Super‐Resolution Optical Microscopy p p py

Bo Huang Light Microscopy May 10, 2010

0.1mm

10µm

d

Naked eye: ~ y 50‐100 μm μ 1595, Zaccharias and Hans Janssen First microscope, 9x magnification Antony Van Leeuwenhoek (1632‐1723), 200x

1µm

 2 NA

Ernst Abbe (1840‐1905) The “physical” The  physical  diffraction limit diffraction limit

100nm

Compound microscope >1000x

10nm

1nm

1600 1Å

1700

1800

1900

2000

The diffraction barrier

10nm

1nm

1Å

S Sub‐cellul ar

Diffraction limit: ~ 250 nm lateral 100nm ~ 600 nm axial    

Moleccular

1µm

Atomicc

10µm

Cellular

0.1mm

1μ μm

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

50 years to extend the resolution y • Confocal microscopy (1957) microscopy (1957) • Near‐field scanning optical microscopy  (1972/1984) • Multiphoton microscopy (1990) microscopy (1990) • 4‐Pi microscopy / I5M (1991‐1995) • Structured illumination microscopy (2000) • Negative refractive index (2006) Negative refractive index (2006)

Near‐field scanning optical microscopy g p py Excitation light i i li h

β2 adrenergic receptor clusters on the plasma membrane

Optical fiber ~ 50 nm 50 nm Aperture

Sample

Ianoul et al., 2005

4‐Pi / I / 5M d NA = n sin 

Major advantage: Similar z resolution as x‐y resolution Similar z resolution as x‐y resolution

 2 NA

Patterned illumination Detector

Detector

=

x

Excitation

Detection

x

Structured Illumination Microscopy (SIM) py ( ) 9 images Reconstruction

WF

SIM

2

= Gustafsson, J Microscopy 2000

The diffraction limit still exists 1  d 2 2 NA 

Confocal

4Pi / I5M

SIM

Breaking the diffraction barrier g

Breaking the diffraction barrier g

Confocal

4Pi / I5M

SIM

Stimulated Emission Depletion (STED) p ( )

FL0

Send to a dark state Send to a dark state

h

Stimulateed   Emission

Excitaation

Fluoresscence

Detector

2h

FL0 FL  1  I STED / I s

0 Is

STED microscopy py Excitation

Detector

Light modulator

Fluorescence

Depletion

Stimulated Emission

Excitation

Excitation

STED pattern p

÷

Effective PSF

=

? Hell 1994, Hell 2000

Saturated depletion p  1  d 1  I / I s 2 NA

STED pattern

ISTED = 100 IS ISISIS 2 10

Saturated Depletion

zero point p

STED images of microtubules g

Wildanger et al., 2009

The “patterned illumination” approach p pp

Multiple cycles

Excitation

Depletion pattern

÷

=

• Ground state Triplet state • Triplet state • Isomerization etc. etc

Saturated SIM FL

Fluorescence saturation

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 g g pp

Single‐Molecule Localization g Image of one fluorescent molecule FWHM ≈ 320 nm

Yildiz et al., Science, 2003

Single‐molecule localization precision g p d

 2 NA

1 photon

10 photons

100 photons 1000 photons

 1 d N 2 NA 

Super‐resolution imaging by localization p g g y Conventional fluorescence

Raw images

STORM Image

Deactivation Activation

Localization

2x real time

Stochastic  Optical Reconstruction Microscopy = STORM Also named as PALM (Betzig et al., Science, 2006) and FPALM (Hess et al., Biophys. J. 2006)

Huang et al., Annu Rev Biochem, 2009

Photoswitching of red cyanine dyes g y y 650 nm

Fluorescent

+

N

360 nm

Cy5 / Alexa 647

Deactivation D

photoactivation

+ thiol

N

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 antibody Alexa 647

FWHM = 24 nm stdev = 10 nm

nts Number of poin

150

100 000 frames 1 502 569 localization points 40 40,000 frames, 1,502,569 localization points

50

-40 40 0 x (nm )

40

500 500 nm

80

)

0 -40 -80

(n m

-80

y

0

80 40

5 μm

The “single‐molecule switching” approach g g pp • Photoswitching • Blinking • Diffusion • Binding etc.

Multiple photons

+

Stochastic Switching

=

STORM probes commercially available or already in your lab 400

500

600

Cyanine dye + thiol system

700 nm Alexa647

Cy5

Cy5.5

Cy7

Bates et al., 2005, Bates et al., 2007, Huang et al., 2008

Rhodamine dye + redox system

Atto565

Alexa488

Atto590

Alexa532

Atto520

Alexa568

Atto655

Atto700

Heilemann et al., 2009

Photoactivatible fluorescent proteins mEosFP2

PA‐GFP PA GFP PS‐CFP2

Dendra2

Dronpa

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 / Astigmatic imaging

(x, y) (x, y, z)

z (nm) ‐400

‐200

0

200

400

Huang et al Science 2008 Huang et al., Science 2008

Bi‐plane imaging SLM

Juette et al., Science 2008

Double‐helical PSF EMCCD EMCCD

z (nm) ‐900 z (nm)

‐500

0

600

1400

Pavani et al., PNAS 2009

3D Imaging of  the Microtubule Network g g z (nm) 600

300

0

Scale bar: 200 nm

5 μm Huang, Wang, Bates and Zhuang, Science, 2008

3D Imaging of  the Microtubule Network g g z (nm) 600

Small, isolated clusters FWHM   22 nm

300 28 nm

55 nm 0

5 μm Huang, Wang, Bates and Zhuang, Science, 2008

The use of two opposing objectives pp g j I5 S

isoSTED

Shal et al., Biophys J 2008

4Pi scheme

iPALM

Schmidt et al., Nano Lett 2009

Near isotropic 3D resolution Shtengel et al., PNAS 2009

3D resolution of super‐resolution methods p x‐y  (nm)

z  (nm)

Opposing  objectives (nm)

Conventional

250

600

4Pi: 120

SIM

100

250

I5S: 120 xyz

STED

~30 30

~100 100

isoSTED: 30 xyz isoSTED: 30 xyz

50‐60

iPALM: 20 xy, 10 z

STORM/PALM 20‐30

Two‐photon Two photon

100 µm deep µm deep

Multi‐color Imaging

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

Multicolor STORM/PALM: Emission / n1 = n = n2

n1

n2

 50% SRA545 + 50% SRA617?  100% SRA577? 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 nm

Fluorescent

Cy5

360 nm

D Deactivation

photoactivation

Cy3

Dark

650 650 nm

Cy5

532 nm

Cy3

█ Cy3 / Alexa 647: Clathrin █ Cy2 / Alexa 647: Microtubule Crosstalk subtracted

Laser sequence A647

C 2 Cy2

A647

457 4

…

532 5

C 3 Cy3

…

1 μm Bates, Huang, Dempsey and Zhuang, Science, 2007

Multicolor imaging g g Multicolor capability Conventional SIM

4 colors in the visible range

STED

2 colors so far 2 colors so far

STORM/PALM 3 activation x 3 emission

Live Cell Imaging

SIM

2 µm 2 µm Kner, Chhun et al., Nat Methods, 2009

STORM/PALM /

STED

S h ff et al., Nat Methods, 2008 Schroff t l N t M th d 2008

Nagerl et al., PNAS, 2008

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

Unbound theoretical resolution d • STORM/PALM

1

S



 2 NA S= N

– 6,000 photons  5 nm – 100,000 photos during Cy5 life time  h d i lif i 