detection, identification and conformational dynamic characterization of single molecules by...

Detection, identification and Detection, identification and conformational dynamic conformational dynamic

characterization of single molecules characterization of single molecules by ultra-sensitive fluorescence by ultra-sensitive fluorescence

spectroscopy techniques.spectroscopy techniques.

Detection, identification and Detection, identification and conformational dynamic conformational dynamic

characterization of single molecules characterization of single molecules by ultra-sensitive fluorescence by ultra-sensitive fluorescence

spectroscopy techniques.spectroscopy techniques.

Jerker WidengrenJerker WidengrenExp. Biomol. PhysicsExp. Biomol. Physics

Dept. PDept. Physicshysics, KTH, KTH

Topics of DiscussionTopics of Discussion

FFluorescence Correlation Spectroscopy (FCS)luorescence Correlation Spectroscopy (FCS)- Concept- Concept- strategies to study molecular kinetics / - strategies to study molecular kinetics /

applicationsapplications

Single-molecule Multi-parameter fluorescence Single-molecule Multi-parameter fluorescence detection (smMFD)detection (smMFD)- Concept- Concept- single-molecule FRET studies- single-molecule FRET studies

Fluorescence Correlation Fluorescence Correlation SpectroscopySpectroscopyFluorescence Correlation Fluorescence Correlation SpectroscopySpectroscopy

Dynamic properties of molecules probed via Dynamic properties of molecules probed via their thermodynamic fluctuationstheir thermodynamic fluctuations

At equilibrium, no perturbationAt equilibrium, no perturbation OOriginal conceptriginal concept

Magde, Elson and Webb, Magde, Elson and Webb, 19721972, , Phys. Rev. Lett. 29, 705Phys. Rev. Lett. 29, 705

Elson and Magde, Elson and Magde, 19741974, , Biopolymers, 13, 1Biopolymers, 13, 1Magde, Elson and Webb, Magde, Elson and Webb, 19741974, , Biopolymers, 13, 29Biopolymers, 13, 29Ehrenberg and Rigler, Ehrenberg and Rigler, 19741974, , Chem. Phys., 4, 390Chem. Phys., 4, 390

FCS set-upFCS set-up

Higher spatial discriminationHigher spatial discrimination Higher spectral discriminationHigher spectral discrimination Enhanced detection efficiencyEnhanced detection efficiency - increased fluor./(mol. x s)- increased fluor./(mol. x s)

- reduced background- reduced background

REF:REF:- Rigler and Widengren, in Rigler and Widengren, in BioscienceBioscience, , Klinge and Owman (Ed.), Klinge and Owman (Ed.), Lund UniversityLund University Press, 180, 1990Press, 180, 1990 - Rigler, Widengren and Mets, in Rigler, Widengren and Mets, in FluorescFluoresc.. SpectroscopySpectroscopy, Wolfbeis (Ed.), , Wolfbeis (Ed.), Berlin:Springer, 13, 1992Berlin:Springer, 13, 1992- Rigler, Mets, Widengren and Kask, Rigler, Mets, Widengren and Kask, Eur. Biophys. J. Eur. Biophys. J. 22, 179, 199322, 179, 1993

t

I(t)



t

I(t)

Fluorescence fluctuations due to translational diffusionFluorescence fluctuations due to translational diffusion

GN D D

D ( ) (/

)(/

) /

1 1

1 4

1

1 41

12

22

1 2

The Autocorrelation function:The Autocorrelation function:

Translational diffusion for a 3D gaussian volume element:

GI t I t

I

I I t I I t

I

I t I t

I( )

( ) ( ) [ ( )][ ( )] ( ) ( )

2 2 21

0.0001 0.001 0.01 0.1 1 100.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

D

1/N

G( )

Correlation time (ms)

The experimental The experimental FCS curveFCS curve for translational diffusion: for translational diffusion:

