Super-Resolution Optical ... Superâ€گResolution Optical Microscopy Bo Huang Light Microscopy May...

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  • Super‐Resolution Optical Microscopyp p py

    Bo Huang Light Microscopy

    May 10, 2010

  • 0.1mm0.1mm Naked eye: ~ 50‐100 μm d

    10µm10µm

    y μ

    1595, Zaccharias and Hans Janssen First microscope, 9x magnification

    d  2 NA

    1µm1µm Antony Van Leeuwenhoek (1632‐1723), 200x Ernst Abbe (1840‐1905)

    The “physical” diffraction limit

    100nm100nm

    The  physical  diffraction limit

    10nm10nm Compound microscope >1000x

    1nm1nm

    1600 1700 1800 1900 2000

    1Å1Å

  • The diffraction barrier

    0.1mm0.1mm

    10µm10µm Ce llu la r

    1µm1µm

    ar

    100nm100nm

    Su b‐ ce llu l

    Diffraction limit: ~ 250 nm lateral ~ 600 nm axial    

    10nm10nm S

    cu la r

    1nm1nm

    c M ol ec

    1 μm

    1Å1Å

    At om

    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

    d 22

    1  

    Confocal

    4Pi / I5M

    SIM

  • Breaking the diffraction barrierg

  • Breaking the diffraction barrierg

    Confocal

    4Pi / I5M

    SIM

  • Stimulated Emission Depletion (STED)

    io n

    p ( ) Detector

    ed     Em

    iss

    2h

    at io n

    sc en

    ce

    St im

    ul at e

    h

    Ex ci ta

    Fl uo

    re s

    sSTED II FLFL

    /1 0

     

    FL0

    Send to a dark state

    sSTED

    0Send to a dark state

    0 Is

  • STED microscopy Detector

    py

    Excitation Fluorescence

    Detector

    Light modulator

    Depletion

    Stimulated Emission

    Excitation

    Excitation STED pattern

    Effective PSF

    ÷ =

    p

    ?

    Hell 1994, Hell 2000

  • Saturated depletionp

    NAII d

    2/1 1 

     

    NAII s 2/1

    STED pattern

    Saturated Depletion

    I II 2 II 10 II 100 I zero point

    ISTED = ISISTED = 2 ISISTED = 10 ISISTED = 100 IS p

  • STED images of microtubulesg

    Wildanger et al., 2009

  • The “patterned illumination” approachp pp

    • Ground state • Triplet stateMultiple cycles Triplet state • Isomerization etcetc.

    Excitation Depletion pattern

    ÷ =

  • Saturated SIM

    FL Fluorescence saturation WF Deconvolution

    IIex 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 approachg g pp

  • Single‐Molecule Localizationg Image 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

    d 2

    1  

  • Super‐resolution imaging by localizationp g g y Raw 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 y 650 nmFluorescent

    Cy5 / Alexa 647

    NN +

    + thiol

    tio n

    on

    ot oa ct iv a

    De ac tiv at i

    ph

    D

    360 nm Dark 650 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

    nt s

    FWHM = 24 nm stdev = 10 nm

    40,000 frames, 1,502,569 localization points

    50

    100

    m be

    r o f p

    oi n

    -80 40

    0 0

    40 80

    N u

    nm )

    5 μm 500

    -40 0

    40 80

    -80 -40 y (

    nm x (nm)

    500 nm

  • The “single‐molecule switching” approachg g pp

    • Photoswitching • Blinking

    Multiple photons Blinking

    • Diffusion • BindingBinding etc.

    Stochastic+ StochasticSwitching =

  • STORM probes commercially available or already in your lab

    400 500 600 700 nm

    Cyanine dye + thiol system Cy5

    Cy5.5 Cy7 Alexa647

    Rhodamine dye + redox system Atto565

    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

    PAmCherry EYFP Reviews:

    Lukyanov et al., Nat. Rev. Cell Biol., 2005 Lippincott‐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 PSF Juette et al., Science 2008

    EMCCD EMCCD

    14006000‐500‐900z (nm) 14006000‐500‐900z (nm) Pavani et al., PNAS 2009

  • 3D Imaging of  the Microtubule Networkg g z (nm)

    600

    300300

    00

    5 μm Scale bar: 200 nm

    Huang, Wang, Bates and Zhuang, Science, 2008

  • 3D Imaging of  the Microtubule Networkg g z (nm)

    600

    Small, isolated clusters 300

    22 nm 28 nm 55 nm 300

    0

    FWHM  

    0

    5 μm

    Huang, Wang, Bates and Zhuang, Science, 2008

  • The use of two opposing objectives I5S

    pp g j

    isoSTED Shal et al., Biophys J 2008

    iPALM

    4Pi scheme

    Schmidt et al., Nano Lett 2009

    Near isotropic 3D 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 2STED 2 color isoSTED resolving

    the inner and outer membrane of 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 wavelength and two detection channels

    Bossi et al., Nano Lett 2008

  • Multicolor STORM/PALM: activation/ 650 nmFluorescent

    Cy5

    Cy3

    tio n

    on

    Cy3

    ot oa ct iv a

    De ac tiv at i

    ph

    D

    360 nm 650

    Dark 650 nm Cy5