Super-Resolution Optical Microscopy

Super-Resolution Optical MicroscopyBo HuangLight MicroscopyMay 10, 2010

1

1600170018001900Naked eye: ~ 50-100 m

1595, Zaccharias and Hans JanssenFirst microscope, 9x magnificationAntony Van Leeuwenhoek(1632-1723), 200x

Ernst Abbe (1840-1905)The physical diffraction limitCompound microscope>1000x2000d 2 NAl

2On September 17, 1683, Leeuwenhoek wrote to the Royal Society about his observations on the plaque between his own teeth, a little white matter, which is as thick as if twere batter. He repeated these observations on two ladies (probably his own wife and daughter), and on two old men who had never cleaned their teeth in their lives. Looking at these samples with his microscope, Leeuwenhoek reported how in his own mouth: I then most always saw, with great wonder, that in the said matter there were many very little living animalcules, very prettily a-moving. The biggest sort. . . had a very strong and swift motion, and shot through the water (or spittle) like a pike does through the water. The second sort. . . oft-times spun round like a top. . . and these were far more in number. In the mouth of one of the old men, Leeuwenhoek found an unbelievably great company of living animalcules, a-swimming more nimbly than any I had ever seen up to this time. The biggest sort. . . bent their body into curves in going forwards. . . Moreover, the other animalcules were in such enormous numbers, that all the water. . . seemed to be alive.

The diffraction barrier

AtomicCellularSub-cellularMolecular

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

1 mDiffraction limit: ~ 250 nm lateral~ 600 nm axial 3Point spread functionLateral as well as axial resolutionBroader pictureLength scale50 years to extend the resolutionConfocal 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 microscopyExcitation lightOptical fiberApertureSample

Ianoul et al., 20052 adrenergic receptor clusters on the plasma membrane~ 50 nm4-Pi / I5Md 2 NAlNA = n sin

Major advantage:Similar z resolution as x-y resolution

Patterned illumination=x

xExcitationDetectionDetectorDetectorStructured Illumination Microscopy (SIM)

9 imagesReconstructionWFSIMGustafsson, J Microscopy 2000

2=

The diffraction limit still existsNAd221lConfocal4Pi / I5MSIM

Breaking the diffraction barrierConfocal4Pi / I5MSIMBreaking the diffraction barrierThe Fluorophore!Stimulated EmissionStimulated Emission Depletion (STED)

Send to a dark stateDetectorh2hExcitationFluorescence

FL00Is

STED microscopyExcitationFluorescenceStimulatedEmission

=ExcitationSTEDpatternEffectivePSFHell 1994, Hell 2000DetectorExcitationDepletionLight modulator?Saturated depletion

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

STEDpatternSaturatedDepletionzero pointNAIIds2/11l+=STED images of microtubules

Wildanger et al., 2009

The patterned illumination approach

ExcitationDepletionpattern Ground state Triplet state Isomerization etc.=

Multiple cyclesSaturated SIMIexFLFluorescencesaturation

Saturation levelSaturated illumination patternSharp zero linesGustaffson, PNAS 2005

WFDeconvolutionSIMSSIM50 nm resolutionSuffers from fast photobleaching under saturated excitation condition

The single-molecule switching approach18Single-Molecule Localization

FWHM 320 nm

Yildiz et al., Science, 2003

Image of one fluorescent molecule19Single-molecule localization precision

1 photon10 photons100 photons1000 photonsd 2 NAlNANd21l=20

Super-resolution imaging by localization

Raw imagesConventional fluorescenceSTORM Image2x real time

ActivationLocalizationDeactivationAlso named as PALM (Betzig et al., Science, 2006) and FPALM (Hess et al., Biophys. J. 2006)Huang et al., Annu Rev Biochem, 2009Stochastic Optical Reconstruction Microscopy = STORM21Photoswitching of red cyanine dyes

photoactivationDeactivation650 nm360 nm650 nmFluorescentDark

Cy5 / Alexa 647NN++ thiolBates eta l., PRL 2005, Bates et al., Science 2007, Dempsey et al., JACS 2009

B-SC-1 cell, anti- tubulinCommercial secondary antibody

5 m

500 nmAlexa 64740,000 frames, 1,502,569 localization points

FWHM = 24 nmstdev = 10 nmTransition to multicolor imaging23The single-molecule switching approach

Multiple photons Photoswitching Blinking Diffusion Binding etc.

+StochasticSwitching=STORM probes commercially available or already in your lab400500600700 nmCyanine dye + thiol systemCy5Cy5.5Cy7Rhodamine dye + redox systemAtto590Alexa568Atto655Atto700Alexa488Heilemann et al., 2009Atto565Bates et al., 2005, Bates et al., 2007, Huang et al., 2008Photoactivatible fluorescent proteinsPA-GFPPS-CFP2DronpamEosFP2Dendra2EYFPReviews:Lukyanov et al., Nat. Rev. Cell Biol., 2005Lippincott-Schwartz et al., Trends Cell Biol., 2009Alexa532Atto520PAmCherryAlexa6473D Imaging

In a 2D worldSatellite image of ???Google maps

3D STED

Harke et al., Nano Lett, 2008

3D STORM/PALMEMCCD(x, y, z)