GN D D

D ( ) (/

)(/

) /

1 1

1 4

1

1 41

12

22

1 2

D D 1

2

4 , where D =

kT

6 R

Change in diffusion propertiesChange in diffusion properties

DF

DB

G tN

Y Y

DF DB

( )/ /

1 1

1 11

Ligand-receptor interactions:Ligand-receptor interactions:A:A: nAChR in solutionnAChR in solution

0,1 1 10

1,0

1,2

1,4

1,6

1,8

2,0

2,2

INCUBATION TIME

0 min

4.5 min

19.5 min

72 hrs

Association of -bungatrotoxin to nAChR

G(

)

correlation time (ms)

10 100

0,0

0,2

0,4

0,6

0,8

1,0 Association kinetics

Free

Bound

Total

Incubation time (min)

1 10 1000,0

0,2

0,4

0,6

0,8

1,0 Dissociation kinetics

total

bound

Incubation time (min)

High sensitivity, ligand-receptor High sensitivity, ligand-receptor interactions at low conc. can be interactions at low conc. can be followedfollowed

low conc. of labelled ligands ---> low conc. of labelled ligands ---> facilitates displacements studiesfacilitates displacements studies

No separation of bound from unboundNo separation of bound from unbound Low quantities of material neededLow quantities of material needed No radioactivity No radioactivity

REF: Rauer, Neumann, Widengren, Rigler 1996, REF: Rauer, Neumann, Widengren, Rigler 1996, Biophys. ChemBiophys. Chem

58, 58, 3-123-12

Change in fluorescence upon chemical reactionChange in fluorescence upon chemical reaction



t

I(t)

0.0001 0.001 0.01 0.1 1 100.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

D

1/N

G(

)

t

I(t)

0.0001 0.001 0.01 0.1 1 100.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

D

1/N

G(

)


Fl X FlXk

k

1

1

t

I(t)

Fl X FlXk

k

1

1

Ion concentration monitoring:Ion concentration monitoring:

Fl H HFlk

k

2

1

1

0.1 1 10 100

1.0

1.2

1.4

1.6

1.8

2.0

2.2

Rh-II in 1 mM EGTA buffers

>100 M

2.4 M

1.1 M

0.64 M

0.41 M

0.18 M

92 nMG(

)

(ms)0.0001 0.001 0.01 0.1 1 10 100 1000

1.0

1.2

1.4

1.6

1.8

2.0

2.2 FITC in 1 mM carbonate bufferspH 7.5

pH 7

pH 6.5

pH 6

pH 5.5

pH 5

G(

)

(ms)

Buffer effectsBuffer effects

Fl H HFl2

k+

k-

B H BH

kass

kdiss

BH Fl B HFl2

k1

k-1

0.01 0.1 1 10

1.00

1.05

1.10

1.15

1.20

1.25

100 M

300 M

1.0 mM

3.0 mM

Buffer dependence (phosphate buffer)

Aut

ocor

rela

tion

Time (ms)

0,0 0,5 1,0 1,5 2,0 2,5 3,00,0

0,1

0,2

0,3

0,4

0,5

0,6

phosphatecitrateHEPESNaCl (/200)

k tot(1

06 s-1)

Concentration (mM)Widengren J, Terry B, Rigler R,Widengren J, Terry B, Rigler R,Chem PhysChem Phys. 249, 259-271, 1999. 249, 259-271, 1999

PhotophysicsPhotophysics

- triplet state transitions - triplet state transitions - electron transfer- electron transfer - trans-cis isomerization- trans-cis isomerization

G GS

SD( ) ( )( )

1

1

1

Triplet state monitoring by FCSTriplet state monitoring by FCS

FFluctuations inluctuations in fluorescence due to singlet-triplet transitionsfluorescence due to singlet-triplet transitions

TS0+S

1TS

0+S

1

k21

k12

=abs

Iexc

k31

k23

S0

S1

T1

)(T

)(1S

)(0

31230

02123k-12k31k21k12

)(

)(1S

)(0

t

t

tS

kk

k

k

tT

t

tS

dt

d

The fluorescence intensity correlation function:The fluorescence intensity correlation function:

