Nanoscopy with Focused Light

Stefan W. Hell

Max Planck Institute for Biophysical Chemistry

Department of NanoBiophotonics

Göttingen

&

German Cancer Research Center (DKFZ)

Optical Nanoscopy Division

Heidelberg

Nobel Lecture in Chemistry, Stockholms universitet

8. December 2014

200 nm

½ wavelength of light

Light microscopy: most popular microscopy technique in life sciences

Light microscopy

Electron microscopy, etc.

80 %

20 %

Fluorescent labels indicate

biomolecule of interest

Excitation

S1

S0

200 nm

½ wavelength of light

200 nm

Wavelength

Verdet (1869)

Abbe (1873)

Helmholtz (1874)

Rayleigh (1874)

Lens

500 nm

sin2nd

Detector

… because of the diffraction barrier:

200 nm

Lens

… because of the diffraction barrier:

Detector

500 nm

Verdet (1869)

Abbe (1873)

Helmholtz (1874)

Rayleigh (1874)

sin2nd

Photomultiplier

or APD

200 nm

Lens

… because of the diffraction barrier:

Detector

500 nm

Verdet (1869)

Abbe (1873)

Helmholtz (1874)

Rayleigh (1874)

sin2nd

Eye

200 nm

Lens

… because of the diffraction barrier:

Detector

500 nm

Verdet (1869)

Abbe (1873)

Helmholtz (1874)

Rayleigh (1874)

sin2nd

Camera

Jena,Germany

Standard (Confocal)STED

“… the resolution limiting effect of diffraction can be overcome (…) by

fully exploiting the properties of the fluorophores. Combined with modern quantum

optical techniques the scanning (confocal) microscope has the potential of

dramatically improving the resolution in far-field light microscopy.”

SWH, Opt. Commun. 106 (1994)

accepted November 1993

What I believed around 1990:

“… the resolution limiting effect of diffraction can be overcome (…) by

fully exploiting the properties of the fluorophores. Combined with modern quantum

optical techniques the scanning (confocal) microscope has the potential of

dramatically improving the resolution in far-field light microscopy.”

What I believed around 1990:

sin2nd

Lens

Keep molecules in a dark state !

S1

S0

S1

off

on

dark

stimulated emission

sin2nd

Lens

Keep molecules in a dark state !

S1

S0

S1

dark

sin2nd

Lens

Keep molecules in a dark state !

S1

S0

S1

off

on

dark

stimulated emission

stimulated emission

STED microscope:

200 nmx

y

sin n 2

λd

d

S1

S0

tfl~ns

tvib< 1ps

fluorescence

excitation

Hell & Wichmann, Opt. Lett. (1994)

off

on

Flu

or.

ab

ilit

y

0

0.5

1.0

2 4 6Is

(S0)(S1)on off

[GW/cm²]

SampleLens

de-excitation

(OFF)

Detector

Laser

excitation

(ON)

PhaseMod

02p

STED microscope:

200 nmx

y

sin n 2

λd

d

S1

S0

tfl~ns

tvib< 1ps

fluorescence

excitation

Hell & Wichmann, Opt. Lett. (1994)

stimulated emissionF

luo

r. a

bilit

y

0

0.5

1.0

2 4 6Is

(S0)(S1)on offon

off

on off

[GW/cm²]

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

STED microscope:

200 nmx

y

Flu

or.

ab

ilit

y

0

0.5

1.0

2 4 6Is

(S0)(S1)on

off

on off

d

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

[GW/cm²]

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:

200 nmx

y

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]I

Flu

or.

ab

ilit

y

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:

200 nmx

y

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]I

Flu

or.

ab

ilit

y

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:

200 nmx

y

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]I

Flu

or.

ab

ilit

y

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:

200 nmx

y

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]I

Flu

or.

ab

ilit

y

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:

200 nmx

y

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]I

Flu

or.

ab

ilit

y

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:

200 nmx

y

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]I

Flu

or.

ab

ilit

y

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:

200 nmx

y

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]I

Flu

or.

ab

ilit

y

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:

200 nmx

y

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]I

Flu

or.

ab

ilit

y

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

sin n 2

λd

STED microscope:

200 nmx

y

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]I

Flu

or.

ab

ilit

y

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:

200 nmx

y

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]I

Flu

or.

ab

ilit

y

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:

200 nmx

y

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]I

Flu

or.

ab

ilit

y

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:

200 nmx

y

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]I

Flu

or.

