Stochastic Optical Reconstruction Microscopy STORM Finding Out the

Stochastic Optical Reconstruction Microscopy (STORM)

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

Many Molecules

The principle of STORM

The principle of STORM

The principle of STORM

The principle of STORM

The principle of STORM

The principle of STORM

The principle of STORM

The principle of STORM

The principle of STORM

The principle of STORM

The principle of STORM

The principle of STORM

A Photo-switchable Probe Imaging laser (657 nm) Activator Cy 3 Reporter Cy 5 Activation laser pulses De Cy 5 fluorescence tiv ac Activation Cy 3 Cy 5 Cy 3 ion at 6000 photons Cy 5 Activation laser (532 nm) 0 5 10 Time (s) 15 20

Fluorescence Cy 3 550 0 5 10 15 20 time (s) Cy 2 490 Alexa 405 400 Activation pulses Fluorescence 532 457 405 nm 665 690 775 Cy 5. 5 Cy 7 Alexa 647 Reporter emission (nm) Bates et al, Science 317, 1749 – 1753 (2007) 0 10 time (s) 20 30 Activation pulses Fluorescence Activator absorption (nm) More Colors Activation pulses

5 μm B-SC-1 cell, Microtubules stained with anti-β tubulin Cy 3 / Alexa 647 secondary antibody

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

500 nm 5 μm

█ Cy 3 / Alexa 647: Clathrin █ Cy 2 / Alexa 647: Microtubule 5 μm Bates et al, Science 317, 1749 – 1753 (2007)

1 μm

Avg = 172 nm 200 nm

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.