Fluorescence Correlation

Spectroscopy
/
I
20050818
sunchul@sogang.ac.kr
Contents
1.What is FCS?
2.Setup
3.Base Theories
4.Whats 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 :
1
By Uncertainty Principle
Focal Volume
3. Based Theories Fluorescence
Fluorescence
light :
2

Molecules are
Fluorescent

1
3. Based Theories Confocal Microscopy
O.M Resolution limit ~ 200nm
But we want to see single
molecule!
-> Confocal Microscopy
3. Based Theories Correlation Function
Time lag ()
4. Whats 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
> < C
G
1
~ ) (t
Be used only Concentration ~ few nM
5. Example Experiment - Diffusion
0 2 4 6 8 10
0
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


N
o
r
m
a
l
i
z
e
d
A
u
t
o
c
o
r
r
e
l
a
t
i
o
n

f
u
n
t
i
o
n
.
Lag time ( )
200pM (norm)
500pM (norm)
1nM (norm)
2nM (norm)
5nM (norm)
10nM (norm)
1E-5 1E-4 1E-3 0.01 0.1
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0


A
u
t
o
c
o
r
r
e
l
a
t
i
o
n

f
u
n
c
t
i
o
n
.
Lag time ( )
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
Reference
P.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 4
th
edition
Saleh&Hahaa, Fundamentals of Photonics 4
th
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 Formula
Molecule Fluctuation Equation of motion : Langevin Equation
) (
int
t F v F
dt
dv
m
ext

+ = o )] 1 ( [
2
1 2 t
e t
kT
x

o

>= <
Use it -> Given Stokes-Einstein relation
R
kT
D
tq 6
=
Diffusion coefficient
q I n
dV t r C q r S r I t F
ex
=
=
}
o k
o o k o
0 0
)) , ( ( ) ( ) ( ) (
Intensity
Supplement Correlation function formula
2
A
( ) ( ) t A A 0
2
A
A
t
t
( ) ( ) ( ) ( ) t t + =
}

t A t dtA
T
A A
T
T 0
1
lim 0
( ) ( ) ( ) ( ) ' '
1
lim 0
0
r r A r drA
R
r A A
R
R
+ =
}

( ) ( ) ( ) ( ) t t + =
}

t B t dtA
T
B A
T
T 0
1
lim 0
Auto correlation function
Cross correlation function
( ) ( ) ( ) ( ) ' '
1
lim 0
0
r r B r drA
R
r B A
R
R
+ =
}

D
D w
r
C
B G
t
t
t
t
t
2
0
0
eff
) ( 1
1
) 1 (
1
V
1
) (
+
+
> <
+ =
D
r
w r V
D
eff
4
2
0
0
2
0
2
3
=
=
t
t
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 Students DATA