ir spectoscopy ppt

7/29/2019 Ir Spectoscopy ppt

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Fluorescence CorrelationSpectroscopy

/

I

20050818 [email protected]


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Contents

1.What is FCS?

2.Setup

3.Base Theories4.Whats FCS used for?

5.Example Experiment - Diffusion

6.Another Example

7.Summary


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1. What is FCS ?

FCS is

Fluorescence/Fluctuation Correlation Spectroscopy

FCS is

Suggested 1972, and Renaissance 1990s

FCS isCalculate correlation function from timedependant intensity fluctuation of dye molecule by

confocal microscopy in single molecule level.


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2. Setup


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3. Based Theories Gaussian Beam

Sample

DichroicMirror

Lens

laser : 1

By Uncertainty Principle

Focal Volume


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3. Based Theories Fluorescence

Fluorescencelight : 2

Molecules are

Fluorescent

2

1


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3. Based Theories Confocal Microscopy

O.M Resolution limit ~ 200nm

But we want to see singlemolecule!

-> Confocal Microscopy


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3. Based Theories Correlation Function

Time lag ()


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4. Whats FCS used for ?

From Correlation Function, seeing

Chemical Dynamics,

Intra-molecular /

Inter-molecular

Dynamics,

Diffusion,

E T C

This Expt.Study Region


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5. Example Experiment - Diffusion

Why does the diffusion create?

-> Brownian Motion !


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Changing concentration

-> Changed Correlation Function CG

1

~)(

Be used only Concentration ~ few nM


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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 0.1

0.0

0.2

0.4

0.6

0.8

1.0

Normalized

Autocorrelationfuntion.

Lag time ( )

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

Autocorrelatio

nfunction.

Lag time ( )

TMR (200pM)

TMR (500pM)

TMR (1nM)

TMR (2nM)

TMR (5nM)TMR (10nM)


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6. Another Example - Chemical Reaction


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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


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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.comNikon Microscopy www.microscopyu.com

Mek&Tosj www.mekentosh.com

Brownian Motion java animation
http://www.rp-photonics.com/http://www.mekentosh.com/http://www.mekentosh.com/http://www.rp-photonics.com/http://www.rp-photonics.com/http://www.rp-photonics.com/


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Supplement Brownian Motion Formula

Molecule Fluctuation Equation of motion : Langevin Equation

)(int tFvFdt

dv

m ext

)]1([2 12 t

et

kT

x

Use it -> Given Stokes-Einstein relation

R

kT

D 6Diffusion coefficient

qIn

dVtrCqrSrItF ex

00

)),(()()()(Intensity


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Supplement Correlation function formula

2A

AA

0

2

A

A

tAtdtA

T

AAT

T 0

1lim0

''1

lim00

rrArdrAR

rAAR

R

tBtdtATBA

T

T 0

1lim0

Auto correlation function

Cross 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

2

0

0

2

0

2

3

We use Autocorrelation Function (bottom)


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Supplement Photon Counter

Counting A Few Photon

PMT

Photomultiplier tube

APD

Avalanche Photodiode


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Supplement FRET

: Fluorescence Resonance Energy Transfer


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Supplement Students DATA

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