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Presented by Artie Wu

Sub-diffraction-limit imaging by Stochastic Optical Reconstruction Microscopy (STORM) Michael J. Rust, Mark Bates, Xiaowei Zhuang Harvard University Published Online August 9, 2006 Nature Methods Vol.3 No.10. Presented by Artie Wu. STORM.

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Presented by Artie Wu

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  1. Sub-diffraction-limit imaging by Stochastic Optical Reconstruction Microscopy (STORM)Michael J. Rust, Mark Bates, Xiaowei ZhuangHarvard UniversityPublished Online August 9, 2006Nature Methods Vol.3 No.10 Presented by Artie Wu

  2. STORM • High-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores • Imaging resolution of 20nm

  3. Outline • Background • fluorescence microscopy • diffraction • Motivation • Fluorescence Microscopy Alternatives • STORM • Results • Conclusions

  4. Background

  5. Background

  6. Motivation I • Resolution limit set by diffraction of light • Fluorescence microscopy widely used in molecular and cell biology

  7. 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)

  8. 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

  9. 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

  10. 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

  11. Resolution • Limited by accuracy of localization of switches • 2d Gaussian fit to PSF used to find centroid position of switch

  12. Centroid position • Fit to pixelated Gaussian function A: background fluorescence level Io: amplitude of peak a,b: widths of Gaussian distribution xo,yo: center coordinates of peak δ: fixed half-width of pixel in object plane

  13. Results I • Linear, dsDNA with 2 switches separated by 135 bps (46nm) • Theoretical dist = 40nm • Experimental dist = 41nm

  14. 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

  15. 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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