GT

TI t I t dt

TI t dt

I I t I t

I

N T D DT T

T

T

eq eq

( )lim

( ) ( )

( )

( ) ( )

( )exp( )

/

1

1

1

11

41

41 1

0

0

2

2

2

12 1

22 1 2

3

1E-4 1E-3 0,01 0,1 1 101,0

1,2

1,4

1,6

1,8

2,0

2,2

Rh6G in Water

Power 2,55 mWPower 350 WPower 48.4 W

G()

(ms)

0,01 0,1 1 100,00,20,40,60,81,01,21,41,61,82,02,2

Rh6G in water

T(s)

Teq

Laser Power (mW)

Tk I

I k k k k

I k k k k

I k

eqexc

exc

Texc

exc

23

23 31 21 31

3

23 31 21 31

21

11

( )

( )

Environmental influence

on the triplet state

Effects of solvents

and quenchers on

the triplet state

1E-4 1E-3 0,01 0,1 1 10

1,0

1,2

1,4

1,6

1,8

2,0

2,2 Rh6G in water

[KI]=5.0 mM[KI]=2.0 mM[KI]=0.2 mM[KI]=0 mM

G()

(ms)

1E-4 1E-3 0,01 0,1 1 10

1,0

1,2

1,4

1,6

1,8

2,0

2,2 Solvent effects of Rh6G

WaterEthanolEthylene glycol

G(

)

(ms)

0.001 0.01 0.1 1 10 100

1.0

1.2

1.4

1.6

1.8

2.0

2.2

argon atm.air atm.oxygen atm.

Rh6g in water at different oxygen conc.

G(

)

(ms)1E-4 1E-3 0,01 0,1 1 10

1,0

1,2

1,4

1,6

1,8

2,0

2,2 FITC in water (pH 9)

Power 568 WPower 95.6 WPower 19.8 W

G()

(ms)

Triplet state

properties of

FITC

Triplet state monitoring: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 REF: - Widengren, Rigler and Mets J. Fluoresc. J. Fluoresc. 4(3), 255-258, 19944(3), 255-258, 1994

- Widengren, Mets and Rigler - Widengren, Mets and Rigler J. Phys. Chem. J. Phys. Chem. 99, 13368-13379, 199599, 13368-13379, 1995

- Mets, Widengren and Rigler - Mets, Widengren and Rigler Phys. Chem. Phys. Chem. 218, 191-198, 1997218, 191-198, 1997

Dual colour FCSDual colour FCS

If D Fl = 0 : 0,

Photon counting histograms (PCH) / Photon counting histograms (PCH) / Fluorescence intensity distribution analysis (FIDA):Fluorescence intensity distribution analysis (FIDA):

Fluorescence brightnessFluorescence brightnessConcentrationConcentration

Photophysical limitations:Photophysical limitations:

- Fluorescence saturation- Fluorescence saturation

- Photodestruction- Photodestruction

Figures of meritFigures of merit::

- number of deteced fluorescence photons

molecule

- number of deteced fluorescence photons

molecule time

Fluorescence saturation:Fluorescence saturation:

PhotobleachingPhotobleaching

0,1 1 10 100

1,0

1,2

1,4

1,6

1,8

2,0

1 kW/cm2

4 kW/cm2

26 kW/cm2

FITC in water, pH 9

G( )

Correlation time (ms)0,001 0,01 0,1 1 10 100

1,0

1,5

2,0

2,5

3,0

3,5

1100 kW/cm2

470 kW/cm2

140 kW/cm2

13 kW/cm2

Rh6G in water

G( )


Widengren J, Rigler R, Widengren J, Rigler R, Bioimaging, Bioimaging, 4, 149-157, 19964, 149-157, 1996Eggeling C, Widengren J, Rigler R, Seidel, C, Eggeling C, Widengren J, Rigler R, Seidel, C, Anal Chem, Anal Chem, 70, 2651-2659, 199870, 2651-2659, 1998

