1. 1.SHRIMP – Super High Resolution IMaging with Photobleaching 2a. PALM – Photoactivated Localization Microscopy b. STORM – Stochastic Optical Reconstruction Microscopy PALM/STORM How to get super-resolution microscopy. Nanometer-scale instead of micron-scale FIONA & Turn on/off dye (accuracy and resolution)

2. 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)

3. 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 center Width (250 nm)  x center = 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

4. SHRImP Super High Resolution IMaging with Photobleaching In vitro Super-Resolution: Nanometer Distances between two (or more) dyes Permanent Photobleaching

5. SHRImP Super High Resolution IMaging with Photobleaching In vitro Super-Resolution: Nanometer Distances between two (or more) dyes 132.9 ± 0.93 nm 72.1 ± 3.5 nm 8.7 ± 1.4 nm Distance 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

6. STORM & PALM Most 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-color secondary antibodies Cy2-Alexa 647 Cy3-Alexa 647

7. Comparison between regular- and super-microscopy Pre-synaptic Bouton Post-synaptic Spine PSD Valtschanoff and Weinberg, 2003 Synapse (30 nm) Zhuang, Neuron, 2010 Regular  scopy STORM  scopy

8. PALM (STORM)- Photo-activated localization super-resolution microscopy 10 - 20 nm resolution (localization precision) The PALM cycle Betzig 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 nm A. B.

9. PALM (STORM)- Photo-activated localization super-resolution microscopy 10 - 20 nm resolution (localization precision) The PALM cycle Betzig 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.

10. “Regular” dyes can be made to blink They are off; then can be made to come on. (Cy3B, Cy5, Alexa 647…)

11. You have measured kinesin moving You will measure the width of microtubules. 24 nm

12. The End