Sub-diffraction-limit imaging by Stochastic Optical Reconstruction
Microscopy (STORM)
Michael J. Rust, Mark Bates, Xiaowei ZhuangHarvard University
Published Online August 9, 2006Nature Methods Vol.3 No.10
Presented by Artie Wu
STORM
• High-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores
• Imaging resolution of 20nm
Outline
• Background– fluorescence microscopy– diffraction
• Motivation• Fluorescence Microscopy Alternatives• STORM• Results• Conclusions
Motivation I
• Resolution limit set by diffraction of light
• Fluorescence microscopy widely used in molecular and cell biology
Fluorescence Microscopy Alternatives
• Lateral resolution of 10s of nanometers– Near-field scanning optical microscopy (NSOM)– Multiphoton fluorscence– Stimulated emission depletion (STED)– Saturated structured-illumination microscopy (SSIM)
Near-field scanning optical microscopy (NSOM)
• Image at interface due to evanescent field
• Study what goes on near membrane– Exocytosis & endocytosis
• Build up point by point• Drawback: low imaging
depth
Motivation II
• Single-molecule detection leads to sub-diffraction-limit spatial resolution
• Stochastic optical reconstruction microscopy (STORM)– Fluorescence image constructed from high-accuracy
localization of individual fluorescent molecules– Imaging resolution: ~20nm using TIRF and
photoswitchable cyanine dye, Cy5
STORM
•Cy5: fluorescent and dark state using different λ•Cy3: secondary dye•Series of imaging cycle•In each cycle
•Only 1-3 switches in FOV are switched ON•Stochastically different subset of fluorophores are ON
•Red: 633nm, 30W/cm2, 2s•Green: 532nm, 1W/cm2, 0.5s•Photobleaching: 230s
Resolution
• Limited by accuracy of localization of switches
• 2d Gaussian fit to PSF used to find centroid position of switch
Centroid position
• Fit to pixelated Gaussian function
A: background fluorescence levelIo: amplitude of peak
a,b: widths of Gaussian distributionxo,yo: center coordinates of peak
δ: fixed half-width of pixel in object plane
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Results I
• Linear, dsDNA with 2 switches separated by 135 bps (46nm)
• Theoretical dist = 40nm• Experimental dist = 41nm
Results II
• Longer DNA with 4 switches spaced 46 nm apart
• Localize large number of switches within diffraction-limited spot by cycling switches on/off
Conclusions
• STORM capable of imaging biological structures with sub-diffraction-limit resolution
• Resolution limited by # photons emitted per switch cycle– Cyanine switch ~3000 photons/cycle
• Theoretical localization accuracy of 4nm• Corresponds to imaging resolution of ~20nm
– Imaging speed improved by increasing switching rate• Stronger excitation or fluorophores with faster switching kinetics
• Valuable tool for high-resolution in situ hybridization and immunofluorescence imaging
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