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

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)

Jablonski Scheme of Fluorescence

S

S

T

0

1

1

Photobleaching

FluorescenceEmission

Excitation

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

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

Fluorescence Correlation Spectroscopy

Confocal Fluorescence Microscopy

Principle of Confocal Detection

Objective

Dichroic mirror

Tube lens

Confocal aperture

Towards detector

Fluorescence Intensity Fluctuations

Fluorescence Intensity Fluctuations:Autocorrelation

Fluorescence Intensity Fluctuations:Autocorrelation

Fluorescence Intensity Fluctuations:Autocorrelation

Structure of an autocorrelation curve

Example: Measured FCS curves of yellow fluorescent protein

Amplitude of an autocorrelation curve

Normalized amplitude of an autocorrelation curve

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

Cover-slide thickness deviation

Refractive index mismatch

Optical saturation

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

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

Laser beam width and detection volume

2-focus confocal system

www.microscopyu.com

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

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

Absolute FCS: two mutually shifted detection volumes

2fFCS of Atto655 in GdHCl: refractive index dependence

2fFCS: optical saturation dependence

“Hard” application of 2fFCS:Ca2+-binding of Calmodulin

Ca2+-binding of Calmodulin:Hydrodynamic radius

Protein folding/unfolding:Tryptophan cage

Measuring fast conformational fluctuationsof biomolecules

Time scale of interest: nanoseconds up to milliseconds

Reporter: (i) Intensity(ii) Lifetime

Probes: Förster resonance energy transferElectron transfer

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

hν hν

k+

k+

k0 k0

k

k

Conformational dynamics of small peptide

W400exc µ=P

mW4exc =P

ns1201 =+kns2671 =−k

Conformational dynamics of small peptide(binding epitope of p53-antibody)

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

FLCSFluorescence lifetime correlation spectroscopy

FLCSFluorescence lifetime correlation spectroscopy

FLCS: Working principle

FLCSFluorescence lifetime correlation spectroscopy

Bi-exponential lifetime of a Cy5-streptavidin conjugate

FLCS of Cy5-Streptavidin

FLCS of Cy5-Streptavidin

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

Single Molecule Imaging

Fluorescing molecule as an electric dipole

Positive charge

Negative charge

OrientationAmplitude

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.

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.

Tunneling of evanescent modes into optically denser medium: Vertical dipole case

upper mediumn1 = 1.33

lower mediumn2 = 1.33

Tunneling of evanescent modes into optically denser medium: Vertical dipole case

Emission into glass from a fluorescent molecule crossing a water/glass interface

Lifetime of fluorescent molecule crossing a water/glass interface

Collection efficiency of oil immersion microscope objective

Angular distribution of single moleculeson glass surface

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

Theoretically calculated patterns

Defocused imaging of single molecules:pattern matching

Emission dipole hopping in a perylene tetrachromophore

Emission dipole hopping in a perylene tetrachromophore

Rotational diffusion of molecules

Rotational diffusion of molecules

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⊥∆ = −

Rotational diffusion of molecules:Correlation analysis

||D D⊥ <<

Motor proteins: myosin V along actin

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

Myosin motion and reorientation

Myosin motion and reorientation

Myosin motion and reorientationN = 97 molecules1151 tilting events

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.

Superresolution microscopy: Overcoming Abbe's resolution limit

Fluorophore distribution(bar = 1µm)

Confocal Laser Scanning Microscope(CLSM)

(A tribute to microscopy pioneer Antoni van Leeuwenhoek)

Spatial resolution limit of standard light microscopy

position [µm]

inte

nsity

Lateral resolution limit of standard light microscopy: Abbe's equation

objective

N.A. = n sinθλ2n sinθ.

θ

.

Laser Scanning Confocal Microscopy (LSCM)

laser beam objective PSF

LSCM with deconvolution is completelyequivalent in resolution power

and photon usagewith structured illumination microscopy

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

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

Back to basics: Physics of fluorescence

S

S

T

0

1

1

Photobleaching

FluorescenceEmission

Excitation

Ground state depletion microscopy:Using saturation of the excited state

Stimulated Emission

S

S

0

1

FluorescenceEmission

Excitation

STE

Stimulated Emission Depletion Microscopy

excitationlaser

PSF

STEDlaser

Stimulated Emission Depletion Microscopy

Stimulated Emission Depletion Microscopy

Temporal behavior of ground state depletion after sudden switch-on of excitation

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

Potential realization of Dynamic Saturation Optical Microscopy:

Potential realization of Dynamic Saturation Optical Microscopy:

Dynamic Saturation Optical Microscopy:Point spread function

Theoretical estimate of DSOM performance

DSOMDSOM

+Besselbeam

Fluorophore distribution(bar = 1µm)

Confocal Laser Scanning Microscope(CLSM)

Complex photophysics of Alexa647

Alexa 647

Combining DSOM and FCS

Alexa 647

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

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

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

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

Publications available at

www.joerg-enderlein.de

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)