1. 1.HW on rough-draft of your 5 minute oral presentation due today at 5pm. 2.Another HW (regular, written assignment due Wednesday in class). “The students simply have to practice a lot and make things very clear and concise.” Today’s Announcements

2. Individual Projects– 5 minute lecture! April 17 th and 19 th If you canNOT make it on April 19 th send me an email with subject title: I must have talk on April 17 th. 1. What is title of paper and talk. 2. Introduction (What is main general subject.) [How things move inside of a cell.] 3. Detail subject : [Molecular motors, in particular,how myosin V moves; Walks or inchworm.] 4. What is major question/problem. [Measure steps that are below the diffraction limit.] 5. What did they do to solve the problem…new techniques? [FIONA] 6. Solution [MyoV walks, hand-over-hand!] 7. One sentence summary of what the audience should take home/ remember. [Technique made that is very general, FIONA, for measuring step mechanism; showed that MyoV walks.] Today’s Announcements

3. Grading 1.Did you follow outline? 2.Were you clear? a. Not too complex, but not too simple. b. How was your eye contact? c. Was your speaking clear? 3. Did audience get a one sentence, main message?

4. 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 nm, 35 nm)

5. Super-Accuracy (Accuracy << 250 nm: 1.5 nm, 1-500 ms) Center can be found much more accurately than width S/N ≈ width /√N ≈ 250/√10 4 ≈ 1.3 nm center width When light gets dim, what happens to your ability to find the center? Fluorescence Imaging with One Nanometer Accuracy

6. Diffraction limited spot: Single Molecule Sensitivity center width Enough photons (signal to noise)…Center determined to ~1.3 nm Dye lasts 5-10x longer -- typically ~30 sec- 1 min. (up to 4 min) Accuracy of Center = width/ S-N = 250 nm / √10 4 = 2.5 nm = ± 1.25nm Thompson, BJ, 2002; Yildiz, Science, 2003 Start of high-accuracy single molecule microscopy Width of /2 ≈ 250 nm Why can such a small step be measured?

7. How well can you localize? Depend on 3 things 1. # of Photons Detected (N) 3. Noise (Background) of Detector (b) (includes background fluorescence and detector noise) 2. Pixel size of Detector (a) = derived by Thompson et al. (Biophys. J.). center width

8. Biomolecular Motors: Intra- & Extra-Cellular Motion Characteristics nm scale Move along tracks intracellular directional movement cell shape changes & extracellular movement Use ATP as energy source Actin,  tubules ATP  mechanical work Nature Reviews Microtubule actin Microtubule  polymer Kinesin Myosin Dynein  Motor ATP-binding heads Cargo binding K D

9. Motility of quantum-dot labeled Kinesin (CENP-E) 8.3 nm/step from optical trap

10. Kinesin (Center-of-Mass) Moving Kinesin moves with 8.4 nm /ATP step size. (8.3 nm/step shown by Optical Traps ≈ 1993)

11. 16 nm qs655 pixel size is 160nm 2 x real time 8.3 nm, 8.3 nm 8.3 nm 16.6 nm 16.6, 0, 16.6 nm, 0… 0 nm 16.6 nm 8.38.3 nm Kinesin: Hand-over-hand or Inchworm? [ATP] (16.6x higher), 125x faster acq. [ATP] = 5  M ; 4 msec exposure time (Originally 0.3  M ; 500 msec exp. time)

12. Kinesin = 16.3 nm y ~ texp(-kt) Takes 16 nm hand-over-hand steps 16 nm 0 nm 16 nm Can you derive this?

13. Dynein Kinesin Yes…in Drosophilia cells, individual kinesin & dynein moving cooperatively (Kural, Science, 2005) We have great x-y accuracy in vitro with fluorescent dyes and quantum dots… Can we get this accuracy in vivo?  r = 1.5 nm  t = 1.1 msec

14. Imaging (Single Molecules) with very good S/N (at the cost of seeing only a thin section very near the surface) Total Internal Reflection (TIR) Microscopy For glass (n=1.5), water (n=1.33): TIR angle = >57° Penetration depth = d p = 58 nm d p =( /4  )[n 1 2 sin 2  i ) - n 2 2 ] -1/2 With d p = 58 nm, can excite sample and not much background. TIR- (  >  c ) Exponential decay

15. 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) Next slides gSHRIMP: > 5-40 molecules ~ 20-100 nm Via 2-photon: ~ 35 nm (next time)

16. Most Super-Resolution Microscopy Inherently a single-molecule technique Huang, Annu. Rev. Biochem, 2009 Bates, 2007 Science STORM STochastic Optical Reconstruction Microscopy PALM PhotoActivation Localization Microscopy (Photoactivatable GFP)

17. PhotoActivation Localization Microscopy (F)PALM (Photoactivatable GFP) 1  m PALMTEM Mitochondrial targeting sequence tagged with mEOS TIRF Patterson et al., Science 2002

18. 3-D (z) resolution

19. 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.