4002000-200-400z (nm)(x, y)Astigmatic imagingHuang et al., Science 2008Bi-plane imagingDouble-helical PSFJuette et al., Science 2008EMCCDSLM

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

5 mHuang, Wang, Bates and Zhuang,Science, 2008Scale bar: 200 nm3D Imaging of the Microtubule Networkz (nm)3000600

No two-layer, just say the whole thing is 400 nm, cannot be resolved by confocal.30

5 mHuang, Wang, Bates and Zhuang,Science, 2008

Small, isolated clusters22 nm28 nm55 nm3D Imaging of the Microtubule Networkz (nm)3000600

FWHM No two-layer, just say the whole thing is 400 nm, cannot be resolved by confocal.31I5SisoSTEDiPALM4Pi schemeThe use of two opposing objectives

Near isotropic3D resolution

Shal et al., Biophys J 2008Schmidt et al., Nano Lett 2009Shtengel et al., PNAS 20093D resolution of super-resolution methodsx-y (nm)z (nm)Opposing objectives (nm)Two-photonConventional2506004Pi: 120SIM100250I5S: 120 xyzSTED~30~100isoSTED: 30 xyz100 m deepSTORM/PALM20-3050-60iPALM: 20 xy, 10 zMulti-color Imaging

Excitation 2STED 2Muticolor STED

ExcitationSTED

2 color isoSTED resolvingthe inner and outer membraneof mitochondriaSchmidt et al., Nat Methods 20081 mMulticolor STORM/PALM: Emission

n1n2

n1 = n2 50% SRA545 + 50% SRA617? 100% SRA577?Single-molecule detection!3-color imaging with one excitation wavelengthand two detection channels

Bossi et al., Nano Lett 2008Multicolor STORM/PALM: activation

photoactivationDeactivation650 nm360 nm650 nmFluorescentDark

Cy5Cy5Cy3Cy3532 nm

1 m

Bates, Huang, Dempsey and Zhuang,Science, 2007 Cy3 / Alexa 647: Clathrin Cy2 / Alexa 647: MicrotubuleCrosstalk subtractedLaser sequence457532Cy3A647Cy2A647Multicolor imagingMulticolor capabilityConventionalSIM4 colors in the visible rangeSTED2 colors so farSTORM/PALM3 activation x 3 emissionLive Cell Imaging

SIMSTORM/PALMSTED2 mKner, Chhun et al., Nat Methods, 2009Nagerl et al., PNAS, 2008

Schroff et al., Nat Methods, 2008

The limit of Super-ResolutionUnbound theoretical resolutionSTORM/PALM6,000 photons 5 nm100,000 photos during Cy5 life time < 1 nmSTED1:100 contrast of the donut 20 nmDiamond defects: 8 nmNAd2S1l=NS =1+ I/IsS =Effective resolution: Probe size matters

100 nmAntibodies: ~ 10 nmLocalization precision:22 nm

Measured width by STORM: 56 nm

Actual microtubule diameter:25 nmSmall fluorophores: ~ 1 nm

100 nmFluorescent Proteins:~ 3 nm

Bacillus subtilis spore500 nm< 1000 photons~ 6000 photons~ 6000 photons44

Conventional imageSTORM: a time-for-space strategySTORM imageTime

Effective resolution: Density matters

Frames for image reconstruction: 2005001,0005,00040,000Point to point distance Feature sizePoint to point distance < Feature sizeThis labeling density limit of resolution applies to all fluorescence microscopy methodsNyquist criteriaEffective resolution: Contrast matters

photoactivationDeactivation650 nm360 nm650 nmFluorescentDarke.g. 1%e.g. 99%1% means

Sparsely labeled sampleDensely labeled sampleEffective resolution: Contrast matters

photoactivationDeactivation650 nm360 nm650 nmFluorescentDarke.g. 1%e.g. 99%Homogeneous sampleMicrotubule

Average point-to-point distance:40 nm14 nm1% meansCommon blinking dyes: >3%Cy5 + mercaptoethylamine: 0.1-0.2%mEosFP: 0.001%Live cell STORM: Density matters100x real time 1 m

Plasma membrane staining of a BS-C-1 cellAssuming: 1 molecule occupies 500 500 nmOn average 0.1 point / 0.25 m2frame70 nm resolution 2000 frames100 fps = 20 sec time resolutionLocalization accuracy. Width of a fillopodia. Explain Nyquist criteria.49Stochastic switching + particle tracking

1 mEffective D = 0.66 m2/s 1000 frames, 10 sec total time-2000200050100 Number of localizationsDisplacement / frame (nm)DiD stained plasma membrane

1 m50Comparison of time resolution2DSpatial resolutionTime resolutionSIMWide-field120 nm9 frames (0.09 sec)STEDScanning60 nm1 x 2 m: 0.03 sec10 x 20 m: 3 secSTORM/PALMWide-field60 nm3000 frames (3 sec)3DSpatial resolutionTime resolutionSIMWide-field120 nm15 frames x 10 (1.5 sec)STEDScanning60 nm1 x 2 x 0.6 m: 0.6 sec10 x 20 x 0.6 m: 60 secSTORM/PALMWide-field60 nm3000