Fluorescence Fluorescence Correlation Correlation SpectroscopySpectroscopy

물리학과물리학과 // 바이오융합기술바이오융합기술바이오융합기술프로젝트바이오융합기술프로젝트 II

20050818 20050818 차선철 차선철 sunchul@sogang.ac.sunchul@sogang.ac.krkr

Contents

1.What is FCS?

2.Setup

3.Base Theories

4.What’s FCS used for?

5.Example Experiment - Diffusion

6.Another Example

7.Summary

1. What is FCS ?

FCS is

Fluorescence/Fluctuation Correlation Spectroscopy

FCS is

Suggested 1972, and Renaissance 1990s

FCS is

Calculate correlation function from time dependant intensity fluctuation of dye molecule by confocal microscopy in single molecule level.

2. Setup

3. Based Theories – Gaussian Beam

Sample

Dichroic Mirror

Lens

laser : λ1

By Uncertainty Principle

Focal Volume

3. Based Theories – Fluorescence

Fluorescence light : λ2

Molecules are Fluorescent

λ2

λ1

3. Based Theories – Confocal MicroscopyO.M Resolution limit ~ 200nm

But we want to see single molecule!

-> Confocal Microscopy

3. Based Theories – Correlation Function

Time lag (τ)

4. What’s FCS used for ?

From Correlation Function, seeing

Chemical Dynamics,

Intra-molecular /

Inter-molecular

Dynamics,

Diffusion,

E T C…

This Expt.

Study Region

5. Example Experiment - Diffusion

Why does the diffusion create?

-> Brownian Motion !

5. Example Experiment - Diffusion

Changing concentration

-> Changed Correlation Function

CG

1~)(

Be used only Concentration ~ few nM

5. Example Experiment - Diffusion

0 2 4 6 8 100

5

10

15

20

25

30

35

1/G(0

)

Concentration (nM)

inverted g(0)

1E-5 1E-4 1E-3 0.01 0.10.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Auto

corr

elat

ion

funt

ion.

Lag time (τ )

200pM (norm) 500pM (norm) 1nM (norm) 2nM (norm) 5nM (norm) 10nM (norm)

1E-5 1E-4 1E-3 0.01 0.11.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2.0

Auto

corr

elat

ion

func

tion.

Lag time (τ )

TMR (200pM) TMR (500pM) TMR (1nM) TMR (2nM) TMR (5nM) TMR (10nM)

6. Another Example - Chemical Reaction

7. Summary

We know theory of FCS

: Uncertainty Principle – Gaussian Beam

: Quantum Mechanics – Fluorescence

: Geometric Optics – Confocal Microscopy

: Statistical Mechanics – Correlation function

We use FCS

Study Brownian Motion – Diffusion

ReferenceP.Schwile and E.Haustein, Fluorescence Correlation Spectroscopy

S.H.Kim, Fluorescence Correlation Spectroscopy

Y.H.Hwang Spectroscopy : Characterization of Material Properties

C.Gell, et al. Handbook of Single Molecule Fluorescence Correlation Spectroscopy

S.Weiss, Fluorescence Spectroscopy of Single Biomolecules

F.Reif, Fundamentals of Statistical and Thermal physics 4th edition

Saleh&Hahaa, Fundamentals of Photonics 4th edition

PerkinElmer, Avalanche Photodiode A User Guide

Hamamatsu, Photomultiplier tubes Basic and Applicaitions

RP Photonics www.rp-photonics.com

Nikon Microscopy www.microscopyu.com

Mek&Tosj www.mekentosh.com

Brownian Motion java animation

Supplement – Brownian Motion FormulaMolecule Fluctuation Equation of motion : Langevin EquationMolecule Fluctuation Equation of motion : Langevin Equation

)(int tFvFdt

dvm ext

)]1([2 12 tetkT

x

Use it -> Given Stokes-Einstein relation Use it -> Given Stokes-Einstein relation

R

kTD

6Diffusion coefficientDiffusion coefficient

qIn

dVtrCqrSrItF ex

00

)),(()()()(IntensityIntensity

Supplement – Correlation function formula

2A

AA 0

2A

A

tAtdtA

TAA

T

T 0

1lim0

''1

lim00

rrArdrAR

rAAR

R

tBtdtA

TBA

T

T 0

1lim0

Auto correlation functionAuto correlation function

Cross correlation functionCross correlation function

''1

lim00

rrBrdrAR

rBAR

R

DD w

rCBG

2

0

0eff)(1

1

)1(

1

V

1)(

D

r

wrV

D

eff

4

20

020

23

We use Autocorrelation Function (bottom)We use Autocorrelation Function (bottom)

Supplement – Photon Counter

Counting A Few Photon

PMT

Photomultiplier tube

APD

Avalanche Photodiode

Supplement – FRET

: Fluorescence Resonance Energy Transfer

Supplement – Student’s DATA