Photobleaching effects in a cell surface Photobleaching effects in a cell surface

Rcell

Pexc

Exposure time: t Excitation power: Pexc

Diffusion coeff: D Radius of cell area: Rcell

0 1 2 3 4 5

0,00,10,20,30,40,50,60,70,80,91,0

0,00,10,20,30,40,50,60,70,80,91,0

D=7x10-10

cm2/s

2.5 W5.0 W10 W25 W50 W100 W250 WN

orm

aliz

ed c

once

ntra

tion

Radial distance (m)

Widengren J submitted to Widengren J submitted to Biophys. J.Biophys. J.

How to maximize fluorescence information from single molecules:

How to maximize fluorescence information from single molecules:

n finfon f

Single-molecule Multi-parameter Fluorescence detection (smMFD)Single-molecule Multi-parameter Fluorescence detection (smMFD)

Cy5

A488

Model systemModel system

500 550 600 650 700

Cy5

exc

itatio

n

A4

88

em

issi

on

Wavelength (nm)

Fluorescence resonance energy transfer

kA10kFRET´

kFRET

kD10DIexc

AD

ER

R RDA

06

06 6

R0=8.7910-5 J() FD n-4 21/6 Å

E(FD, FA, rD, rA, FA, FD)

FRET and Coincidence AnalysisFRET and Coincidence Analysis

E (1F

FG

R

) 1

-1 0 1 2-1

0

1

2

Alexa 488 - Cy5z:/singlem/dec99/12/HD1HD5_DA_g/smd/bi4/HD1HD5_DA_g_vortrag.opj

log

I R [

kH

z]

log IG [kHz]

0

5

10

0.1 1 10 100

IG [kHz]

fre

qu

en

cy

0 100.1

1

10

100

Ex 496 nm

5 bp

IR [kHz]

frequency

-1 0 1 2-1

0

1

2

Alexa 488 - Cy5z:/singlem/dec99/12/HD1HD5_DA_gr/smd/bi4/HD1HD5_DA_gr_vortrag.opj

log

I R [

kH

z]

log IG [kHz]

0

10

20

300.1 1 10 100

IG [kHz]

fre

qu

en

cy

0 20 400.1

1

10

100

Ex 496 nmEx=647 nm

5 bp

IR [kHz]

frequency

-1 0 1 2-1

0

1

2


log

I R [

kH

z]

log IG [kHz]

010203040

0.1 1 10 100

IG [kHz]

fre

qu

en

cy

0 20 400.1

1

10

100

Ex 496 nm

18 bp

IR [kHz]

frequency

-1 0 1 2-1

0

1

2


log

I R [

kH

z]

log IG [kHz]

0102030

0.1 1 10 100

IG [kHz]

fre

qu

en

cy

0 20 400.1

1

10

100

Ex 496 nmEx=647 nm

18 bp

IR [kHz]

frequency

-1 0 1 2-1

0

1

2


log

I R [

kH

z]log IG [kHz]

0

10

20

300.1 1 10 100

IG [kHz]

fre

qu

en

cy

0 20 400.1

1

10

100

Ex 496 nm

10 bp

IR [kHz]

frequency

-1 0 1 2-1

0

1

2


log

I R [

kH

z]

log IG [kHz]

0

10

20

300.1 1 10 100

IG [kHz]

fre

qu

en

cy

0 200.1

1

10

100

Ex 496 nmEx=647 nm

10 bp

IR [kHz]

frequency

E (1F

FG

R

) 1

E 1 DA

D

0

100

0 2 4 6

g [ns]

freq

uen

cy

0 1500,1

1

10

100

Fg/F

r

frequency0 2 4 6

0,1

1

10

100

Mixture: bp

:5; 9; 13; 15 bp

Fg/F

r

g [ns]

E (1F

FG

R

) 1

E 1 DA

D

Conformation-based identificationConformation-based identification

:

Fit to a structural model of DNAFit to a structural model of DNA

2 4 6 8 10 12 14 16 18 20 22 24

0,0

0,2

0,4

0,6

0,8

1,0

E via kISOtot

E via cps/mol fit to model E via DA

Effi

cien

cy E

bp

FRET studies withFRET studies withsmMFDsmMFD::-High sensitivity, precision andHigh sensitivity, precision and accuracyaccuracy

-resolution better than 1 nmresolution better than 1 nm

- identification based on- identification based on conformationaconformational l propertiesproperties (”conformational fingerprints”)(”conformational fingerprints”)

- range: 10-100 range: 10-100 ÅÅngströmngström

- Detection and selective analysis of subpopulationsDetection and selective analysis of subpopulations

em , F, r, F

1 10 100 10000,01

0,1

1

647 nm exc 496 nm exc

k ISO

tot(1

06 s-1

)

Exc Int (kW/cm2)

1E-4 1E-3 0,01 0,1 1 10 1001,0

1,5

2,0

2,5

3,0

3,5

86 kW/cm20.4 kW/cm2

0.8 kW/cm2

1.6 kW/cm2

2.9 kW/cm2

1

3

*

PNG

()


Photodynamics of Cy5Photodynamics of Cy5

N

N P

P

PN

1

3 3

1

11

0

1 1

0

11

k

k

k

k

k k

1

= I = I

k k

k k

P N

T

IS C

N 0 1

N 0 1 P 0 1

P e rp

Pe x c e x c

N p e rp P p e rp

P 0 1 P 1 0

k N 1 0

Trans-cis Trans-cis isomerization isomerization of Cy5of Cy5

Widengren J. & Schwille P. Widengren J. & Schwille P. J. Phys. Chem. J. Phys. Chem. 104(27), 6416-104(27), 6416-6428, 20006428, 2000Widengren J. & Seidel C. Widengren J. & Seidel C. Phys. Chem. Chem. Phys. Phys. Chem. Chem. Phys. 2, 2, 3435-3441, 20003435-3441, 2000

N

-O3S SO3

-

N

ON

O

OO

Cy5-NHS

FRET-mediatedFRET-mediatedexcitation:excitation:

kA10kFRET´

kFRET

kD10DIexc

AD

excBISODISODBISOISOISOtot IPENEkkk )()(

FRET-mediated excitationFRET-mediated excitation of Cy5 of Cy5

0,01 0,1 1 10 1001,0

1,5

2,0

1.0 kW/cm2

1.9 kW/cm2

3.2 kW/cm2

Acceptor 1st bp, Donor 14th bp

G()



0,01 0,1 1 10 1001,0

1,5

2,0

2,5

A-D distance:4 bp13 bp18 bp

Excitation intensity 3.2 kW/cm2

G()


0,01 0,1 1 10 1001,0

1,5

2,0

1.0 kW/cm2

1.9 kW/cm2

3.2 kW/cm2


G()



0,01 0,1 1 10 1001,0

1,5

2,0

2,5



G()


0,01 0,1 1 10 1001,0

1,5

2,0

1.0 kW/cm2

1.9 kW/cm2

3.2 kW/cm2


G()


0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,00

5

10

15

20

25

30

35

40

45

50

bp

=4

bp=11

bp

=13

bp=18

bp

=22

k ISO

tot(

103 s-1

)

Excitation Intensity (kW/cm2)


0,01 0,1 1 10 1001,0

1,5

2,0

2,5



G()


0,01 0,1 1 10 1001,0

1,5

2,0

1.0 kW/cm2

1.9 kW/cm2

3.2 kW/cm2


G()


0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,00

5

10

15

20

25

30

35

40

45

50

bp

=4

bp=11

bp

=13

bp=18

bp

=22

k ISO

tot(

103 s-1

)

Excitation Intensity (kW/cm2)

k FRETk direct

E N E PISOtot

ISOtot

( )( )

