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

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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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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