1. Stochastic Optical Reconstruction Microscopy (STORM)

2. Finding Out the Position of a Molecule σ  σ PSF / N 1/2 Yildiz et al., Science, 2003 Gordon et al., PNAS, 2004Lagerholm et al., Biophys. J., 2006 FIONA Localization by sequential photobleaching Thompson at al., Biophys. J., 2002 Qu et al., PNAS, 2004 Lydke et al., Optics Exp., 2005 or QD blinking A few molecules

3. Many Molecules

4. The principle of STORM

16. A Photo-switchable Probe Activation Deactivation Imaging laser (657 nm) Activation laser (532 nm) Cy3Cy5 Cy3Cy5 Cy3Cy5 ActivatorReporter 6000 photons

17. More Colors Activator absorption (nm) Reporter emission (nm) Alexa 405 Cy2 Cy3 Cy5 Cy5.5 Cy7 665690 775 550 490 400 Alexa 647 20151050 time(s) 3020100 time(s) Activation pulses Fluorescence 405 nm532457 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 Bates et al, Science 317, 1749 – 1753 (2007)

22. 1 μm

23. 200 nm Avg = 172 nm

24. Class evaluation 1.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.