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: sens@mail.jnu.ac.in sobhan.sen@gmail.com

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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