1. Review of FIONA/PALM/STORM How to make photoactivatable fluorophores How to get 3-D Measurements Intro to STED (?)

2. 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

3. Normal- vs. Super- resolution ~ 250 nm Let’s say you have a single fluorophore (a few nanometers in size, much less than the diffraction limit) Does it matter if your excitation light is focused to a (diffraction-limited spot) or you are exciting with wide-field? In a microscope, what does its emission look like?? No, either one produces a diffraction-limited spot.

4. 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? ~ 250 nm A. B. 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.

5. 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.

6. Fig. 1. The principle behind PALM. A sparse subset of PA-FP molecules that are attached to proteins of interest and then fixed within a cell are activated (A and B) with a brief laser pulse at λ act = 405 mm and then imaged at λ exc = 561 mm until most are bleached (C). This process is repeated many times (C and D) until the population of inactivated, unbleached molecules is depleted. Summing the molecular images across all frames results in a diffraction- limited image (E and F). E Betzig et al. Science 2006;313:1642-1645 Published by AAAS PALM Use photoswitchable GFP Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to ~2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a super-resolution image.

7. PhotoActivation Localization Microscopy (F)PALM (Photoactivatable GFP) Mitochondrial targeting sequence tagged with mEOS (an photoactivatable Fluorescent Protein) 1  m PALM TEM (Transmission Electron Microscopy) TIRF (reg. diff’n limit) Patterson et al., Science 2002

8. Jennifer Lippincott-Schwartz, and George H. Patterson Science 2003;300:87-91 Published by AAAS The Wild Type Green Fluorescent Protein (wtGFP) consisting of a cyclized tripeptide made of Ser 65, Tyr 66, and Gly 67.

9. Absorbance spectra of purified wtGFP before (A) and after (B) photoactivation with ∼ 400-nm light. Jennifer Lippincott-Schwartz, and George H. Patterson Science 2003;300:87-91 Published by AAAS Before activation After activation A cell expressing PA-GFP was imaged with the use of 488-nm excitation (Pre) before and after ∼ 1-s photoactivation of the nuclear pool with 413-nm laser light. Photoactivatable GFP Thr 203 → His 203

10. 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

11. 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

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

13. 3-D (z) resolution

14. Fig. 2. Three-dimensional STORM imaging of microtubules in a cell. B Huang et al. Science 2008;319:810-813 Published by AAAS Conventional indirect immunofluorescence image of microtubules 3D section (color coded) C-E zoom in of box in B (Notice box)

15. 3D Movie B Huang et al. Science 2008;319:810-813

16. The End