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Super‐Resolution Optical Microscopyp p py
Bo HuangLight Microscopy
May 10, 2010
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0.1mm0.1mmNaked eye: ~ 50‐100 μm d
10µm10µm
y μ
1595, Zaccharias and Hans JanssenFirst microscope, 9x magnification
d 2 NA
1µm1µmAntony Van Leeuwenhoek(1632‐1723), 200x Ernst Abbe (1840‐1905)
The “physical” diffraction limit
100nm100nm
The physical diffraction limit
10nm10nmCompound microscope>1000x
1nm1nm
1600 1700 1800 1900 2000
1Å1Å
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The diffraction barrier
0.1mm0.1mm
10µm10µm Cellular
1µm1µm
ar
100nm100nm
Sub‐cellul
Diffraction limit: ~ 250 nm lateral~ 600 nm axial
10nm10nmS
cular
1nm1nm
cMolec
1 μm
1Å1Å
Atom
ic
http://www.3dchem.com; http://cs.stedwards.edu; http://cvcweb.ices.utexas.edu; Fotin et al., Nature 2004; http://hrsbstaff.ednet.ns.ca; http://www.ebi.ac.uk
μ
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50 years to extend the resolutiony
• Confocal microscopy (1957)Confocal microscopy (1957)• Near‐field scanning optical microscopy (1972/1984)
• Multiphoton microscopy (1990)Multiphoton microscopy (1990)• 4‐Pi microscopy / I5M (1991‐1995)• Structured illumination microscopy (2000)• Negative refractive index (2006)Negative refractive index (2006)
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Near‐field scanning optical microscopyg p py
i i li h β adrenergic receptor clustersExcitation light β2 adrenergic receptor clusters on the plasma membrane
Optical fiber
~ 50 nm
Aperture
50 nm
Sample Ianoul et al., 2005
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4‐Pi / I5M/
d
d 2 NA
NA = n sin
Major advantage:Similar z resolution as x‐y resolutionSimilar z resolution as x‐y resolution
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Patterned illumination
Detector DetectorDetector Detector
= x
Excitation Detection
x
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Structured Illumination Microscopy (SIM)py ( )9 images
ReconstructionReconstruction
WF SIMWF SIM
22
=
Gustafsson, J Microscopy 2000
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The diffraction limit still exists
1 NA
d22
1
Confocal
4Pi / I5M
SIM
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Breaking the diffraction barrierg
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Breaking the diffraction barrierg
Confocal
4Pi / I5M
SIM
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Stimulated Emission Depletion (STED)
ion
p ( )Detector
ed Em
iss
2h
ation
scen
ce
Stim
ulate
h
Excita
Fluo
res
sSTED IIFLFL
/10
FL0
Send to a dark state
sSTED
0Send to a dark state
0Is
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STED microscopyDetector
py
ExcitationFluorescence
Detector
Light modulator
Depletion
StimulatedEmission
Excitation
Excitation STEDpattern
EffectivePSF
÷ =
p
?
Hell 1994, Hell 2000
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Saturated depletionp
NAIId
2/11
NAII s 2/1
STEDpattern
SaturatedDepletion
I II 2 II 10 II 100 Izero point
ISTED = ISISTED = 2 ISISTED = 10 ISISTED = 100 ISp
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STED images of microtubulesg
Wildanger et al., 2009
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The “patterned illumination” approachp pp
• Ground state• Triplet stateMultiple cycles Triplet state• Isomerizationetcetc.
