Fluorescence Lifetime Correlation Spectroscopy … Correlation Spectroscopy (FCS) in Chemistry and Biology Exact determination of concentrations Molecular diagnostics Monitoring reaction

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© PicoQuant GmbH, 2014

Fluorescence Lifetime Correlation Spectroscopy (FLCS) - A Powerful Tool for Measuring

Diffusion, Concentrations and Interactions

Webtalk February 2014

Volker Buschmann, Peter Kapusta, Felix Koberling, Marcelle König, Benedikt Krämer, Steffen Rüttinger, Sebastian Tannert, Manoel Veiga, Rainer Erdmann, Uwe Ortmann

© PicoQuant GmbH Webtalk FLCS 2

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Fluorescence Correlation Spectroscopy (FCS) in Chemistry and Biology

Exact determination of concentrations● Molecular diagnostics ● Monitoring reaction kinetics

Diffusion behavior ● Folding / unfolding of proteins● Lipid dynamics in model and cell

membranes● Hydrodynamic radius of complexes like

molecules with their solvation shell

Molecular interactions● Protein-protein interactions● Association / dissociation processes● Surface adsorption / desorption ● Complex formation, stoichiometry ● Formation of micelles (size, heterogeneity)● Polymerization through monitoring viscosity

Molecular dynamics● Conformational dynamics● Binding kinetics● Protonation dynamics / equilibrium● FRET

Study of dynamic chemical or biomolecular processes causing fluctuations in the fluorescence intensity:


Principle of Fluorescence Correlation Spectroscopy

Diffusion of a single molecule through the laser focus

0 2 4 60

10

20

30

40

Co

un

t ra

te [

kcp

s]

Time [s]

Intensity bursts

Curve fitting:number of molecules in the focal volume → (+ focus size) → molecule concentrationdiffusion time through focus → (+ focus size) → diffusion coefficient

10-3 10-2 10-1 100 101 1020,0

0,2

0,4

0,6

Tacc.

=60 s

No

rm.

Au

toco

rre

latio

n

Time [ms]

G (τ)=⟨δF (t)⋅δ F (t+ τ)⟩

⟨F (t)⟩2=

1V eff ⟨C ⟩

⋅1

1+ ττD

⋅1

√1+κ2⋅ ττD

G(τ) = Autocorrelation functionτ = Correlation timeF(t) = Fluorescence at time tVeff = Effective volume

⟨C⟩ = Concentrationτ D = Diffusion timeκ² = z/xy – Focus extention

G(τ) = Autocorrelation functionτ = Correlation timeF(t) = Fluorescence at time tVeff = Effective volume

⟨C⟩ = Concentrationτ D = Diffusion timeκ² = z/xy – Focus extention


Limitations of FCS with cw Excitation

Dual colour FCCS: spectral crosstalk➔adds a wrong

cross correlation

SPAD afterpulsing adds a fluctuating component in the µs regime

Autofluorescence + background in the same spectral region➔change the extracted amplitude

= concentration

FCCS with dual colour excitation:➔different excitation volumes

and perhaps positions➔difficult to be calibrated and to be

described with a fit function


Time-Tagged Time-Resolved (TTTR) Single Photon DetectionAllows for FLCS

recorded TTTR data streamTTTR data file

t

T

CH

T

M

t

T

CH

start

laser pulse laser pulse laser pulse

photon photon

photon time 1 photon time 2

start-stop-time 1

start-stop-time 2

synchronization marker

5 ns

250 ms

detector 2

7 ns

381 ms

detector 3

310 ms

start line

M

marker time 1

Time

Cor

rela

tion

Time

Cou

nts

Photonfiltering

Photoncorrelation

Lifetime

Scanner integration


FLCS Principle

photon filtering

photonauto- correlation

raw data (single photon records)

photon arrival time distribution= fluorescence decay of Ref A

fluorescence decay of Ref B

t

T

t

t

T

T

Sub-ensemble FCS curves

photonauto- correlation

photon subsets


Principles of FLCS

Separated FCS curves for different species● Removal of background ● Removal of afterpulsing● More accurate results for concentrations and

diffusion constants● Separation of different fluorescent species

0 100 200 300 4000

3000

6000

9000

12000

Re

lativ

e p

ho

ton

de

tect

ion

pro

ba

bili

ty

Delay time [channel number, i ]

