Sub-diffraction-limitimaging by stochasticoptical reconstructionmicroscopy (STORM)

Michael J Rust, Mark Bates & Xiaowei Zhuang, Nature 2006

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Theoretical Resolution Limit for Optical Microscopy = 0.61λ~200nm

Optical Microscopy is Resolution-Limited by the Wavelength of Light

20.309 Lecture Notes: 10

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Theoretical Resolution Limit for Optical Microscopy = 0.61λ~200nm

However, it is still possible to measure position of a point to much greater accuracy by finding the centroid

Optical Microscopy is Resolution-Limited by the Wavelength of Light

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Readout

vv

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Readout

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Centroid of Point Spread Function

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Readout

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Readout

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Readout

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~20nm

Resolution is Now Limitedby Shot Noise (i.e. by number of photons), not by Wavelength of Light

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STORM uses a cyanine switch

Cy5

Cy3

• Red light causes fluorescence followed by a stable dark state

• Green light causes a return to fluorescent state

• Recovery rate depends on proximity to Cy3

• Can be used hundreds of times before photobleaching occurs

• Approx 3000 photons/s -theoretical position accuracy 4nm (8nm measured)

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Imaging a single switch attached to DNA

Cy5

Cy3

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Image processing reveals a resolution limit of 18nm.

Before Drift Correction After Drift Correction

FWHM = 18nmFriday, December 3, 2010

STORM visualizes switches on a plasmid.

46nm

Inter-switch distance (nm)

Frac

tion

of M

olec

ules

46nm

- - - Theoretical distance Measured Distance using STORM

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Storm Imaging Compared to TIRM

Total internal reflection

microscope

STORM

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Whole-cell 3D STORM reveals interactions between cellular structures

with nanometer-scale resolutionBo Huang, Sara A Jones, Boerries Brandenburg &

Xiaowei Zhuang (2008)

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Possible Future Directions• Imaging speed can be improved by increasing

switching rate

• High-resolution fluorescence in situ hybridization and immunofluorescence imaging

• Potentially allows for high-resolution, live-cell imaging

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Questions?

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Acknowledgements

Michael J Rust Harvard University University Mark Bates Harvard University

Xiaowei Zhuang Harvard University, Howard Hughes Medical Institute

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Switching of Cy5-Cy3-labeled antibody.

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