Nanoscopy with Focused Light Stefan W. Hell Max Planck Institute - - PowerPoint PPT Presentation

▶
nanoscopy with focused light
SMART_READER_LITE
LIVE PREVIEW

Nanoscopy with Focused Light Stefan W. Hell Max Planck Institute - - PowerPoint PPT Presentation

Jan 20, 2023 22 likes •736 views

Nanoscopy with Focused Light Stefan W. Hell Max Planck Institute for Biophysical Chemistry Department of NanoBiophotonics Gttingen & German Cancer Research Center (DKFZ) Optical Nanoscopy Division Heidelberg Nobel Lecture in

slide-1
SLIDE 1

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
slide-2
SLIDE 2

200 nm

½ wavelength of light

slide-3
SLIDE 3

Light microscopy: most popular microscopy technique in life sciences

Light microscopy

Electron microscopy, etc.

80 %

20 %

slide-4
SLIDE 4

Fluorescent labels indicate biomolecule of interest

Excitation

S1 S0

slide-5
SLIDE 5

200 nm

½ wavelength of light

slide-6
SLIDE 6

200 nm

Wavelength

Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874)

Lens

500 nm

  sin 2n d 

Detector

… because of the diffraction barrier:

slide-7
SLIDE 7

200 nm

 Lens

… because of the diffraction barrier:

Detector

500 nm

Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874)

  sin 2n d 

Photomultiplier

  • r APD
slide-8
SLIDE 8

200 nm

 Lens

… because of the diffraction barrier:

Detector

500 nm

Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874)

  sin 2n d 

Eye

slide-9
SLIDE 9

200 nm

 Lens

… because of the diffraction barrier:

Detector

500 nm

Verdet (1869) Abbe (1873) Helmholtz (1874) Rayleigh (1874)

  sin 2n d 

Camera

slide-10
SLIDE 10

Jena,

Germany

Standard (Confocal) STED

slide-11
SLIDE 11

“… the resolution limiting effect of diffraction can be overcome (…) by fully exploiting the properties of the fluorophores. Combined with modern quantum

  • ptical 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:

slide-12
SLIDE 12

“… the resolution limiting effect of diffraction can be overcome (…) by fully exploiting the properties of the fluorophores. Combined with modern quantum

  • ptical techniques the scanning (confocal) microscope has the potential of

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

What I believed around 1990:

slide-13
SLIDE 13

  sin 2n d 

Lens

Keep molecules in a dark state !

S1 S0 S1

  • ff
  • n

dark

stimulated emission

slide-14
SLIDE 14

  sin 2n d 

Lens

Keep molecules in a dark state !

S1 S0 S1

dark

slide-15
SLIDE 15

  sin 2n d 

Lens

Keep molecules in a dark state !

S1 S0 S1

  • ff
  • n

dark

stimulated emission

slide-16
SLIDE 16

stimulated emission

STED microscope:

200 nm

x y

 sin n 2 λ  d

d

S1 S0

tfl~ns

tvib< 1ps

fluorescence

excitation

Hell & Wichmann, Opt. Lett. (1994)

  • ff
  • n
  • Fluor. ability

0.5 1.0 2 4 6

Is

(S0) (S1)

  • n
  • ff

[GW/cm²]

Sample Lens

de-excitation

(OFF) Detector Laser

excitation

(ON)

PhaseMod

2p

slide-17
SLIDE 17

STED microscope:

200 nm

x y

 sin n 2 λ  d

d

S1 S0

tfl~ns

tvib< 1ps

fluorescence

excitation

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

  • Fluor. ability

0.5 1.0 2 4 6

Is

(S0) (S1)

  • n
  • ff
  • n
  • ff
  • n
  • ff

[GW/cm²]

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-18
SLIDE 18

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

STED microscope:

200 nm

x y

  • Fluor. ability

0.5 1.0 2 4 6

Is

(S0) (S1)

  • n
  • ff
  • n
  • ff

d

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

[GW/cm²]

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-19
SLIDE 19

STED microscope:

200 nm

x y

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

I

  • Fluor. ability

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-20
SLIDE 20

STED microscope:

200 nm

x y

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

I

  • Fluor. ability

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-21
SLIDE 21

STED microscope:

200 nm

x y

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

I

  • Fluor. ability

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-22
SLIDE 22

STED microscope:

200 nm

x y

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

I

  • Fluor. ability

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-23
SLIDE 23

STED microscope:

200 nm

x y

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

I

  • Fluor. ability

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-24
SLIDE 24

STED microscope:

200 nm

x y

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

I

  • Fluor. ability

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-25
SLIDE 25

STED microscope:

200 nm

x y

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

I

  • Fluor. ability

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-26
SLIDE 26

STED microscope:

200 nm

x y

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

I

  • Fluor. ability

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

Sample Lens OFF Detector Laser ON

PhaseMod

2p

 sin n 2 λ  d

slide-27
SLIDE 27

STED microscope:

200 nm

x y

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

I

  • Fluor. ability

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-28
SLIDE 28

STED microscope:

200 nm

x y

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

I

  • Fluor. ability

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-29
SLIDE 29

STED microscope:

200 nm

x y

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

I

  • Fluor. ability

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-30
SLIDE 30

STED microscope:

200 nm

x y

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

I

  • Fluor. ability

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Hell & Wichmann, Opt. Lett. (1994)

stimulated emission

Sample Lens OFF Detector Laser ON

PhaseMod

2p

slide-31
SLIDE 31

250nm

Nuclear pore complex

Protein assemblies in cell

Göttfert, Wurm et al Biophys J (2013)

Standard (Confocal)

slide-32
SLIDE 32

250nm

Nuclear pore complex

STED

Protein assemblies in cell

Göttfert, Wurm et al Biophys J (2013)

slide-33
SLIDE 33

1 2 3 4 5 6 7 8

150nm

1 2 3 4 5 6 7 8

150nm

STED

Göttfert, Wurm et al Biophys J (2013)

Protein assemblies in cell

250nm

Nuclear pore complex

slide-34
SLIDE 34

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

STED STED

slide-35
SLIDE 35

Standard (Confocal) snapshot

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

Synaptic vesicles in axon of living hippocampal neuron

Synaptotagmin immunostained

Scale: 300 nm

slide-36
SLIDE 36

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

slide-37
SLIDE 37

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

STED

Neurophysiology

in living mouse brain

Berning et al, Science (2012)

slide-38
SLIDE 38

The resolution

slide-39
SLIDE 39

STED microscope:

200 nm x y

Fluorescence

0.5 1.0 2 4 6

(S0) (S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

I

 sin 2n λ d 

Is

Sample Lens

OFF

Detector Laser

ON

PhaseMod

2p

slide-40
SLIDE 40

STED microscope:

200 nm x y

Fluorescence

0.5 1.0 2 4 6

(S0) (S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

 sin 2n λ d 

Is I

Sample Lens

OFF

Detector Laser

ON

PhaseMod

2p

slide-41
SLIDE 41

STED microscope:

200 nm x y

Fluorescence

0.5 1.0 2 4 6

(S0) (S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

 sin 2n λ d 

Is I

Sample Lens

OFF

Detector Laser

ON

PhaseMod

2p

slide-42
SLIDE 42

STED microscope:

200 nm x y

Fluorescence

0.5 1.0 2 4 6

(S0) (S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

 sin 2n λ d 

Is I

Sample Lens

OFF

Detector Laser

ON

PhaseMod

2p

slide-43
SLIDE 43

STED microscope:

200 nm x y

Fluorescence

0.5 1.0 2 4 6

Is

(S0) (S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

s

1 sin 2 I I n λ d   

I

Sample Lens

OFF

Detector Laser

ON

PhaseMod

2p

slide-44
SLIDE 44

STED microscope:

200 nm x y

Fluorescence

0.5 1.0 2 4 6

(S0) (S1)

[GW/cm²]

s

1 sin 2 I I n λ d   

d

stimulated emission

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

Is I

Sample Lens

OFF

Detector Laser

ON

PhaseMod

2p

slide-45
SLIDE 45

STED microscope:

200 nm x y

Fluorescence

0.5 1.0 2 4 6

(S0) (S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

s

1 sin 2 I I n λ d   

Is I

Sample Lens

OFF

Detector Laser

ON

PhaseMod

2p

slide-46
SLIDE 46

STED microscope:

200 nm x y

Fluorescence

0.5 1.0 2 4 6

(S0) (S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

s

1 sin 2 I I n λ d   

Is I

Sample Lens

OFF

Detector Laser

ON

PhaseMod

2p

slide-47
SLIDE 47

STED microscope:

200 nm x y

Fluorescence

0.5 1.0 2 4 6

(S0) (S1)

[GW/cm²]

d

stimulated emission

fluorescence

excitation S1 S0

tfl~ns

tvib< 1ps

  • n
  • ff
  • n
  • ff

s

1 sin 2 I I n λ d   

Is I

Sample Lens

OFF

Detector Laser

ON

PhaseMod

2p

slide-48
SLIDE 48

200 nm x y

d

4.2 nm 2.4 nm

(1-dimensional) N V

NV- in diamond

Color centers

Material sciences, magnetic sensing, quantum information

 = 775 nm

slide-49
SLIDE 49

GSD (metastable dark state)

S1 S0 T1

  • n
  • ff

Hell, Kroug Appl Phys B (1995) fluoresc.

