Transcript FCS - System and Instrumentation Physics

Detection, identification and
conformational dynamic
characterization of single molecules
by ultra-sensitive fluorescence
spectroscopy techniques.
Jerker Widengren
Exp. Biomol. Physics
Dept. Physics, KTH
Topics of Discussion

Fluorescence Correlation Spectroscopy (FCS)
- Concept
- strategies to study molecular kinetics /
applications

Single-molecule Multi-parameter fluorescence
detection (smMFD)
- Concept
- single-molecule FRET studies
Fluorescence Correlation
Spectroscopy
Dynamic properties of molecules probed via
their thermodynamic fluctuations
 At equilibrium, no perturbation
 Original concept

Magde, Elson and Webb, 1972, Phys. Rev. Lett. 29, 705
Elson and Magde, 1974, Biopolymers, 13, 1
Magde, Elson and Webb, 1974, Biopolymers, 13, 29
Ehrenberg and Rigler, 1974, Chem. Phys., 4, 390
FCS set-up
Higher spatial discrimination
 Higher spectral discrimination
 Enhanced detection efficiency
 - increased fluor./(mol. x s)
- reduced background

REF:
- Rigler and Widengren, in Bioscience,
Klinge and Owman (Ed.), Lund University
Press, 180, 1990
- Rigler, Widengren and Mets, in Fluoresc.
Spectroscopy, Wolfbeis (Ed.),
Berlin:Springer, 13, 1992
- Rigler, Mets, Widengren and Kask,
Eur. Biophys. J. 22, 179, 1993
Fluorescence fluctuations due to translational diffusion
I(t)
<I>
t
I(t)
<I>
t
The Autocorrelation function:
G( ) 
 I ( t ) I ( t   )   [  I   I ( t )][  I   I ( t   )] 
 I ( t ) I ( t   ) 


1

 I 2
 I 2
 I 2
Translational diffusion for a 3D gaussian volume element:
1
1
1
1/ 2
G D ( )  (
)(
)
1
2
2
N 1  4D /  1 1  4D /  2
The experimental FCS curve for translational diffusion:
2.6
2.4
2.2
2.0
1/N
G()
1.8
1.6
D
1.4
1.2
1.0
0.8
0.0001
0.001
0.01
0.1
1
10
Correlation time (ms)
2
1
1
1
1/ 2

G D ( )  (
)(
)  1  D  1 , where D = kT
2
2
N 1  4D /  1 1  4D /  2
4D
6R
Change in diffusion properties

DF

DB
1  1 Y
Y 
G (t )  

 1
N 1  / 
1  /  
DF
DB
Ligand-receptor interactions:
A: nAChR in solution
Association kinetics
1,0
2,2
0,8
2,0
Association of -bungatrotoxin to nAChR
INCUBATION TIME
0 min
4.5 min
19.5 min
72 hrs
1,8
G()
1,6
1,4
0,6
Free
0,4
Bound
0,2
Total
0,0
10
100
Incubation time (min)
1,0
Dissociation kinetics
0,8
1,2
0,6
0,4
1,0
total
0,2
0,1
1
correlation time (ms)
bound
10
0,0
1
10
Incubation time (min)
100
 High
sensitivity, ligand-receptor
interactions at low conc. can be
followed
 low conc. of labelled ligands --->
facilitates displacements studies
 No separation of bound from unbound
 Low quantities of material needed
 No radioactivity
REF: Rauer, Neumann, Widengren, Rigler 1996, Biophys. Chem
58, 3-12
Change in fluorescence upon chemical reaction
I(t)
<I>
t
2.6
2.4
2.2
2.0
1/N
G()
1.8
1.6
D
1.4
1.2
1.0
0.8
0.0001
0.001
0.01
0.1
Correlation time (ms)
1
10
Change in fluorescence upon chemical reaction
I(t)
Fl  X


k1


k 1
FlX
<I>
t
2.6
2.4
2.2
2.0
1/N
G()
1.8
1.6
D
1.4
1.2
1.0
0.8
0.0001
0.001
0.01
0.1
Correlation time (ms)
1
10
Change in fluorescence upon chemical reaction
I(t)
Fl  X