ab

ilit

y

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

250nmNuclear pore complex

Protein assemblies in cell

Göttfert, Wurm et al Biophys J (2013)

Standard (Confocal)

250nmNuclear pore complex

STED

Protein assemblies in cell

Göttfert, Wurm et al Biophys J (2013)

1

2

3

45

6

7

8

150nm

1

2

3

45

6

7

8

150nm

STED

Göttfert, Wurm et al Biophys J (2013)

Protein assemblies in cell

250nmNuclear pore complex

Confocal STED

300nm

100nm

Confocal STED

100nm

HIV (Vpr.eGFP) Env (Ab 2G12)

HIV Envelope protein on single virions

Env

immature mature

Viral infection

J Chojnacki,..,SWH, HG Kräusslich, Science (2012)

Insight: Env proteins are assembled in mature HIV

STEDSTED

Standard (Confocal)

snapshot

Westphal, Rizzoli, Lauterbach, Jahn, SWH, Science (2008)

Synaptic vesicles in axon of living hippocampal neuron

Synaptotagmin

immunostained

Scale: 300 nm

Scale: 300 nm

Westphal, Rizzoli, Lauterbach, Jahn, SWH, Science (2008)

Video rate STED

Westphal, Rizzoli, Lauterbach, Jahn, SWH, Science (2008)

Synaptotagmin

immunostained

28 frames/ second

Synaptic vesicles in axon of living hippocampal neuron

1 µm

~20 µm deep

2 µm

23 x 18 x 3 µm, 10µs / px, 800 x 600 x 5 px, interval 5 min

YFP

Cortical neurons expressing

cytoplasmic EYFP

STEDNeurophysiology

in living mouse brain

Berning et al, Science (2012)

The resolution

STED microscope:200 nm

x

y

Flu

ore

sc

en

ce

0

0.5

1.0

2 4 6

(S0)(S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

I

sin 2n

λd

Is

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:200 nm

x

y

Flu

ore

sc

en

ce

0

0.5

1.0

2 4 6

(S0)(S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

sin 2n

λd

Is I

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:200 nm

x

y

Flu

ore

sc

en

ce

0

0.5

1.0

2 4 6

(S0)(S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

sin 2n

λd

Is I

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:200 nm

x

y

Flu

ore

sc

en

ce

0

0.5

1.0

2 4 6

(S0)(S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

sin 2n

λd

Is I

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:200 nm

x

y

Flu

ore

sc

en

ce

0

0.5

1.0

2 4 6Is

(S0)(S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

s1sin 2 IIn

λd

I

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

STED microscope:200 nm

x

y

Flu

ore

sc

en

ce

0

0.5

1.0

2 4 6

(S0)(S1)

[GW/cm²]

s1sin 2 IIn

λd

d

stimulated emission

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

Is I

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

0

STED microscope:200 nm

x

y

Flu

ore

sc

en

ce

0

0.5

1.0

2 4 6

(S0)(S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

s1sin 2 IIn

λd

Is I

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

0

STED microscope:200 nm

x

y

Flu

ore

sc

en

ce

0

0.5

1.0

2 4 6

(S0)(S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

s1sin 2 IIn

λd

Is I

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

0

STED microscope:200 nm

x

y

Flu

ore

sc

en

ce

0

0.5

1.0

2 4 6

(S0)(S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation

S1

S0

tfl~ns

tvib< 1ps

on

off

on off

s1sin 2 IIn

λd

Is I

SampleLens

OFF

Detector

LaserON

PhaseMod

02p

0

200 nmx

y

d

4.2 nm2.4 nm

(1-dimensional)

N

V

NV- in diamond

Color centers

Material sciences, magnetic sensing, quantum information

= 775 nm

0

GSD (metastable dark state)

S1

S0

T1on

off

Hell, Kroug Appl Phys B (1995)

fluoresc.

STED

fluoresc.

S1

S0 off

on

Hell, Wichmann, Opt Lett (1994)

offon

Hell, Nat Biotech (2003)

transcis

RESOLFT

Principle: Discern by ON / OFF states in the sample

t ~ ms

t ~ ns

t ~ ms

s1sin 2 IIn

λd

0

0.5

1.0

2 4 6Is

(S0)offon ~ 1/t

GSD (metastable dark state)

S1

S0

T1on

off

Hell, Kroug Appl Phys B (1995)

fluoresc.

STED

fluoresc.