STED

fluoresc.

S1 S0

  • ff
  • n

Hell, Wichmann, Opt Lett (1994)

  • ff
  • n

Hell, Nat Biotech (2003)

trans cis

RESOLFT

Principle: Discern by ON / OFF states in the sample

t ~ ms t ~ ns t ~ ms

s

1 sin 2 I I n λ d   

0.5 1.0 2 4 6

Is

(S0)

  • ff
  • n

~ 1/t

slide-50
SLIDE 50

GSD (metastable dark state)

S1 S0 T1

  • n
  • ff

Hell, Kroug Appl Phys B (1995) fluoresc.

STED

fluoresc.

S1 S0

  • ff
  • n

Hell, Wichmann, Opt Lett (1994)

  • ff
  • n

Hell, Nat Biotech (2003)

trans cis

RESOLFT

Principle: Discern by ON / OFF states in the sample

t ~ ms t ~ ns t ~ ms

MW/cm2 kW/cm2 W/cm2

s

1 sin 2 I I n λ d   

0.5 1.0 2 4 6

Is

(S0)

  • ff
  • n

~ 1/t

slide-51
SLIDE 51

RESOLFT:

many ‚doughnuts‘ (zeros) in parallel

… because of low intensity operation.

  • ff
  • n

 sin n 2 λ 

slide-52
SLIDE 52

RESOLFT:

many ‚doughnuts‘ (zeros) in parallel

… because of low intensity operation.

  • ff
  • n
slide-53
SLIDE 53

n pixels n pixels

RESOLFT:

many ‚doughnuts‘ (zeros) in parallel

… because of low intensity operation.

  • ff
  • n
slide-54
SLIDE 54

RESOLFT

recorded with

>100,000

‚doughnuts‘

in

2 seconds

Keratin filaments in living kidney epithelial cells

Scale bar: 10 µm

Chmyrov et al, Nature Meth (2013)

  • ff
  • n
slide-55
SLIDE 55

What does it take to get the best resolution ?

slide-56
SLIDE 56

Object

20th century:

… separate features by focusing light

Good lenses !

Detector

slide-57
SLIDE 57

Object … separate features by focusing light

Detector

20th century:

slide-58
SLIDE 58

Object

S1 S0

  • ff
  • n

S1 S0 T1

  • n
  • ff

trans cis

  • ff
  • n

etc.

Detector

… separate by molecular (on/off) states

Solution:

slide-59
SLIDE 59

S1 S0

  • ff
  • n

S1 S0 T1

  • n
  • ff

trans cis

  • ff
  • n

etc. … separate by molecular (on/off) states

STED, GSD, SSIM, RESOLFT,…

Detector

slide-60
SLIDE 60

S1 S0

  • ff
  • n

S1 S0 T1

  • n
  • ff

trans cis

  • ff
  • n

etc. … separate by molecular (on/off) states

Detector

STED, GSD, SSIM, RESOLFT,… X,Y,Z

controlled

by incident light pattern

slide-61
SLIDE 61

S1 S0

  • ff
  • n

S1 S0 T1

  • n
  • ff

trans cis

  • ff
  • n

etc. … separate by molecular (on/off) states

Detector

STED, GSD, SSIM, RESOLFT,… X,Y,Z

controlled

by incident light pattern

slide-62
SLIDE 62

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

controlled

by incident light pattern

Detector

slide-63
SLIDE 63

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

controlled

by incident light pattern

Detector

? stochastic

slide-64
SLIDE 64

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

centroid

  • f emitted

light pattern

Detector

X,Y,Z

controlled

by incident light pattern

slide-65
SLIDE 65

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 ~

s

I I

? stochastic

and … many photons for X,Y,Z precision

slide-66
SLIDE 66

single molecule !

Camera

Detector

  • ff
  • n

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,…

slide-67
SLIDE 67
  • ff
  • n

Superresolution

separates features using (at least) 2 molecular states

fluorescent non-fluorescent

slide-68
SLIDE 68
  • ff
  • n

B A

fluorescent absorbing scattering spin up … non-fluorescent non-absorbing non-scattering spin down …

Superresolution

separates features using (at least) 2 molecular states

slide-69
SLIDE 69
  • ff
  • n

B A

fluorescent absorbing scattering spin up … non-fluorescent non-absorbing non-scattering spin down …

Superresolution

separates features using (at least) 2 molecular states

slide-70
SLIDE 70

Former lab members Current lab members

1993-1996 since 1997 since 2003

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

slide-71
SLIDE 71

Standard STED

ON OFF

… down to molecular scale.