single molecule spectroscopy and imaging...single molecule spectroscopy and imaging ingo gregor,...
TRANSCRIPT
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Single Molecule Spectroscopy and Imaging
Ingo Gregor, Thomas Dertinger, Iris von der Hocht, Jan Sykora, Luru Dai, Jörg Enderlein
Institute for Biological Information Processing 1Forschungszentrum Jülich
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Motivation
Cellular and molecular biology studies(cell signaling, membrane dynamics)
Distribution functions of molecular parameters (photo-physics, enzymatic
activity, binding affinity)
Ultra-sensitive chemical analysis (drug screening, medical diagnostics)
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Jablonski Scheme of Fluorescence
S
S
T
0
1
1
Photobleaching
FluorescenceEmission
Excitation
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Main challenge of single molecule detection: Raman and Rayleigh scattering
High-efficientoptical filters
Minimizing de-tection volume
Background ~ V
Long wave-length dyes
Background ~ λ-4
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Tryptophan
Thyrosin
Collagen
Elastin
Flavins
Furan Coumarine Fluorescein Rhodamine Oxazine Cyanine
Courtesy: Christoph Zander. 1999 Uni GH Siegen
300 400 500 600 700
Coproporphyrine / Protoporphyrine
Chlorophyll
ηfl
Wavelength (nm)
Absoprtion Spectra of Standard Dyes and Autofluorescent Biomolecules
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Fluorescence Correlation Spectroscopy
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Confocal Fluorescence Microscopy
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Principle of Confocal Detection
Objective
Dichroic mirror
Tube lens
Confocal aperture
Towards detector
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Fluorescence Intensity Fluctuations
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Fluorescence Intensity Fluctuations:Autocorrelation
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Fluorescence Intensity Fluctuations:Autocorrelation
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Fluorescence Intensity Fluctuations:Autocorrelation
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Structure of an autocorrelation curve
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Example: Measured FCS curves of yellow fluorescent protein
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Amplitude of an autocorrelation curve
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Normalized amplitude of an autocorrelation curve
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Ideal molecule detection function
Molecule detectionfunction (1/e2 isosurface)
NA = 1.2
wd = 3 mmtubelens = 180 mm
n0 = 1.33
λex
= 635 nmω = 4.9 mm
focus pos. = 10 µmλ
em = 670 nm
magn. = 60pinhole radius = 50 µm
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Cover-slide thickness deviation
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Refractive index mismatch
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Optical saturation
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Intensity dependence of FCS (Alexa633)
10-5 10-4 10-3 10-2 10-10
0.2
0.4
0.6
0.8
1
time [s]
auto
corr
elat
ion
[a.u
.]30 µW100 µW300 µW
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Pulsed versus cw-excitation (Alexa633)
0 200 400 600 800 10000.8
1
1.2
1.4
1.6
1.8
2
2.2
2.4 x 10-6
cw excitation power [µW]
appa
rent
diff
usio
n [c
m2 /s
]pulsed excitation @ 635 nmcw excitation @ 647 nm
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Laser beam width and detection volume
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2-focus confocal system
www.microscopyu.com
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Time-tagged time-resolved mode of photon counting
-5 0 5 10 15
Laser pulse
Freq
uenc
y
Decay time (ns)
Fluorescence decay curve
τ τ τ τ τ τ τ τ1 2 3 4 5 6 7 8
Data: t1 t t t t t t t2 3 4 5 6 7 8
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PIE: Pulsed interleaved excitation
0 5 10 15 20 25Time [ns]
Phot
on c
ount
s [a.
u.]
1 3 5 7 ....
2 4 6 8 ....