Species A

Mixture of species A and B

Species B

Photons most likely emitted by A

Photons most likely emitted by B

Fluorescence lifetime histograms Filter functions (“weights”)Separated FCS curves

for species A and B

0 100 200 300 400

-1

0

1

2

Filt

er

valu

e [

we

igh

t]

Delay time [channel number, i ]10-3 10-2 10-1 100 101

0

1

2

3

4

Co

rre

latio

n a

mp

litu

de

Lag time [ms]


(1) Separation of Different Fluorescent Species with FLCS:Unmixing of 4 Components

Equimolar mixture of Cy5 and ATTO 655:

Graphs courtesy of Steffen Ruettinger, former member of PTB, Berlin, Germany

1k

2k

1

2

3

1

2

3

10-3 10-2 10-1 100 101

1

2

3

Co

rre

latio

n

Lag time [ms]

-5

0

5

-5

0

5

-5

0

5

0 5 10 15 20

-5

0

5

We

igh

ting

fa

cto

rs

Time [ns]

102

104

106

102

104

106

102

104

106

0 5 10 15 20

102

104

106

Co

un

ts

Time [ns]

Separated FCS curves Filter functions

(“weighting scheme”) Fluorescence decay pattern

Background, afterpulsing

Scattering0.4 ns FWHM

ATTO 6551.8 ns

Cy50.9 ns

Background, afterpulsing

Scattering

ATTO 655

Cy5


10-4 10-3 10-2 10-1 100 1010,0

0,5

1,0

1,5

2,0

2,5

10-4 10-3 10-2 10-1 100 1010,0

0,5

1,0

1,5

2,0

2,5

10-4 10-3 10-2 10-1 100 1010,0

0,5

1,0

1,5

2,0

2,5

G(τ

)

τ [ms]

++ not influenced by detector

afterpulsing++ only 1 detector necessary- - afterpulsing

(2) FLCS to Eliminate Detector Afterpulsing

++ only 1 detector necessary++ not influenced by detector

afterpulsing

high fluorophore concentration (ATTO 655 in water c = 1 nM) low background contribution

Crosscorrelation Autocorrelation FLCS

0,0 0,5 1,00

50

100

Co

un

tra

te [

kcp

s]

Time [s]Time [ns]

SPAD afterpulsing


10-4 10-3 10-2 10-1 100 1010

5

10

15

20

25

10-4 10-3 10-2 10-1 100 1010

5

10

15

20

25

10-4 10-3 10-2 10-1 100 1010

5

10

15

20

25

G(τ

)

τ [ms]

(3) FLCS to Extract Accurate Concentrations

++ <N> background corrected→ accurate concentration

Crosscorrelation Autocorrelation FLCS

0,0 0,5 1,00

50

100

Co

un

tra

te [

kcp

s]

Time [s]

low fluorophore concentration (ATTO 655 in water c = 0.05 nM) strong background contribution

Scattered excitationlight

Time [ns]


Cytoplasm Nucleus0

2

4

6

8

10

FCS FLCS FCS FLCS

N (

Mo

lecu

les

in F

ocu

s)

● Concentration of GFP-Ago2 can be determined in different cellular compartments.

