Stochastic Optical

Reconstruction Microscopy

(STORM)

Finding Out the Position of a Molecule

σ σPSF / N1/2

Yildiz et al., Science, 2003

Gordon et al., PNAS, 2004

Lagerholm 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 blinkingA few molecules

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

ActivationDeactivation

Imaging laser (657 nm)

Activation laser (532 nm)

Cy3 Cy5

Cy3 Cy5Cy3 Cy5

20151050

Activation laser pulses

Cy5 fluorescence

Time (s)

Activator Reporter

6000 photons

More ColorsA

ctiv

ato

r ab

sorp

tion (

nm

)

Reporter emission (nm)

Alexa 405

Cy2

Cy3

Cy5 Cy5.5 Cy7

665 690 775

550

490

400

Alexa 647

20151050

time (s)

3020100time (s)

Activation pulses

Activation pulses

Activation pulses

Flu

ore

scen

ceFl

uore

scen

ceFl

uore

scen

ce

405 nm532 457

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

5 μm

B-SC-1 cell, Microtubules stained with anti-β tubulinCy3 / Alexa 647 secondary antibody

5 μm

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

500 nm

5 μm

5 μm

█ Cy3 / Alexa 647: Clathrin

█ Cy2 / Alexa 647: Microtubule

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

1 μm

200 nm

Avg = 172 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.