Rust et al, Nat Methods (2006) -- STORM Betzig et al, Science (2006) -- PALM Hess et al, Biophys. J (2006) -- FPALM
Single-molecule localization
σ ≈ σPSF / N1/2
z
100 nm
y x
Pit 4
z x x
y x
z x
100 nm
y x
z x
Nyquist resolution: ~20 nm Time resolution: ~1 sec Frame rate: 500 Hz – 1kHz
Jones, Shim et al, Nat Methods (2011)
Ke et al, Nat Methods (in press)
Actin
Ventral layer
x,y -200 nm Z Dorsal layer
2 µm 350 nm
Conventional
STORM dorsal layer
STORM ventral layer
200 nm
Localization precision (SD): xy ~ 4 nm; z ~ 8 nm Resolution (FWHM): xy < 10 nm; z < 20 nm
“Here we demonstrate the concept of STORM using the cyanine switch, but in principle any suitable optically switched fluorophore could be used.” "The STORM concept is also applicable to other photoswitchable fluorophores and fluorescent proteins.”
RecA
Rust, Bates et al, Nat Methods (2006)
Conventional
STORM
Bates et al, Science (2007)
Photo-switchable cyanine dyes
Red light
Cy5
UV light
Red
UV
Bates et al, PRL (2005) Dempsey et al, JACS (2009)
5 µm
Huang et al, Science (2008)
2D STORM
3D STORM
Huang et al, Science (2008)
2D STORM
3D STORM
y x
100 nm
y x
z x
Localization precision (SD): xy ~ 8 nm; z ~ 20 nm Resolution (FWHM): xy < 20 nm; z < 50 nm
Photo-switchable cyanine dyes
Red light
Cy5
UV light
+
Red UV
Dark state
Dempsey et al, JACS (2009)
Photo-switchable cyanine dyes
Red light
Cy5
UV light
+
Red
Dark state
3 µm
Actin
x,y -200 nm Z
2 µm 350 nm
Conventional
STORM dorsal layer
STORM ventral layer
200 nm
Localization precision (SD): xy ~ 4 nm; z ~ 8 nm Resolution (FWHM): xy < 10 nm; z < 20 nm
Huang et al, Science (2008)
EM
STORM
STORM
200 nm
Clathrin FBP17
Dynamin FBP17
Wu et al, Nat Cell Biol (2010)
Huang, Jones et al, Nat. Meth. (2008)
Endoplasmic Reticulum
Dempsey et al, JACS (2009)
Single red laser Commercial Alexa 647 (Cy5 analog) labeled antibody
Conventional
500 nm
STORM
5 µm
Zhuang, Nature Photon. (2009)
STochastic Optical Reconstruction Microscopy
σ ≈ σPSF / N1/2
Rust et al, Nat Methods (2006) -- STORM Betzig et al, Science (2006) -- PALM Hess et al, Biophys. J (2006) -- FPALM
Light microscopy
LM 100 µm
10 µm
1 µm
100 nm
10 nm
Alberts et al, Molecular Biology of the Cell
1 nm
Light microscopy
LM 100 µm
10 µm
1 µm
~5 nm
100 nm
10 nm
Alberts et al, Molecular Biology of the Cell
Resolution of light microscopy
Abbe resolution (diffraction) Limit: A few hundred nanometers To resolve molecules: A few nanometers Discrepancy: Two orders of magnitude
Gelles et al, Nature (1988) Thompson et al, Biophys J (2002) Yildiz, Selvin et al, Science (2003)
Molecular motor (Myosin V)
Yildiz et al, Science (2003)
Single-­‐molecule-­‐localization based approach
Stochastic Optical Reconstruction Microscopy -- STORM (Fluorescence) Photoactivation Localization microscopy --(F)PALM
Dempsey, Vaughan, Chen et al, Nat Methods (2011)
3D STORM
(x, y, z)
In focus
z (nm) 400 200 0 -200
2γ
EMCCD
-400
Huang et al, Science (2008)
z (nm)
800 600 400 200 0
Conventional image STORM image
45 nm
Rust, Bates et al, Nat Methods (2006)
STochastic Optical Reconstruction Microscopy
Conventional image STORM image
45 nm
Ke et al, Nat Methods (in press)
Live-cell 3D STORM
NEB Pit 1 Pit 2
Clathrin Transferrin
Alexa 647clathrin Alexa 647clathrin Alexa 568transferrin
y x
Pit 3
STochastic Optical Reconstruction Microscopy
Conventional image STORM image
45 nm
Rust, Bates et al, Nat Methods (2006)
STochastic Optical Reconstruction Microscopy
Live cell imaging with different probes
Alexa 647
Fluorescent proteins tdEos mEos2
Cell permeable dyes Oregon green TMR Atto655
Jones, Shim et al, Nat Methods (2011)
Super-resolution fluorescence imaging with STORM
Xiaowei Zhuang, Harvard University , HHMI USTC imaging course, 2011
Bio-imaging at various lengths scales
1 nm
10 nm
100 nm1 µmFra bibliotek10 µm
100 µm
1 mm
10 mm 100 mm PET
1m
MRI CT Light Microscopy EM, SPM X-ray crystallography, NMR, EM