Excitation Depletionpattern
÷ =
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Saturated SIM
FLFluorescencesaturation WF Deconvolution
IIexSIM SSIM
Saturation level
Saturated illumination pattern 50 nm resolution
Suffers from fast photobleachingunder saturated excitation condition
Sharp zero linesGustaffson, PNAS 2005
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The single‐molecule switching approachg g pp
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Single‐Molecule LocalizationgImage of one fluorescent molecule
FWHM ≈ 320 nm
Yildiz et al., Science, 2003
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Single‐molecule localization precisiong p
d 2 NA
1 photon
2 NA
10 photons 100 photons 1000 photons
1 NAN
d2
1
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Super‐resolution imaging by localizationp g g yRaw imagesConventional fluorescence STORM Image
Activation LocalizationDeactivationActivation LocalizationDeactivation
2x real time
Also named as PALM (Betzig et al., Science, 2006) and FPALM (Hess et al., Biophys. J. 2006)
Stochastic Optical Reconstruction Microscopy = STORM
Huang et al., Annu Rev Biochem, 2009
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Photoswitching of red cyanine dyesg y y650 nmFluorescent
Cy5 / Alexa 647
NN+
+ thiol
tion
on
otoactiva
Deactivati
ph
D
360 nm Dark650 nm
Bates eta l., PRL 2005, Bates et al., Science 2007, Dempsey et al., JACS 2009
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B‐SC‐1 cell, anti‐β tubulin
Commercial secondary antibodyAlexa 647
FWHM = 24 nm
40 000 frames 1 502 569 localization points
150
nts
FWHM = 24 nmstdev = 10 nm
40,000 frames, 1,502,569 localization points
50
100
mbe
r of p
oin
-8040
00
4080
Nu
nm)
5 μm500
-400
4080
-80-40 y (
nmx (nm)
500 nm
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The “single‐molecule switching” approachg g pp
• Photoswitching• Blinking
Multiple photonsBlinking
• Diffusion• BindingBindingetc.
Stochastic+ StochasticSwitching =
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STORM probes commercially available or already in your lab
400 500 600 700 nm
Cyanine dye + thiol systemCy5
Cy5.5 Cy7Alexa647
Rhodamine dye + redox systemAtto565
Bates et al., 2005, Bates et al., 2007, Huang et al., 2008
Atto590
Alexa568
Atto655 Atto700Alexa488
Heilemann et al., 2009
Alexa532
Atto520
Photoactivatible fluorescent proteins
PA‐GFP mEosFP2PA GFP
PS‐CFP2
Dronpa
Dendra2
PAmCherryEYFP Reviews:
Lukyanov et al., Nat. Rev. Cell Biol., 2005Lippincott‐Schwartz et al., Trends Cell Biol., 2009
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In a 2D world…Satellite image of ???
Google maps
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3D STED
Harke et al., Nano Lett, 2008
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3D STORM/PALM/(x, y, z)(x, y)
Astigmatic imaging
4002000‐200‐400z (nm)Huang et al Science 2008Huang et al., Science 2008
Bi‐plane imaging
SLMSLM
Double‐helical PSFJuette et al., Science 2008
EMCCDEMCCD
14006000‐500‐900z (nm) 14006000‐500‐900z (nm)Pavani et al., PNAS 2009
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3D Imaging of the Microtubule Networkg gz (nm)
600
300300
00
5 μmScale bar: 200 nm
Huang, Wang, Bates and Zhuang,Science, 2008
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3D Imaging of the Microtubule Networkg gz (nm)
600
Small, isolated clusters300
22 nm 28 nm 55 nm300
0
FWHM
0
5 μm
Huang, Wang, Bates and Zhuang,Science, 2008
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The use of two opposing objectivesI5S
pp g j
isoSTEDShal et al., Biophys J 2008
iPALM
4Pi scheme
Schmidt et al., Nano Lett 2009
Near isotropic3D resolution
Shtengel et al., PNAS 2009
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3D resolution of super‐resolution methodsp
x‐y z Opposing Two‐photon(nm) (nm) objectives (nm) Two photon
Conventional 250 600 4Pi: 120
SIM 100 250 I5S: 120 xyz
STED ~30 ~100 isoSTED: 30 xyz 100 µm deepSTED 30 100 isoSTED: 30 xyz 100 µm deep
STORM/PALM 20‐30 50‐60 iPALM: 20 xy, 10 z
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Multi‐color Imaging
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Muticolor STED
Excitation 2Excitation
STED 2STED2 color isoSTED resolving
the inner and outer membraneof mitochondria
1 µm
Schmidt et al., Nat Methods 2008
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Multicolor STORM/PALM: Emission/
n = n
n1 n2
n1 = n2 50% SRA545 + 50% SRA617? 100% SRA577?1 2 100% SRA577?
Single‐molecule detection!