k1


k 1
FlX
<I>
t
Ion concentration monitoring:
Fl  H
2


k1



k 1
HFl

2.2
FITC in 1 mM carbonate buffers
2.0
Rh-II in 1 mM EGTA buffers
pH 7.5
2.0
>100 M
pH 7
pH 6.5
G()
1.8
0.64 M
pH 5.5
1.4
1.1 M
1.6
pH 6
1.6
2.4 M
1.8
0.41 M
G()
2.2
0.18 M
1.4
92 nM
pH 5
1.2
1.2
1.0
1.0
0.0001 0.001
0.01
0.1
(ms)
1
10
100
1000
0.1
1
 (ms) 10
100
1.25
Buffer dependence (phosphate buffer)
Buffer effects
Autocorrelation
1.20
k
Fl 2   H 
+


300 M
1.15
1.0 mM
3.0 mM
1.10
1.05
HFl 

k -
100 M
1.00
0.01
0.1
Time (ms)
1
10
k
BH  Fl
2
k1


k -1
0,6
0,5
6 -1


k diss
BH
ktot(10 s )
B  H 
ass



B  HFl

0,4
phosphate
citrate
HEPES
NaCl (/200)
0,3
0,2
0,1
Widengren J, Terry B, Rigler R,
Chem Phys. 249, 259-271, 1999
0,0
0,0
0,5
1,0
1,5
2,0
Concentration (mM)
2,5
3,0
Photophysics
S1 ( )
G ( )  G D ( )
1
S1
-
triplet state transitions
 - electron transfer
 - trans-cis isomerization
Triplet state monitoring by FCS
k23
S1
k12=absIexc
T1
k21
k31
S0
 S0 (t )   k12
 
d
S
(
t
)

 k
dt  1   12
 T (t )   0
S +S
0
1
T
S +S
0
1

k 21
- k 23  k 21
k 23

k 31  S 0 (t ) 


0  S1(t ) 
 k31  T(t ) 
T
Fluctuations in fluorescence due to singlet-triplet transitions
The fluorescence intensity correlation function:
2,2
Rh6G in Water
2,0
1,8
Power 48.4 W
Power 350 W
Power 2,55 mW
G()
1,6
T
G( ) 
lim
T 
1
I( t )I( t   )dt
T 0
1 T

I
(
t
)
dt
 

T 0

1
2
1,4
 I  2   I( t )I( t   ) 


 I 2

1
 12    2 2 

1 
 1 

N (1  T) 
4D  
4D 
Teq 
1,0
1E-4
1/ 2
1  T
eq

 Teq exp(  3 )  1
k 23I exc
I exc ( k 23  k 31 )  k 21k 31
 I ( k  k 31 )  k 21k 31 
T  
  exc 23

