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Stochastic Optical Reconstruction Microscopy (STORM)

Stochastic Optical Reconstruction Microscopy (STORM). Finding Out the Position of a Molecule. FIONA. σ  σ PSF / N 1/2. Thompson at al., Biophys. J. , 2002. Yildiz et al., Science , 2003. Localization by sequential photobleaching. or QD blinking. A few molecules.

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Stochastic Optical Reconstruction Microscopy (STORM)

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  1. Stochastic Optical Reconstruction Microscopy (STORM)

  2. Finding Out the Position of a Molecule FIONA σ σPSF / N1/2 Thompson at al., Biophys. J., 2002 Yildiz et al., Science, 2003 Localization by sequential photobleaching or QD blinking A few molecules Gordon et al., PNAS, 2004 Qu et al., PNAS, 2004 Lagerholm et al., Biophys. J., 2006 Lydke et al., Optics Exp., 2005

  3. Many Molecules

  4. The principle of STORM

  5. The principle of STORM

  6. The principle of STORM

  7. The principle of STORM

  8. The principle of STORM

  9. The principle of STORM

  10. The principle of STORM

  11. The principle of STORM

  12. The principle of STORM

  13. The principle of STORM

  14. The principle of STORM

  15. The principle of STORM

  16. A Photo-switchable Probe Cy3 Cy5 Activation laser pulses Cy3 Cy5 Cy3 Cy5 Cy5 fluorescence 0 5 1 0 1 5 2 0 Time (s) Imaging laser (657 nm) Activator Reporter Deactivation 6000 photons Activation Activation laser (532 nm)

  17. More Colors 0 5 1 0 1 5 2 0 t i m e ( s ) 0 1 0 2 0 3 0 t i m e ( s ) Activation pulses Fluorescence 550 Cy3 532 457 405 nm Activation pulses 490 Cy2 Activator absorption (nm) Fluorescence 400 Alexa 405 775 665 690 Cy5 Cy5.5 Cy7 Activation pulses Alexa 647 Fluorescence Reporter emission (nm) Bates et al, Science 317, 1749 – 1753 (2007)

  18. 5 μm B-SC-1 cell, Microtubules stained with anti-β tubulin Cy3 / Alexa 647 secondary antibody

  19. 5 μm Bates et al, Science 317, 1749 – 1753 (2007)

  20. 500 nm 5 μm

  21. █Cy3 / Alexa 647: Clathrin █Cy2 / Alexa 647: Microtubule 5 μm Bates et al, Science 317, 1749 – 1753 (2007)

  22. 1 μm

  23. Avg = 172 nm 200 nm

  24. Class evaluation • What was the most interesting thing you learned in class today? • 2. What are you confused about? • 3. Related to today’s subject, what would you like to know more about? • 4. Any helpful comments. Answer, and turn in at the end of class.

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