( ) ( ) 2

2 4 6 8 10 12 14 16 18 20 22 24

0,0

0,2

0,4

0,6

0,8

1,0

E via kISOtot

E via cps/mol fit to model E via DA

Eff

icie

ncy

E

bp

Determination of E via trans-cis isomerization of Determination of E via trans-cis isomerization of the acceptor dyethe acceptor dye

- Interference with other relaxation processes- Interference with other relaxation processes

* Photodynamic reaction to excitation monitored on acceptor side* Photodynamic reaction to excitation monitored on acceptor side

+ Independent read-out:+ Independent read-out: * donor-fluorescence cross-talk* donor-fluorescence cross-talk * background* background * labelling efficiencies* labelling efficiencies * absolute concentrations* absolute concentrations * absolute fluorescence and detection Q.Y.* absolute fluorescence and detection Q.Y.

+ Calibration on same sample possible+ Calibration on same sample possible

+ wide range, good precision+ wide range, good precision

**P:P: * lower than expected* lower than expected Widengren, Schweinberger, Berger, and SeidelWidengren, Schweinberger, Berger, and Seidel

* non-constant* non-constant J. Phys. Chem. AJ. Phys. Chem. A 105, 6851-6866, 2001 105, 6851-6866, 2001

0

100

0 2 4 6

g [ns]

freq

uen

cy

0 1500,1

1

10

100

Fg/F

r

frequency0 2 4 6

0,1

1

10

100

Mixture: bp

:5; 9; 13; 15 bp

Fg/F

r

g [ns]1E-4 1E-3 0,01 0,1 1 10

0

1

2

3

4

5

bp=18 bpbp=15 bpbp=13 bp

G(

)Correlation time (ms)

Selective FCS:Selective FCS:

Traditional fluorescence Traditional fluorescence parametersparameters

four dimensions:- excitation and fluorescence spectra: E, F

- quantum yield: F

- lifetime: - anisotropy: r

FluctuationFluctuationparametersparameters

Acknowledgements:Acknowledgements:

Dept. Med. Biophysics, MBB, KarolinskaDept. Med. Biophysics, MBB, KarolinskaInsitutet, StockholmInsitutet, Stockholm::Ylo Mets, Per Thyberg, Petra Schwille, Aladdin Pramanik, Rudolf RiglerMPI f. Biophys.MPI f. Biophys. Chem. Göttingen, Germany:Chem. Göttingen, Germany:Enno Schweinberger, Christian Eggeling, Jörg Schaffer, Sylvia Berger, Matthew Antonik, Claus Seidel, Martin Margittai, Reinhard Jahn

Financial Financial Support: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:Prospects for the future:

-Basic research: Reveal structures and dynamics of molecules Basic research: Reveal structures and dynamics of molecules beyond ensemble averagingbeyond ensemble averaging

-Ultrasensitve diagnostics: Detection and identification of sparse Ultrasensitve diagnostics: Detection and identification of sparse amounts of disease-specific molecules on/inside cells or in amounts of disease-specific molecules on/inside cells or in body fluids.body fluids.

-Ultrasensitive characterization of disease specific molecules or Ultrasensitive characterization of disease specific molecules or target molecules for drug therapiestarget molecules for drug therapies

-High-throughput-screening (small sample volumes, low High-throughput-screening (small sample volumes, low concentrations, fast read-out)concentrations, fast read-out)

The Experimental Biomolecular Physics groupThe Experimental Biomolecular Physics group

Senior researchers / post docs:Senior researchers / post docs:

Anders HedqvistAnders HedqvistPer ThybergPer Thybergvacantvacant

PhD students:PhD students:

Per-Åke LöfdahlPer-Åke Löfdahlvacantvacantvacantvacant

detection, identification and conformational dynamic characterization of single molecules by...

Documents

kth slide

solution slide

translational diffusion

triplet state monitoring

chemical reaction slide

triplet state lifetimes

triplet state buildup

fluorescence fluctuations