3 
I exc  k 21

1
1,2
1
2,2
2,0
1,8
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
1E-3
0,01
 (ms)
0,1
1
10
Rh6G in water
T(s)
Teq
0,01
0,1
1
Laser Power (mW)
10
Environmental influence
on the triplet state
2,2
2,0
1,8
properties of
1,2
1,0
1E-4
1E-3
0,01
 (ms)
0,1
1
10
FITC
2.2
[KI]=0 mM
[KI]=0.2 mM
[KI]=2.0 mM
[KI]=5.0 mM
1,8
2.0
Rh6g in water at different oxygen conc.
1.8
1.6
1,6
1,8
1,4
1,2
1.2
1,2
1,0
1.0
1,0
1E-4
1E-3
 (ms) 0,1
0,01
1
10
FITC in water (pH 9)
Power 19.8 W
Power 95.6 W
Power 568 W
1,6
1.4
1,4
2,2
2,0
oxygen atm.
air atm.
argon atm.
G( )
G()
1,4
Rh6G in water
2,0
Triplet state
1,6
G()
2,2
G()
Effects of solvents
and quenchers on
the triplet state
Solvent effects of Rh6G
Ethylene glycol
Ethanol
Water
0.001
0.01
0.1
 (ms)
1
10
100
1E-4
1E-3
0,01
 (ms)
0,1
1
10
Triplet state monitoring:
• Distortion of FCS curves at high excitation intensities can
to a large extent be attributed to triplet state build-up.
• By FCS it is possible to measure intersystem crossing
rates, triplet state lifetimes and excitation cross sections.
• The environmental sensitivity of the triplet parameters
suggests the use of FCS for micro-environmental probing.
• Knowledge of triplet parameters important for
optimization of fluorescence
REF: - Widengren, Rigler and Mets J. Fluoresc. 4(3), 255-258, 1994
- Widengren, Mets and Rigler J. Phys. Chem. 99, 13368-13379, 1995
- Mets, Widengren and Rigler Phys. Chem. 218, 191-198, 1997
If D  0, Fl = 0 :
Dual colour FCS
Photon counting histograms (PCH) /
Fluorescence intensity distribution analysis (FIDA):
Fluorescence brightness
Concentration
Figures of merit:
number of deteced fluorescence photons
molecule
number of deteced fluorescence photons
molecule  time
Photophysical limitations:
- Fluorescence saturation
- Photodestruction
Fluorescence saturation:
Photobleaching
3,5
2,0
Rh6G in water
3,0
FITC in water, pH 9
2
1100 kW/cm
1,8
2
1 kW/cm2
470 kW/cm
140 kW/cm2
2,0
13 kW/cm2
1,6
1,4
1,5
1,2
1,0
1,0
0,001
0,01
0,1
1
Correlation time (ms)
10
100
4 kW/cm2
G()
G()
2,5
26 kW/cm2
0,1
1
10
100
Correlation time (ms)
Widengren J, Rigler R, Bioimaging, 4, 149-157, 1996
Eggeling C, Widengren J, Rigler R, Seidel, C, Anal Chem, 70, 2651-2659, 1998
Photobleaching effects in a cell surface
Exposure time: t
Diffusion coeff: D
Excitation power: Pexc
Radius of cell area: Rcell
-10
2
Pexc
Rcell
Normalized concentration
D=7x10 cm /s
1,0
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,0
2.5 W
5.0 W
10 W
25 W
50 W
100 W
250 W
0
Widengren J submitted to Biophys. J.
1,0
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0,0
1
2
3
4
Radial distance (m)
5
How to maximize
fluorescence information
from single molecules:
nf
info
nf
Single-molecule Multi-parameter Fluorescence detection (smMFD)
Model system
A488 emission
Cy5 excitation
Cy5
500
550
600
650
Wavelength (nm)
A488
700
Fluorescence resonance energy transfer
DIexc
kFRET
kD10
kFRET´
kA10
A
D
6
R0
E 6
6
R 0  R DA
R0=8.7910-5 J() FD n-4 21/6 Å
E(FD, FA, rD, rA, FA, FD)
FRET and Coincidence Analysis
FG 1
E  (1  
)
FR
10
100
IG [kHz]
Ex 496 nm
5
1
10
0
1
log IR [kHz]
100
1
10
0
1
10
10
0
frequency
100
IG [kHz]
Ex 496 nm
Ex=647 nm
10
0
0.1
30
0
1
20
10
z:/singlem/dec99/12/HD3HD5_DA_g/smd/bi4/HD3HD5_DA_g_vortrag.opj
0
20
100
IG [kHz]
Ex 496 nm
Ex=647 nm
10
IR [kHz]
2
100
1
10
0
1
100
1
10
0
1
1
2
log IG [kHz]
0
20
frequency
-1
-1
0
1
2
log I G [kHz]
0.1
40
frequency
100
Ex 496 nm
Ex=647 nm
IR [kHz]
100
1
10
0
1
Alexa 488 - Cy5
z:/singlem/dec99/12/HD5HD6_DA_gr/smd/bi4/HD5HD6_DA_gr_vortrag.opj
