
Laser: It give a light of wavelength 532nm (spl-532-LN-002T)
Mirrors: Light obtained from the laser is aligned and directed into a proper direction
with the help of mirrors.
Lens: Two lenses of focal lengths 2.54cm and 5cm are used for broadening of the
laser beam. These two lenses are placed at a distance of 7.54 cm so that they have
common focus point .The light focused by lens of focal length 2.54cm get diverged
and again this beam is collimated by the second lens and we get a parallel beam of
double size.
Iris: It is used to maintain the size of the light beam.
OD filter: It is used to maintain the intensity of the laser beam.
Dichroic: It transmit the beam of suitable wavelength and reflect the unwanted light
of different wavelength i.e, it reflect the light of wavelength less than 532nm.
Objective: A water immersion objective of Numerical Aperture =1.20 and Cover glass
thickness 0.13-0.20 is used to focus the laser beam in the sample to form the confocal
volume. The same objective is used to collect the fluorescence emission and to focus
that into the optical fibre tube.
Fibre Optics: High power fused silica multimode fibre patch cord is used to transmit
the fluorescence signal.
Avalanche Photo Diode (APD): It detects the signal of even a single photon.
Correlator Card: It autocorrelated the fluctuation in the signal which can be analysed
using Igor Pro.
Detail schematic diagram of FCS set up is shown below (figure 6).
Objective Lens
M4
Pinhole Focusing
M2 Iris Iris
Sample
OD Filter
APD
Correlator
LASER
Optical Fibre
Computer
Dichroic
7.5 cm
Fig 6: Schematic diagram of FCS Setup
L1 L2
2.4 Application of FCS: Measurement of Number of Molecules inside
Focal Volume.
2.4.1Preparation of sample:
Rhodamine 6G is a dark red colour dye of molecular formula C28H30N2O3.HCL (figure
7). In this experiment, we prepare solutions of different concentration 20nM, 15nM,
10nM, 5nM from stock solution having 100nm concentration by using equation
M1V1=M2V2 (5)
2.4.2 result and discussion:
Fig. 7: Structure of Rhodamine 6G
The data obtained from FCS is analysed using Igor Programme. By fitting the
autocorrelation curve obtained from FCS data with equation (2), we get the values of
N, . Figure8 shown below is the fitted graph of Rh6g of different concentration, i.e,
5nM, 10nM, 15nM, 20nM.
Table1
Sample Concentration(nM)
G(0)
Number of
Molecule in
focal volume
Fig. 8: Correlation curve measured for different concentration of Rh6G with fitted
curve.
(1/ G(0))
Rh6G 5 63.708 1.5958 0.62664
Rh6G 10 62.99 1.1491 0.87026
Rh6G 15 62.498 0.9629 1.0385
Rh6G 20 64.786 0.7603 1.3153
Therefore we summarised the result obtained from the fitting of correlation curve as,
From the table1, we can observe that as the concentration of the rhodamine6G
increases the number of particle inside the confocal volume increase whiles the value
of is almost constant.
Therefore, as the concentration in (nM scale) from 20 to 5 is decreased we are
approaching to detect a single molecule in confocal volume as this technique is called
as single molecule detection.
Curve Fitting
We study various physical processes using various experimental setup and
instruments such as study of fluctuation in fluorescence using Fluorescence
Correlation Spectroscopy. These physical processes follows certain pattern with
change in various physical parameters. These variation in physical parameters effect
the dynamics and other chemical or physical properties of the process or particle
under study. Therefore we model the experimental data obtained from any such
instrument i.e, we fit the curve obtained from these data which some mathematical
function( which could be previously known or newly generated depending upon the
kind of pattern or shape which the curve will take. In curve fitting we approximate the
curve obtained from the data with the fitting function i.e, we minimise the error or
standard deviation to get more accurate values of the variables under study. For
example if we have a data which takes approximately shape of a straight line then we
will fit that data with the equation of staright line and get an exact straight line with
same standard deviation . Now this fitted line line represent a more accurate data with
lowest possible error. Similarly we fit the data obtained from FCS with
autocorrelation function to get more accurate values of (diffusion time i.e, time
taken to cross the confocal volume ), N(number of molecules in confocal volume) etc.
Following are some fitted function :-
Fig(9):A line fitting
Fig(10): A Gaussian fitting
Fig(11): A lorentzian fitting
References:
1. Joseph R. Lakowicz, Principles of Fluorescence Spectroscopy, Third
addition (Springer).
2. K.K Rohatagi-Mukherjee, Fundamentals of Photochemistry, Revised
Second Edition (New Age International Publishers).
3. Petra Schwille and Elke Haustein, Fluorescence Correlation
Spectroscopy: An Introduction To its Concepts and Applications, “
http://www.biophysics.org/Portals/1/PDFs/Education/schwille.pdf ”
4. Lis a J. Carlson, Principles of Fluorescence Correlation Spectroscopy,
http://www.optics.rochester.edu/workgroups/novotny/courses/OPT463/
STUDENT_PAPERS/fcs.pdf
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