A
B
A B
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Absolute FCS: two mutually shifted detection volumes
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2fFCS of Atto655 in GdHCl: refractive index dependence
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2fFCS: optical saturation dependence
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“Hard” application of 2fFCS:Ca2+-binding of Calmodulin
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Ca2+-binding of Calmodulin:Hydrodynamic radius
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Protein folding/unfolding:Tryptophan cage
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Measuring fast conformational fluctuationsof biomolecules
Time scale of interest: nanoseconds up to milliseconds
Reporter: (i) Intensity(ii) Lifetime
Probes: Förster resonance energy transferElectron transfer
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Tryptophan induced fluorescence quenching of dye Atto655
N O
N
N
OHO
0 10 20 30 40 50 60
20
15
10
5
0
2.0
1.5
1.0
Trp [mM]
I /I/0
0τ τ
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hν hν
k+
k+
k0 k0
k
k
Conformational dynamics of small peptide
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W400exc µ=P
mW4exc =P
ns1201 =+kns2671 =−k
Conformational dynamics of small peptide(binding epitope of p53-antibody)
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Time-tagged time-resolved mode of photon counting
-5 0 5 10 15
Laser pulse
Freq
uenc
y
Decay time (ns)
Fluorescence decay curve
τ τ τ τ τ τ τ τ1 2 3 4 5 6 7 8
Data: t1 t t t t t t t2 3 4 5 6 7 8
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FLCSFluorescence lifetime correlation spectroscopy
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FLCSFluorescence lifetime correlation spectroscopy
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FLCS: Working principle
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FLCSFluorescence lifetime correlation spectroscopy
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Bi-exponential lifetime of a Cy5-streptavidin conjugate
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FLCS of Cy5-Streptavidin
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FLCS of Cy5-Streptavidin
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τ = 1.7 ns τ = 0.7 ns
A > 90 % A < 10 %
dark state dark state
1.2 sµ
0.91 sµ
1.2 sµ
0.91 sµ
0.23 sµ 3.5 sµ 0.23 sµ 3.5 sµ
FLCS of Cy5-Streptavidin
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Single Molecule Imaging
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Fluorescing molecule as an electric dipole
Positive charge
Negative charge
OrientationAmplitude
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The electric dipole:Near field, far field, and virtual photons
Oscillating dipole is surroun-ded by virtual photons that are damped with increasing distance from the dipole. During return to the ground state, a propagating photons is emitted carrying away the excited state energy.
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Angular distribution of emission
Angular distributionof emitted radiation
is given by the classical sin2θ law.
In the quantum mechanical picture, the classical angular distribution of radiation corresponds to a probability of
emitting a photon into a given direction.
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Tunneling of evanescent modes into optically denser medium: Vertical dipole case
upper mediumn1 = 1.33
lower mediumn2 = 1.33
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Tunneling of evanescent modes into optically denser medium: Vertical dipole case
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Emission into glass from a fluorescent molecule crossing a water/glass interface
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Lifetime of fluorescent molecule crossing a water/glass interface
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Collection efficiency of oil immersion microscope objective
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Angular distribution of single moleculeson glass surface
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Defocused imaging of single molecules
Dichroic Mirror
CCD
Microscope Table
PiFoc
KrAr450-700 nm
Oil Immersion1.4 NA, 100 x
Tube Lens
EmissionFilter Excitation/
PolarizationFilter
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Theoretically calculated patterns
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Defocused imaging of single molecules:pattern matching
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Emission dipole hopping in a perylene tetrachromophore
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Emission dipole hopping in a perylene tetrachromophore
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Rotational diffusion of molecules
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Rotational diffusion of molecules
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Symmetric top Brownian rotator
( ) ( )( )
2 2
0 0 0
2
cos cos cos sin sin cos
sin , , , cos cos sin sin cos
t t
D t
d d d G t
e ⊥
π π π
− +∆
Θ = φ ψ − φ ψ θ
= θ φ ψ θ φ θ ψ φ ψ − φ ψ θ
=
∫ ∫ ∫
( )6 462 1 1 1cos3 6 2
D tD t
te e ⊥⊥ − + ∆−Θ = + +
( ) ( ) ( )2 12 12 93 3 3 1cos5 20 4
D t D t D t
te e e⊥ ⊥ ⊥− +∆ − +∆ − + ∆Θ = + +
( ) ( ) ( )6 4 20 4 20 166 204 1 1 9 3 1 1cos5 7 280 7 14 8
D t D t D tD t D t
te e e e e⊥ ⊥ ⊥⊥ ⊥ − + ∆ − + ∆ − + ∆− −Θ = + + + + +
||D D⊥∆ = −
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Rotational diffusion of molecules:Correlation analysis
||D D⊥ <<
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Motor proteins: myosin V along actin
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Myosin V moving along actin filament
1.45 oil immersion objective
160 x magnification
10 ms exposure time / frame
defocusing 500 nm
Measurement by Erdal Toprak, UIUC
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Myosin motion and reorientation
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Myosin motion and reorientation
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Myosin motion and reorientationN = 97 molecules1151 tilting events
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Myosin motion and reorientation
We observe that there is a consistent fluctuation of β between two well defined angles as myosin V steps.