0.1
40
20
2
Alexa 488 - Cy5
z:/singlem/dec99/12/HD1HD5_DA_gr/smd/bi4/HD1HD5_DA_gr_vortrag.opj
10
0
18 bp
2
Alexa 488 - Cy5
1
2
log I G [kHz]
0.1
1
30 I [kHz]
G
20
10
0
IR [kHz]
10 bp
log IR [kHz]
5 bp
0
frequency
0
0
10
Alexa 488 - Cy5
1
2
log I G [kHz]
1
IR [kHz]
1
-1
-1
0.1
40
frequency
1
0
Ex 496 nm
100
z:/singlem/dec99/12/HD6HD5_DA_g/smd/bi4/HD5HD6_DA_g_vortrag.opj
frequency
0.1
30
1
2
log IG [kHz]
100
2
Alexa 488 - Cy5
-1
-1
0.1
10
18 bp
2
z:/singlem/dec99/12/HD1HD5_DA_g/smd/bi4/HD1HD5_DA_g_vortrag.opj
0
0.1
1
40
30 IG [kHz]
20
10
0
IR [kHz]
log IR [kHz]
log IR [kHz]
10
Alexa 488 - Cy5
frequency
Ex 496 nm
10 bp
100
-1
-1
log IR [kHz]
100
0
2
-1
-1
10
IG [kHz]
20
IR [kHz]
5 bp
20
1
log IR [kHz]
0
0.1
30
frequency
1
frequency
frequency
0.1
10
z:/singlem/dec99/12/HD3HD5_DA_gr/smd/bi4/HD3HD5_DA_gr_vortrag.opj
0
20
frequency
0.1
-1
-1
0
1
2
log I G [kHz]
0
20
0.1
40
frequency
FG 1
E  (1  
)
FR
 DA
E  1
D
Conformation-based identification
 DA
E  1
D
2
4
6
g [ns]
100
0
100
Fg/Fr
FG 1
E  (1  
)
FR
frequency
0
100
10
10
1
1
Mixture: bp:
5; 9; 13; 15 bp
0,1
0
2
4
g [ns]
6
Fg/Fr
0
0,1
150
frequency
Fit to a structural model of DNA
1,0
E via kISOtot
E via cps/mol
fit to model
E via DA
Efficiency E
0,8
0,6
0,4
:
0,2
0,0
2
4
6
8
10
12
bp
14
16
18
20
22
24
FRET studies with
smMFD:
em , F, r,  F
-High sensitivity, precision and accuracy
-resolution better than 1 nm
- identification based on conformational properties
(”conformational fingerprints”)
- range: 10-100 Ångström
- Detection and selective analysis of subpopulations
Photodynamics of Cy5
86 kW/cm
2
2
0.4 kW/cm
3,0
6 -1
kISOtot(10 s )
1
3,5
647 nm exc
496 nm exc
2
0.8 kW/cm
2
1.6 kW/cm
0,1
G()
2,5
0,01
1
10
100
1000
2,0
2
2.9 kW/cm
3
2
Exc Int (kW/cm )
1
N P
1,5
*
1,0
1E-4
1E-3
0,01
0,1
1
Correlation time (ms)
10
100
-
O3S
SO3
N
Trans-cis
isomerization
of Cy5
N
O
N
O
Cy5-NHS
O
O
kNperp
1
1N
kPperp
1
1P
kISC
3
1N
kN10
Widengren J. & Schwille P.
J. Phys. Chem. 104(27), 64166428, 2000
Widengren J. & Seidel C.
Phys. Chem. Chem. Phys. 2,
3435-3441, 2000
-
3
1P
1Perp
kT
kP01
kN01
kP10
kPN
1
0N
kN01 = I exc
1
0P
kP01 = Iexc
P
FRET-mediated
excitation:
DIexc
kFRET
kD10
kFRET´
D
kA10
A
k ISOtot  k ISO  k BISO   D E ( N ) ISO   D E ( P) BISOI exc
FRET-mediated excitation of Cy5
Acceptor 1st bp, Donor 14th bp
G()
2,0
1,5
1.0 kW/cm
2
1.9 kW/cm
2
3.2 kW/cm
2
1,0
0,01
0,1
1
10
Correlation time (ms)
100
FRET-mediated excitation of Cy5
Acceptor 1st bp, Donor 14th bp
G()
2,0
1,5
1.0 kW/cm
2
1.9 kW/cm
2
3.2 kW/cm
2
1,0
0,01
0,1
1
10
100
Correlation time (ms)
2,5
Excitation intensity 3.2 kW/cm
2
G()
2,0
1,5
A-D distance:
4 bp
13 bp
18 bp
1,0
0,01
0,1
1
Correlation time (ms)
10
100
FRET-mediated excitation of Cy5
50
Acceptor 1st bp, Donor 14th bp
bp=4
bp=11
bp=13
bp=18
bp=22
45
2,0
G()
3 -1
kISOtot(10 s )
40
1,5
1.0 kW/cm
2
1.9 kW/cm
2
3.2 kW/cm
2
0,01
0,1
1
10
100
Correlation time (ms)
2,5
25
20
15
Excitation intensity 3.2 kW/cm
2
A-D distance:
4 bp
13 bp
18 bp
1,0
0,01
0,1
1
Correlation time (ms)
10
5
0
0,0
2,0
G()
30
10
1,0
1,5
35
100
0,5
1,0
1,5
2,0
2,5
3,0
3,5
2
Excitation Intensity (kW/cm )
4,0
FRET-mediated excitation of Cy5
50
Acceptor 1st bp, Donor 14th bp
bp=4
bp=11
bp=13
bp=18
bp=22
45
2,0
G()
3 -1
kISOtot(10 s )
40
1,5
1.0 kW/cm
2
1.9 kW/cm
2
3.2 kW/cm
2
0,01
0,1
1
10
100
Correlation time (ms)
2,5
25
20
15
5
0
0,0
Excitation intensity 3.2 kW/cm
2
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
2
Excitation Intensity (kW/cm )
k ISOtot ( FRET) E ( N )  E ( P)