● FLCS removes background contributions. → more accurate concentration determinations

++

1µm

ER293-cells with constant expression of GFP-Ago2

Excitation: 470 nm, 32 MHzEmission: 520/40 band-pass filterObjective: C-Apochromat 40x 1.2 W10 curves with 30 s acquisition time are averagedLSM Upgrade Kit

0,01 0,1 1 10 1000,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

av_curve fit

G(τ

)

τ in ms

Cytoplasm without FLCS with FLCSNucleus without FLCS with FLCS

Cytoplasm without FLCS with FLCSNucleus without FLCS with FLCS

FCS FLCS FCS FLCS

FCS FLCS FCS FLCS

Courtesy of M. Gärtner, J. Mütze, P. Schwille, TU Dresden, Germanysee also: T. Ohrt, J. Mütze et al., Nucl. Acids Res. 2008

Concentration Measurements in vivo: GFP-tagged Proteins in Living Cells (Example)


10-3 10-2 10-1 100 101 1020.0

0.1

0.2

0.3

G(τ

)

Time [ms]

Studying Molecular Interactions with Fluorescence Cross Correlation Spectroscopy (FCCS)

diffusion of molecules with 2 fluorescent labels through the laser focus

Autocorrelations green channel / red channel → molecule concentration green / red molecules

Crosscorrelation→ concentration of double labeled species

⟨C gr⟩=G x(0)

Gg (0)⋅G r(0)⋅V eff

Cgr = concentration doublelabeled species

Gg(0) = Amplitude autocorrelationgreen channel

Gr(0) = Amplitude autocorrelationred channel

Gx(0) = Amplitude Crosscorrelation

Veff = effective volume

Cgr = concentration doublelabeled species

Gg(0) = Amplitude autocorrelationgreen channel

Gr(0) = Amplitude autocorrelationred channel

Gx(0) = Amplitude Crosscorrelation

Veff = effective volume

0.0 0.5 1.0 1.5 2.00

10

20

30

40

Co

un

t ra

te [

kcp

s]

Time [s]

Intensity bursts in two spectral channels

Autocorrelation green channelAutocorrelation red channelCrosscorrelation green/red channel



Limitations of Dual Color FCCS: Spectral Crosstalk and Excitation Volumes

● Focus size depends on the excitation wavelength.

● Additionally, imperfections of the optics & alignment can cause a non – perfect overlay of the different colors.

→ The overlapping (effective) volume needs to be calibrated.

→ Necessary is a positive control.

● Emission Spectra are usually broader than the spectral window of the detection channels.

● Especially the green dye's emission spectrum reaching into the red dye's detection window results in spectral bleedthrough.

→ Spectral bleedthrough causes a false positive crosscorrelation.

→ Necessary is a negative control.

greenchannel

red channel


Calibration Measurements using Dual Color FCCS (without Lifetime Analysis)

Sample courtesy of IBA, see:http://www.iba-lifesciences.com/FCCS_Standards_Products.html

Sample: In vitro fluorescence crosscorrelation standard; (FCCS standard) 488-543 nm from IBAData acquired with: LSM Upgrade Kitλ

Exc 488-dye: 485 nm, 20 MHz, λ

Exc 547-dye: 559 nm, cw

UPLAPO 60x, NA 1.2Emission bandpass: Det. 2: HQ520/40, Det. 1: HQ620/60DM488/559/635 + 570 nm dichroic

488 +488

547

independently moving molecules

double labeled molecules

547

False positive crosscorrelation due to spectral bleedthrough






Sample: In vitro fluorescence crosscorrelation standard; (FCCS standard) 488-543 nm from IBA Data acquired with: LSM Upgrade Kitλ



UPLAPO 60x, NA 1.2Emission bandpass: Det. 2: HQ520/40, Det. 1: HQ620/60DM488/559/635 + 570 nm dichroic

● Idea: Excite the samples with a pulsed 485 nm and a cw 559 nm laser.● The fluorescence decay pattern in respect to the laser pulse is very distinct in both

channels:

