STochastic Optical Reconstruction Microscopy
Conventional image STORM image
45 nm
Rust, Bates et al, Nat Methods (2006)
STochastic Optical Reconstruction Microscopy
Hess et al, Biophys. J (2006) -- FPALM
STochastic Optical Reconstruction Microscopy
Conventional image STORM image
45 nm
Rust, Bates et al, Nat Methods (2006)
Stimulated Emission Depletion Microscopy – STED Reversible saturable optical fluorescence transition miBiblioteka roscopy – RESOLFT
Klar& Hell, Opt Lett (1999) Hell, Science (2007)
Abbe resolution (diffraction) Limit: A few hundred nanometers ~Wavelength/2NA
Resolution of light microscopy
Abbe resolution (diffraction) Limit: A few hundred nanometers
Conventional image STORM image
45 nm
RecA
Rust, Bates et al, Nat Methods (2006)
Conventional
STORM
Bates et al, Science (2007)
Photo-switchable cyanine dyes
LM
~5 nm
Alberts et al, Molecular Biology of the Cell
Diffraction-limited resolution
Diffraction-limited resolution
dd == 23456705500 nnmm
Resolution of light microscopy
100 µm 10 µm 1 µm 100 nm 10 nm 1 nm
Light microscopy
LM
Alberts et al, Molecular Biology of the Cell
100 µm 10 µm 1 µm 100 nm 10 nm 1 nm
Light microscopy
Molecular motor (Myosin V)
Yildiz et al, Science (2003)
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
Single-molecule localization
σ ≈ σPSF / N1/2
Gelles et al, Nature (1988) Thompson et al, Biophys J (2002) Yildiz, Selvin et al, Science (2003)
To resolve molecules: A few nanometers
Discrepancy: Two orders of magnitude
Sub-diffraction limit imaging
Patterned illumination based approaches
Stochastic Optical Reconstruction Microscopy -- STORM (Fluorescence) Photoactivation Localization microscopy --(F)PALM
Rust et al, Nat Methods (2006) -- STORM Betzig et al, Science (2006) -- PALM
(Saturated) Structured Illumination Microscopy – (S)SIM
Gustafsson, PNAS (2005)
Sub-diffraction limit imaging
Single-­‐molecule-­‐localization based approach
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 nm 1 µm 10 µm 100 µm 1 mm 10 mm 100 mm 1 m
EM, SPM
MRI CT
PET Light Microscopy
X-ray crystallography, NMR, EM
Light microscopy
Antonie van Leeuwenhoek (1632-1723)
Red light
Cy5
UV light
Red
UV
Bates et al, PRL (2005) Dempsey et al, JACS (2009)
Photo-switchable cyanine dyes
Red light
Cy5
UV light
Red
+
UV
Dark state
Dempsey et al, JACS (2009)