3‐color imaging with one excitation wavelengthand two detection channels
Bossi et al., Nano Lett 2008
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Multicolor STORM/PALM: activation/650 nmFluorescent
Cy5
Cy3
tion
on
Cy3
otoactiva
Deactivati
ph
D
360 nm650
Dark650 nm Cy5
Cy3532 nm
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█ Cy3 / Alexa 647: Clathrin
█ Cy2 / Alexa 647: Microtubule
Crosstalk subtracted
Laser sequenceC 3 A647 C 2 A647
457
532
Cy3 A647 Cy2 A647
45 ……
1 μm
Bates, Huang, Dempsey and Zhuang,Science, 2007
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Multicolor imagingg g
Multicolor capability
ConventionalConventionalSIM 4 colors in the visible range
STED 2 colors so farSTED 2 colors so far
STORM/PALM 3 activation x 3 emission
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Live Cell Imaging
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SIM
/2 µm STORM/PALM2 µmKner, Chhun et al., Nat Methods, 2009
STED
S h ff t l N t M th d 2008Schroff et al., Nat Methods, 2008
Nagerl et al., PNAS, 2008
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Th li it f “S R l ti ”The limit of “Super‐Resolution”
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Unbound theoretical resolution
1 NA
d2S
1
• STORM/PALM
SNS =STORM/PALM
– 6,000 photons 5 nmh d i lif i
NS
– 100,000 photos during Cy5 life time < 1 nm
• STED 1+ I/IsS =– 1:100 contrast of the donut 20 nmDiamond defects: 8 nm
1 I/Is
– Diamond defects: 8 nm
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Effective resolution: Probe size mattersAntibodies:
~ 10 nmSmall fluorophores:
~ 1 nmFluorescent Proteins:
~ 3 nm~ 10 nm ~ 1 nm~ 3 nm
Localization precision: 22 nm
Measured width by STORM: 56 nm
Actual microtubule diameter: 25 nm100 nm
Bacillus subtilis spore
100 nm500 nm
1000 h t 6000 h t6000 h t < 1000 photons ~ 6000 photons~ 6000 photons
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Effective resolution: Density mattersy
• Localization precisionDiD stained plasma membrane p– 1000 photons ‐> 20 nm
• Localization density
p
Localization density– Nyquist criteria
The labeling density limit of resolution applies
1 μm
to all fluorescence microscopy methods
1 μm
1000 frames 10 sec total time
Diameter: 62 nm
1000 frames, 10 sec total time
Point to point distance ≈ Feature sizePoint to point distance < ½ Feature sizePoint to point distance ≈ Feature sizePoint to point distance < ½ Feature size
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Effective resolution: contrast matters650 nmFluorescent 1% means…
1% Homogeneous sample Microtubule
1% means…tio
n
on
Common dye blinking: >3%Cy5 MEA 0 1 0 2%
otoactiva
Deactivati Cy5‐MEA: 0.1‐0.2%
mEos2: 0.001%
ph
D
360 nm DarkAverage point‐to‐point distance:
40 nm 14 nmNo such limit for non‐single methods of SIM and STED650 nm
99%40 nm 14 nmmethods of SIM and STED
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Time resolution: density matters
Typical Localization accumulation:28 points / μm2∙s
Effective resolution:70 nm at 25 sec integration time
Now as fast as 2 sec time resolution
25 sec time resolution, 100x real time3 M t th l i
with 1000 frames / sec camera
3 mMmercaptoethylamine1 μm
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Comparison of time resolutionp
2D Spatial l ti Time resolutionresolution
SIM Wide‐field 120 nm 9 frames (0.09 sec)
1 x 2 µm: 0 03 secSTED Scanning 60 nm 1 x 2 µm: 0.03 sec10 x 20 µm: 3 sec
STORM/PALM Wide‐field 60 nm 3000 frames (3 sec)( )
3D Spatial Time resolution3D 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 sec10 x 20 x 0.6 µm: 60 sec
STORM/PALM Wide‐field 60 nm 3000 frames (3 sec) – no scan!STORM/PALM Wide field 60 nm 3000 frames (3 sec) no scan!
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With the creation of new tools…
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