1.shrimp – super high resolution imaging with photobleaching 2a. palm – photoactivated...
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1. SHRIMP – Super High Resolution IMaging with Photobleaching
2a. PALM – Photoactivated Localization Microscopy b. STORM – Stochastic Optical Reconstruction Microscopy
PALM/STORMHow to get super-resolution microscopy.Nanometer-scale instead of micron-scale
FIONA & Turn on/off dye(accuracy and resolution)
For visible microscopy,Resolution is limited to ~250 nm
Ernst Abbe & Lord Rayleigh
Recent microscopy: 1-100 nm,
Ernst Abbe
How fine can you see? The Limits of Microscopy
Here we present techniques which are able to get super-accuracy (1.5 nm) and/or super-resolution (<10 – 25 nm)
Super-Accuracy: Nanometer Distances w Single Molecules
Center can be found much more accurately than width
W.E. Moerner, Crater Lake
Fluorescence Imaging with One Nanometer Accuracy
1.5 nm accuracy 1-500 msec
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Prism-type TIR 0.2 sec integration
Z-Data from Columns 1-21
center
Width(250 nm)
Dxcenter = width /√N ≈ 250/√10k = 1.3 nm
Yildiz et al, Science, 2003
Resolved!
Super-Resolution: PALM/STORM. between (activatable) molecules
Betzig, Zhuang
Good for dynamics
SHRImP Super High Resolution IMaging with Photobleaching
In vitro
Super-Resolution: Nanometer Distances between two (or more) dyes
Permanent Photobleaching
SHRImP Super High Resolution IMaging with Photobleaching
In vitro
Super-Resolution: Nanometer Distances between two (or more) dyes
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132.9 ± 0.93 nm
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8.7 ± 1.4 nmDistance can be found much more accurately than width (250 nm)
Resolution now:Between 2-5 molecules: <10 nm
(Gordon et al.; Qu et al, PNAS, 2004)
Have shown that you can get ~5-15 dyes get ~ 20-100 nm
STORM & PALMMost Super-Resolution Microscopy
Inherently a single-molecule technique
Huang, Annu. Rev. Biochem, 2009
Zhuang, 2007 Science
STochastic Optical Reconstruction Microscopy
PhotoActivation Localization Microscopy
Betzig, 2006 Science
2-colorsecondary antibodies
Cy2-Alexa 647
Cy3-Alexa 647
Comparison between regular- and super-microscopy
Pre-synaptic Bouton
Post-synaptic Spine
PSD
Valtschanoff and Weinberg, 2003
Synapse (30 nm)
Zhuang, Neuron, 2010
Regular mscopy STORM mscopy
PALM (STORM)- Photo-activated localization super-resolution microscopy
10 - 20 nm resolution (localization precision)
The PALM cycleBetzig et al. Science 2006
You have PALM spelled out with really tiny molecules separated by a tiny distance—such that each letter is less than a diffraction limit apart.How to see what is written? First you try regular fluorescence, labeling it with some fluorescent dye and shine light to make it fluoresce. What do you see? Each dye emits with a diffraction-limited (i.e., about 250 nm) size. The result is B. It’s not well resolved.However, if you can make each fluorescent molecule emit one at a time, then you can determine where the dye is by doing FIONA—taking the SEM (instead of the Standard Deviation), where you can determine it’s position to within a few nanometers. Then you repeat this measurements many many times, until you get the entire image. See next page.
~ 250 nmA.
B.
PALM (STORM)- Photo-activated localization super-resolution microscopy
10 - 20 nm resolution (localization precision)
The PALM cycleBetzig et al. Science 2006
After many cycles
Read out with visible light
Weak near-UV light
Activate with weak near UV-light; Once activated, shine visible light to get out fluorescence. Locate each fluorphore to within a few nanometers by taking the center of the emission (rather than the diffraction-limited width). Record the position of these molecules, Then repeat, until you get all of the position of all of the fluorophores.
“Regular” dyes can be made to blink They are off; then can be made to come on.
(Cy3B, Cy5, Alexa 647…)
You have measured kinesin movingYou will measure the width of microtubules.
24 nm
The End