stochastic optical reconstruction microscopy (storm)
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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.