11/1/2011
Super‐Resolution Microscopy
Structured Illumination
Bo Huang
Department of Pharmaceutical Chemistry, UCSF
CSHL Quantitative Microscopy, 10/31/2011
50 years to extend the resolution
• Confocal microscopy (1957)
• Near‐field scanning optical microscopy (1972/1984)
• Multiphoton microscopy (1990)
• 4‐Pi microscopy / I5M (1991‐1995)
• Structured illumination microscopy (2000)
• Negative refractive index (2006)
Near‐field scanning optical microscopy
Excitation light
β2 adrenergic receptor clusters
on the plasma membrane
Optical fiber
~ 50 nm
Aperture
Sample
Ianoul et al., 2005
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4‐Pi / I5M
d

2 NA
NA = n sin

Major advantage:
Similar z resolution as x‐y resolution
Patterned illumination
Detector
Detector
=
x
Excitation
Detection
x
Structured Illumination Microscopy (SIM)
9 images
Reconstruction
WF
SIM
2
=
Gustafsson, J Microscopy 2000
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Being (slightly) more rigorous about SIM
Fourier transform and spatial frequencies
?
=
Fourier transform and spatial frequencies
+
Summed image
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Fourier transform and spatial frequencies
+
+
Summed image
Fourier transform and spatial frequencies
Discrete spatial frequencies
+
+
+
+ …
Summed image
…
G(x) = Σ F(k) sin(k x)
Fourier transform and spatial frequencies
y
ky
x
Original Image (real space)
kx
Fourier transform (frequency space)
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Fourier optics and microscope resolution
Sample
f
Objective
f
Back focal plane
k = f sinα
α
Fourier optics and microscope resolution
Sample
f
Objective
f
Back focal plane
A(k)
k
α
x
I(x)
Phase delay from the mid‐point
Δφ = x sinα / 2πλ = x k/2πλf
Light intensity at the sample plane
assuming refractive index = 1
Fourier Transform!
I(x) = ΣA(k) sin(Δφ) = Σ A(k) sin (x k/2πλf)
Fourier optics and microscope resolution
Sample
f
Objective
f
Back focal plane
kmax
α
Spatial frequency = k / 2πλf
Size of the back focal plane
kmax = f sinαmax = f · NA
Resolution = λ / 2NA
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Extending the measurable freq. range
Excitation(x) × Sample(x) = Observed Signal(x)
x
Freq = 30
=
Freq = 25
Freq = 55 & 5
sinA · sinB = (cos (A – B) – cos(A + B)) / 2
Extending the measurable freq. range
Excitation(x) × Sample(x) = Observed Signal(x)
x
Freq = 30
=
Freq = 25
Freq = 55 + 5
sinA · sinB = (cos (A + B) – cos(A – B)) / 2
Extending the measurable freq. range
ky
kmax
kex ≤ kmax
k – kex ≤ kmax
ky
kmax
kex
kx
kx
k + kex ≤ kmax
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Extending the measurable freq. range
ky
kmax
kex ≤ kmax
k – kex ≤ kmax
ky
kmax
kex
kx
kx
Gustaffson et al., J. Microscopy, 2000
k + kex ≤ kmax
Generating the illumination pattern
Sample plane
Objective
Back focal plane
Grating
Spatial‐light modulator
3D SIM: better resolution + optical sectioning Schermellech et al., Science 2008, Gustafsson et al., Biophys J. 2008
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Multicolor SIM
Same as conventional fluorescence microscopy!
Live imaging with SIM
Kner, Chhun et al., Nat Methods, 2009
Shao et al., Nat Methods, 2011
The diffraction limit still exists
d
1 
2 2 NA

Confocal
4Pi / I5M
SIM
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Breaking the diffraction barrier
Breaking the diffraction barrier
Confocal
4Pi / I5M
SIM
Stimulated Emission Depletion (STED)
FL0
Send to a dark state
0
h
FL 
Stimulated Emission
Excitation
Fluorescence
Detector
2h
FL0
1  I STED / I s
Is
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STED microscopy
Excitation
Detector
Light modulator
Fluorescence
Depletion
Stimulated
Emission
Excitation
Excitation
STED
pattern
÷
Effective
PSF
?
=
Hell 1994, Hell 2000
Saturated depletion
ISTED = I210
100
S ISISIS
D

1

1  I / I s 2 NA
STED
pattern
Saturated
Depletion
zero point
STED images of microtubules
Wildanger et al., 2009
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3D STED
Harke et al., Nano Lett, 2008
Muticolor STED
Excitation
Excitation 2
STED
STED 2
2 color isoSTED resolving
the inner and outer membrane
of mitochondria
1 µm
Schmidt et al., Nat Methods 2008
Live STED
Westaphl et al., Science, 2008
Nagerl et al., PNAS, 2008
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The use of two opposing objectives
I5S
Shal et al., Biophys J 2008
isoSTED
4Pi scheme
Schmidt et al., Nano Lett 2009
iPALM
Near isotropic
3D resolution
Shtengel et al., PNAS 2009
Super‐resolution optical microscopy
Hell, Science, 2007; Hell, Nat Methods, 2008
Rust, Bates & Zhuang, Nat Methods, 2006
Betzig et al., Science, 2006
Hess, Girirajan and Mason, Biophys. J., 2006
Gustafsson, PNAS., 2005
STED
SSIM
STORM/(F)PALM
The “patterned illumination” approach
Multiple cycles
Excitation
• Ground state
• Triplet state
• Isomerization
etc.
Depletion
pattern
÷
=
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Saturated SIM
Fluorescence
saturation
FL
WF
Deconvolution
Iex
SIM
SSIM
Saturation level
Saturated illumination pattern
50 nm resolution
Suffers from fast photobleaching
under saturated excitation condition Sharp zero lines
Gustaffson, PNAS 2005
The “single‐molecule switching” approach
(STORM/PALM etc.)
• Photoswitching
• Blinking
• Diffusion
• Binding
etc.
Multiple photons
+
Stochastic
Switching
=
Super resolution microscopy
spec sheets
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11/1/2011
3D spatial resolution
x‐y (nm)
z (nm)
Opposing objectives (nm)
Conventional
250
600
4Pi: 120
SIM
100
250
I5S: 120 xyz
STED
~30
STORM/PALM 20‐30
Two‐photon
500 µm deep
~100
isoSTED: 30 xyz
100 µm deep
50‐60
iPALM: 20 xy, 10 z
10 µm
Multicolor imaging
Multicolor capability
Conventional
SIM
4 colors in the visible range
STED
2 colors so far
STORM/PALM 3 activation x 3 emission
Time resolution
Spatial resolution
2D
Time resolution
SIM
Wide‐field
120 nm
9 frames (0.09 sec)
STED
Scanning
60 nm
1 x 2 µm: 0.03 sec
10 x 20 µm: 3 sec
STORM/PALM Wide‐field
60 nm
3000 frames (6 sec)
3D
Spatial resolution
Time resolution
SIM
Wide‐field
120 nm
15 frames x 10 (1.5 sec)
STED
Scanning
60 nm
1 x 2 x 0.6 µm: 0.6 sec
10 x 20 x 0.6 µm: 60 sec
60 nm
3000 frames (6 sec) – no scan!
STORM/PALM Wide‐field
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Practical issues
SIM
STED
Fluorophore limitation
‐
x
STORM/PALM
x
Instrument complexity
xx
xxx
x
Data analysis
xxx
‐
xx
Cost (rapidly changing)
xx
xxx
x
With the creation of new tools…
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