k ISOtot (direct )
2
2,0
A-D distance:
4 bp
13 bp
18 bp
1,0
E via kISOtot
E via cps/mol
fit to model
E via DA
0,8
Efficiency E
G()
30
10
1,0
1,5
35
0,6
0,4
0,2
1,0
0,01
0,1
1
Correlation time (ms)
10
100
0,0
2
4
6
8
10
12
bp
14
16
18
20
22
24
Determination of E via trans-cis isomerization of
the acceptor dye
- Interference with other relaxation processes
* Photodynamic reaction to excitation monitored on acceptor side
+ Independent read-out:
* donor-fluorescence cross-talk
* background
* labelling efficiencies
* absolute concentrations
* absolute fluorescence and detection Q.Y.
+ Calibration on same sample possible
+ wide range, good precision
*P:
* lower than expected
* non-constant
Widengren, Schweinberger, Berger, and Seidel
J. Phys. Chem. A 105, 6851-6866, 2001
Selective FCS:
frequency
0
2
4
6
5
g [ns]
100
bp=18 bp
bp=15 bp
bp=13 bp
4
0
100
100
10
10
1
1
Mixture: bp:
5; 9; 13; 15 bp
0,1
0
2
4
g [ns]
6
G()
Fg/Fr
3
2
1
Fg/Fr
0
0,1
150
frequency
0
1E-4
1E-3
0,01
0,1
Correlation time (ms)
1
10
Traditional fluorescence
parameters
four dimensions:
- excitation and
fluorescence spectra: E, F
- quantum yield: F
- lifetime: 
- anisotropy: r
Fluctuation
parameters
Acknowledgements:
Dept. Med. Biophysics, MBB, Karolinska
Insitutet, Stockholm:
Ylo Mets, Per Thyberg, Petra Schwille, Aladdin Pramanik,
Rudolf Rigler
MPI f. Biophys. Chem. Göttingen, Germany:
Enno Schweinberger, Christian Eggeling, Jörg Schaffer, Sylvia
Berger, Matthew Antonik, Claus Seidel, Martin Margittai,
Reinhard Jahn
Financial Support:
- Swedish Foundation for Cooperation in Higher Education and Research (STINT)
- BMBF-Biofuture Program
- VW-Stiftung
- The Swedish Research Council (Medicine)
- Magnus Bergwall Foundation
- The Swedish Society of Medicine
- Karolinska Intitutet Research Funds
Prospects for the future:
-Basic research: Reveal structures and dynamics of molecules
beyond ensemble averaging
-Ultrasensitve diagnostics: Detection and identification of sparse
amounts of disease-specific molecules on/inside cells or in
body fluids.
-Ultrasensitive characterization of disease specific molecules or
target molecules for drug therapies
-High-throughput-screening (small sample volumes, low
concentrations, fast read-out)
The Experimental Biomolecular Physics group
Senior researchers / post docs:
Anders Hedqvist
Per Thyberg
vacant
PhD students:
Per-Åke Löfdahl
vacant
vacant