Green channel Red channel

Fluorescence green dye

Background

Bleedthroughgreen dye

Fluorescence red dye


FLCCS: Fluorescence Decay based Crosstalk Identification


From decay pattern

calculate

time – dependentweighting factors

(FLCS filter functions)for each spectral channel

Sample courtesy of IBA, see: http://www.iba-lifesciences.com/FCCS_Standards_Products.html

Green channel

Fluorescence green dye

Background

Red channel

Bleedthroughgreen dye

Fluorescence red dye+

Background

FLCCS: Fluorescence Decay based Crosstalk Removal



Sample: In vitro fluorescence crosscorrelation standard; (FCCS standard) 488-543 nm from IBA Data acquired with: LSM Upgrade Kitλ



FCCS

FLCCS

FLCCS almost completely removes false positive cross correlation caused by spectral bleedthrough

FLCCS: Results for False Positive Crosstalk Removal

488 +

independently moving molecules

488

547double labeled molecules

547

FCCS

FLCCS


FLCCS Principle

photon arrival time distribution= fluorescence decay of Ref A

fluorescence decay of Ref B

t

T

t

t

T

photon subsets

ACFACF

ACF

photon cross-

correlation

photon filtering

raw data (single photon records)


0,01 0,1 1 10 1000,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

G(τ

)

τ in ms

Sample: double stranded DNA oligonucleotidelabeled with Alexa 633 and Alexa 647Acquisition time: 10 minExcitation: 635 nm, 20 MHz, 3.21 µWEmission: HQ685/70 band-pass filterObjective: C-Apochromat 40x 1.2 WLSM Upgrade Kit

Diffusion times:● dsDNA + Alexa 633: (222.4 ± 6.3) µs● dsDNA + Alexa 647: (215.1 ± 7.8) µs● dsDNA + Alexa 633 + Alexa 647: (244.4 ± 7.8) µs

Crosscorrelation: (69.8 ± 2.0) %

single color excitationsingle channel detection

Alexa 647 τL1 = (1.07 ± 0.01) ns

Emission max: 668 nm

Alexa 633 τL2 = (3.13 ± 0.03) ns

Emission max: 647 nm

Courtesy of M. Gärtner, P. Schwille, TU Dresden, Germany

— Alexa 647— Alexa 633— Cross-Correlation

Alexa 647 Alexa 633 Cross correlation

Alexa 647 Alexa 633 Cross correlation

Example for FLCCS: Cross Correlation between Two Spectrally Inseparable Dyes


Summary: FLCS Applications

Remove SPAD afterpulsing➔ In a single channel setup!

Remove scattered excitation light and parasitic fluorescence➔ quantitative results

Separate pulsed and cw excitation

Separate species withdifferent lifetimes ➔ but overlapping fluorescence spectra

X

Decay pattern FCS curve

Time [ns] Time [ms]


Further Information

See specifications on our website:http://www.picoquant.com/applications/category/life-science/fluorescence-lifetime-correlation-spectroscopy-flcsand the application notehttp://www.picoquant.com/images/uploads/page/files/7272/appnote_flcs.pdf

Check our website for training courses on FLIM, FCS and Time-Correlated Single Photon Counting: http://www.picoquant.com/events/workshops-and-courses

Share your experiences with the scientific community in the PicoQuant forum at:http://forum.picoquant.com/

Please contact PicoQuant at [email protected] for further information on:➔ Applications➔ Possible configurations➔ Prices

http://www.picoquant.com/applications/category/life-science/fluorescence-lifetime-correlation-spectroscopy-flcs

http://www.picoquant.com/applications/category/life-science/fluorescence-lifetime-correlation-spectroscopy-flcs

http://www.picoquant.com/images/uploads/page/files/7272/appnote_flcs.pdf

http://www.picoquant.com/events/workshops-and-courses

http://forum.picoquant.com/

mailto:[email protected]

Documents

Fluorescence Lifetime Correlation Spectroscopy … Correlation Spectroscopy (FCS) in Chemistry and Biology Exact determination of concentrations Molecular diagnostics Monitoring reaction