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 University Shantiniketan(West Bengal)

Under the Supervision of Dr. Sobhan Sen Assistant professor School of Physical Sciences Jawaharlal Nehru University New Delhi 110067

JAWAHARLAL NEHRU UNIVERSITY SCHOOL OF PHYSICAL SCIENCES

New Delhi - 110067 Dr. Sobhan Sen Assistant Professor E-mail: sens@mail.jnu.ac.in sobhan.sen@gmail.com Phone: +91-11-26738803 +91-9999191840 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

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 SHARMA

SALONI

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-Beers 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 h = E0 - E1 where h is plancks constant and is frequency of the photon. Since, c= E= hc/

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 energystate. 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 Beers Law: Quantity of light absorbed or rate of absorption by

any substance is based on Lambert Beers Law. It is combination of two laws which areLamberts 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. Beers 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 = ()CL Where log (I0/I) is called as absorption density or OD and () is the molar extinction coefficient which is dependent.

I0

dl
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

Fig.2: Franck-Condon energy

giving the width of absorption band. The intensity diagram of the transition is given by the square of the Franck-Condon overlap integral.

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

Fig.3: Jablonski diagram

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 nonradiative 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 Stocks Shift as shown in the figure (3)

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.

Fig.3: Fluorescence Stock Shift

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 Beers law to get absorption spectrum. Detailed ray diagram of the spectrophotometer is shown in figure (4).

Grating

Beam Splitter Sample Cell

Mirror

Slit Light Source

Detector

Mirror

Reference Cell

Chopper

Computer

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

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-Beers Law as follows,
OD = CL

Here, C = Unknown

530= 10.5 10 4 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,
G ( ) =

f ( t )f ( t )
f (t )

(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,
1 G ( ) = N 1 + d
1 2 r 1 + l d

1 2

(2)

Where, N=average number of particle in the confocal volume

d =time taken by the molecule to cross the confocal volume

The diffusion constant can be calculated with the help of following equation
2 d = r

4D

(3)

Then from Stock Einsteins equation given as,

D= KT 6Rh

(4)

Where,
Rh is hydrodynamic radius,

=viscosity of the medium, K= boltzmans 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). M2 Iris L1 L2 Iris

7.5 cm Sample LASER Computer Objectiv e Lens

Dichroic Correlator APD

OD Filter Pinhole Focusing

M4

Optical Fibre Fig 6: Schematic diagram of FCS Setup

2.4 Application of FCS: Measurement of Number of Molecules inside Focal Volume. 2.4.1Preparation of sample:

Fig. 7: Structure of Rhodamine 6G

Rhodamine 6G is a dark red colour dye of molecular formula C 28H30N2O3.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:

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, d . Figure8 shown below is the fitted graph of Rh6g of different concentration, i.e, 5nM, 10nM, 15nM, 20nM.

Fig. 8: Correlation curve measured for different concentration of Rh6G with fitted curve.

Table1 Number of Sample Concentration(nM)

G(0)

Molecule in focal volume

Rh6G Rh6G Rh6G Rh6G

5 10 15 20

63.708 62.99 62.498 64.786

1.5958 1.1491 0.9629 0.7603

(1/ G(0)) 0.62664 0.87026 1.0385 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 d 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 d (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/STUDEN T_PAPERS/fcs.pdf 5