Super‐Resolution Optical Microscopyp p py

Bo HuangLight Microscopy

May 10, 2010

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

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

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)

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

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

Patterned illumination

Detector DetectorDetector Detector

= x

Excitation Detection

x

Structured Illumination Microscopy (SIM)py ( )9 images

ReconstructionReconstruction

WF SIMWF SIM

22

=

Gustafsson, J Microscopy 2000

The diffraction limit still exists

1 NA

d22

1

Confocal

4Pi / I5M

SIM

Breaking the diffraction barrierg

Breaking the diffraction barrierg

Confocal

4Pi / I5M

SIM

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

STED microscopyDetector

py

ExcitationFluorescence

Detector

Light modulator

Depletion

StimulatedEmission

Excitation

Excitation STEDpattern

EffectivePSF

÷ =

p

?

Hell 1994, Hell 2000

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

STED images of microtubulesg

Wildanger et al., 2009

The “patterned illumination” approachp pp

• Ground state• Triplet stateMultiple cycles Triplet state• Isomerizationetcetc.

Excitation Depletionpattern

÷ =

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

The single‐molecule switching approachg g pp

Single‐Molecule LocalizationgImage of one fluorescent molecule

FWHM ≈ 320 nm

Yildiz et al., Science, 2003

Single‐molecule localization precisiong p

d 2 NA

1 photon

2 NA

10 photons 100 photons 1000 photons

1 NAN

d2

1

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

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

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

The “single‐molecule switching” approachg g pp

• Photoswitching• Blinking

Multiple photonsBlinking

• Diffusion• BindingBindingetc.

Stochastic+ StochasticSwitching =

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

In a 2D world…Satellite image of ???

Google maps

3D STED

Harke et al., Nano Lett, 2008

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

3D Imaging of  the Microtubule Networkg gz (nm)

600

300300

00

5 μmScale bar: 200 nm

Huang, Wang, Bates and Zhuang,Science, 2008

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

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

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

Multi‐color Imaging

Muticolor STED

Excitation 2Excitation

STED 2STED2 color isoSTED resolving

the inner and outer membraneof mitochondria

1 µm

Schmidt et al., Nat Methods 2008

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

Multicolor STORM/PALM: activation/650 nmFluorescent

Cy5

Cy3

tion

on

Cy3

otoactiva

Deactivati

ph

D

360 nm650

Dark650 nm Cy5

Cy3532 nm

█ 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

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

Live Cell Imaging

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

Th li it f “S R l ti ”The limit of “Super‐Resolution”

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

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

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

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

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

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!

With the creation of new tools…