S1

S0 off

on

Hell, Wichmann, Opt Lett (1994)

offon

Hell, Nat Biotech (2003)

transcis

RESOLFT

Principle: Discern by ON / OFF states in the sample

t ~ ms

t ~ ns

t ~ ms

MW/cm2

kW/cm2

W/cm2

s1sin 2 IIn

λd

0

0.5

1.0

2 4 6Is

(S0)offon ~ 1/t

RESOLFT: many ‚doughnuts‘ (zeros) in parallel

… because of low intensity operation.offon

sin n 2

λ

RESOLFT: many ‚doughnuts‘ (zeros) in parallel

… because of low intensity operation.offon

n pixels

n p

ixe

ls

RESOLFT: many ‚doughnuts‘ (zeros) in parallel

… because of low intensity operation.offon

RESOLFT

recorded with

>100,000‚doughnuts‘

in

2 seconds

Keratin filaments in living kidney epithelial cells

Scale bar: 10 µmChmyrov et al, Nature Meth (2013)

offon

What does it take to get the best resolution ?

Object

20th century:

… separate features by focusing light

Good lenses !

Detector

Object

… separate features by focusing light

Detector

20th century:

Object

S1

S0 off

on S1

S0

T1on

off transcis

offonetc.

Detector

… separate by molecular (on/off) states

Solution:

S1

S0 off

on S1

S0

T1on

off transcis

offonetc.

… separate by molecular (on/off) states

STED, GSD, SSIM, RESOLFT,…

Detector

S1

S0 off

on S1

S0

T1on

off transcis

offonetc.

… separate by molecular (on/off) states

Detector

STED, GSD, SSIM, RESOLFT,…

X,Y,Zcontrolledby incident

light

pattern

S1

S0 off

on S1

S0

T1on

off transcis

offonetc.

… separate by molecular (on/off) states

Detector

STED, GSD, SSIM, RESOLFT,…

X,Y,Zcontrolledby incident

light

pattern

single

molecule !Betzig et al (2006)Rust et al (2006)

Hess et al (2006)

Moerner et al (1989)Orrit et al (1990)

STED, GSD, SSIM, RESOLFT,…

PALM, STORM, PAINT, GSDIM,…

Detector

X,Y,Z

controlledby incident

light

pattern

Detector

Betzig et al (2006)Rust et al (2006)

Hess et al (2006)

Moerner et al (1989)Orrit et al (1990)

STED, GSD, SSIM, RESOLFT,…

PALM, STORM, PAINT, GSDIM,…

single

molecule !

Detector

X,Y,Z

controlledby incident

light

pattern

Detector

? stochastic

Betzig et al (2006)Rust et al (2006)

Hess et al (2006)

Moerner et al (1989)Orrit et al (1990)

STED, GSD, SSIM, RESOLFT,…

PALM, STORM, PAINT, GSDIM,…

single

molecule !

? stochastic

Camera

x,y,z

centroidof emitted

light

pattern

Detector

X,Y,Z

controlledby incident

light

pattern

Betzig et al (2006)Rust et al (2006)

Hess et al (2006)

Moerner et al (1989)Orrit et al (1990)

STED, GSD, SSIM, RESOLFT,…

PALM, STORM, PAINT, GSDIM,…

single

molecule !

Camera

Detector

x,y,z

1~n

X,Y,Z

1~

sII

? stochastic

and… many photons for X,Y,Z precision

single

molecule !

Camera

Detector

offon separates the features

Betzig et al (2006)Rust et al (2006)

Hess et al (2006)

Moerner et al (1989)Orrit et al (1990)

PALM, STORM, PAINT, GSDIM,…

STED, GSD, SSIM, RESOLFT,…

offon

Superresolution

separates features using (at least) 2 molecular states

fluorescent non-fluorescent

offon BA

fluorescent

absorbing

scattering

spin up

…

non-fluorescent

non-absorbing

non-scattering

spin down

…

Superresolution

separates features using (at least) 2 molecular states

offon BA

fluorescent

absorbing

scattering

spin up

…

non-fluorescent

non-absorbing

non-scattering

spin down

…

Superresolution

separates features using (at least) 2 molecular states

Former lab members Current lab members

1993-1996since 1997

since 2003

Collaborators: P. Hänninen, E. Soini, R. Jahn, F. Barrantes, S. Sigrist, H.G. Kräusslich, S. Rizzoli

StandardSTED

ON

OFF

0

… down to molecular scale.