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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.
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