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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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5. Example Experiment - Diffusion
Changing concentration
-> Changed Correlation Function CG
1
~)(
Be used only Concentration ~ few nM
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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 0.1
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/7/29/2019 Ir Spectoscopy ppt
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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