This is consistent with the lever arm hypothesis. Unlike β, the change in α shows no consistent or recognizable patternwhich is an evidence for diffusional binding of myosin V.
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Superresolution microscopy: Overcoming Abbe's resolution limit
Fluorophore distribution(bar = 1µm)
Confocal Laser Scanning Microscope(CLSM)
(A tribute to microscopy pioneer Antoni van Leeuwenhoek)
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Spatial resolution limit of standard light microscopy
position [µm]
inte
nsity
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Lateral resolution limit of standard light microscopy: Abbe's equation
objective
N.A. = n sinθλ2n sinθ.
θ
.
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Laser Scanning Confocal Microscopy (LSCM)
laser beam objective PSF
LSCM with deconvolution is completelyequivalent in resolution power
and photon usagewith structured illumination microscopy
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Axial resolution limit of standard light microscopy
objective
θ0nk =λ
, cosznk θ = θλ
( ),02
0 ,2 2coszik zik zze e k k zθ
θ + = + −
( )1 cosnλ
− θ
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4π microscopy
laser beam 1st objective
PSF
standing wave generation by counter-propagatingfocusing of two coherent laser beams
laser beam2nd objective
( )41 cos 2n nπ
λ λ=− θ
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Back to basics: Physics of fluorescence
S
S
T
0
1
1
Photobleaching
FluorescenceEmission
Excitation
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Ground state depletion microscopy:Using saturation of the excited state
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Stimulated Emission
S
S
0
1
FluorescenceEmission
Excitation
STE
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Stimulated Emission Depletion Microscopy
excitationlaser
PSF
STEDlaser
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Stimulated Emission Depletion Microscopy
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Stimulated Emission Depletion Microscopy
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Temporal behavior of ground state depletion after sudden switch-on of excitation
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Converting temporal into spatial information:Dynamic Saturation Optical Microscopy
0 0.1 0.2 0.3 0.4 0.50
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
10.0 sµ
0.1 sµ
0.2 sµ
3.2 sµ6.4 sµ
1.6 sµ
0.8 sµ
0.4 sµ
rel.
ampl
itude
x [ m]µ
0 2 4 6 8 100
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
time [ s]µ
rel.
ampl
itude
40 n
m
0 nm
80 n
m
160
nm
320
nm
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Potential realization of Dynamic Saturation Optical Microscopy:
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Potential realization of Dynamic Saturation Optical Microscopy:
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Dynamic Saturation Optical Microscopy:Point spread function
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Theoretical estimate of DSOM performance
DSOMDSOM
+Besselbeam
Fluorophore distribution(bar = 1µm)
Confocal Laser Scanning Microscope(CLSM)
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Complex photophysics of Alexa647
Alexa 647
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Combining DSOM and FCS
Alexa 647
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Ground state depletion into triplet state
( ) ( )( )
( ) ( ){ }, expph iscph isc
ph isc ph isc
k k fs t k k f t
k k f k k fτ
= + − + τ + τ + τr
r rr r
( ) ( )( )1
af
a=
+ τr
rr
S
S
T
0
1
1Fluorescence
Emission
Excitation
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Ground state depletion into metastable state(switchable chromophores)
( ) ( ){ }, exp transs t k f t= −τr r ( ) ( )( )1
af
a=
+ τr
rr
S
S
M
0
1
FluorescenceEmission
Excitation
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Ground state depletion into first excited state
( ) ( )( ) ( ){ }{ }1
1, 1 expa
s t a ta
−−
= − − τ + τ +r
r rr
S
S
0
1
FluorescenceEmission
Excitation
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Summary of DSOM
Relatively simple: one laser only
employing a standard CLSM
pure electronic data evaluation
relatively robust against aberration
can be combined with 4π or other techniques
Drawback: resolution enhancement limited to ca. 5 times
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Publications available at
www.joerg-enderlein.de
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Acknowledgements/CooperationsIngo Gregor
Digambara PatraJan SykoraLuru Dai
Thomas DertingerIris von der Hocht
Jörg FitterThomas GenschBenjamin Kaupp
(FZ Jülich)
Markus Sauer(Univ. Bielefeld)
Hiroshi Uji-i, Johan Hofkens(Katholieke Universiteit Leuven)
Erdal Toprak, Paul Selvin(Univ. Illinois
Urbana-Champaign)