fluorescence lifetime correlation spectroscopy … correlation spectroscopy (fcs) in chemistry and...
TRANSCRIPT
Copyright of this document belongs to PicoQuant GmbH.No parts of it may be reproduced, translated or transferred to third parties without written permission of PicoQuant GmbH.
© 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
Please note ...
We at PicoQuant always try to present our latest findings to the scientific community as quickly as possible.
The creation of suited presentation material with in-depth information is, however, not always possible in a short time frame.
We have therefore decided that it would be beneficial to the scientific community to make our presentations or parts of presentations, that were given on conferences, available to the public. As a consequence, it might be possible that information is missing to understand all information included in a slide.
Thus, please don't hesitate to contact us in case you have any questions or need more information. We hope for your understanding and looking forward to hearing from you.
Your PicoQuant team
© PicoQuant GmbH Webtalk FLCS 3
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:
© PicoQuant GmbH Webtalk FLCS 4
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
© PicoQuant GmbH Webtalk FLCS 5
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
© PicoQuant GmbH Webtalk FLCS 6
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
© PicoQuant GmbH Webtalk FLCS 7
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
© PicoQuant GmbH Webtalk FLCS 8
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]
© PicoQuant GmbH Webtalk FLCS 9
(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
© PicoQuant GmbH Webtalk FLCS 10
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
© PicoQuant GmbH Webtalk FLCS 11
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]
© PicoQuant GmbH Webtalk FLCS 12
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)
© PicoQuant GmbH Webtalk FLCS 13
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
Autocorrelation green channelAutocorrelation red channelCrosscorrelation green/red channel
© PicoQuant GmbH Webtalk FLCS 14
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
© PicoQuant GmbH Webtalk FLCS 15
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
Autocorrelation green channelAutocorrelation red channelCrosscorrelation green/red channel
Autocorrelation green channelAutocorrelation red channelCrosscorrelation green/red channel
Autocorrelation green channelAutocorrelation red channelCrosscorrelation green/red channel
Autocorrelation green channelAutocorrelation red channelCrosscorrelation green/red channel
© PicoQuant GmbH Webtalk FLCS 16
Sample: In vitro fluorescence crosscorrelation standard; (FCCS standard) 488-543 nm from IBA Data 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
● 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
Sample courtesy of IBA, see:http://www.iba-lifesciences.com/FCCS_Standards_Products.html
FLCCS: Fluorescence Decay based Crosstalk Identification
© PicoQuant GmbH Webtalk FLCS 17
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
© PicoQuant GmbH Webtalk FLCS 18
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 IBA Data acquired with: LSM Upgrade Kitλ
Exc 488-dye: 485 nm, 20 MHz, λ
Exc 547-dye: 559 nm, cw
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
© PicoQuant GmbH Webtalk FLCS 19
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)
© PicoQuant GmbH Webtalk FLCS 20
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
© PicoQuant GmbH Webtalk FLCS 21
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]
© PicoQuant GmbH Webtalk FLCS 22
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