HW on rough-draft of your 5 minute oral presentation due today at 5pm.

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

2. Today’s Announcements Individual Projects– 5 minute lecture! April 17th and 19th If you canNOT make it on April 19th send me an email with subject title: I must have talk on April 17th. 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.]

3. Grading • Did you follow outline? • 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. How fine can you see? The Limits of Microscopy For visible microscopy, Resolution is limited to ~250 nm Ernst Abbe & Lord Rayleigh Ernst Abbe Recent microscopy: 1-100 nm, Here we present techniques which are able to get super-accuracy (1.5 nm) and/or super-resolution (<10 nm, 35 nm)

5. FIONA Fluorescence Imaging with One Nanometer Accuracy Super-Accuracy (Accuracy << 250 nm: 1.5 nm, 1-500 ms) center width Center can be found much more accurately than width S/N ≈ width /√N ≈ 250/√104 ≈ 1.3 nm When light gets dim, what happens to your ability to find the center?

6. center width Why can such a small step be measured? Diffraction limited spot: Single Molecule Sensitivity Accuracy of Center = width/ S-N = 250 nm / √104= 2.5 nm = ± 1.25nm Width of l/2 ≈250 nm 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) Start of high-accuracy single molecule microscopy Thompson, BJ, 2002; Yildiz, Science, 2003

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

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 D K Actin, mtubules ATP-binding heads Nature Reviews ATP  mechanical work Cargo binding Kinesin Myosin Dynein  Motor Microtubule actinMicrotubule polymer

9. Streptavidin Quantum Dot Streptavidin conjugate Biotinylated Anti-Pentahis antibody Six-histidine tag Leucine zippered CENP-E dimer w/ six histidine-tag Axoneme or microtubule + - 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. Kinesin: Hand-over-hand or Inchworm? 8.3 nm 8.3 8.3 nm qs655 16.6 nm 16.6 nm 8.3 nm, 8.3 nm 16 nm 0 nm 16.6, 0, 16.6 nm, 0… [ATP] = 5 mM ; 4 msec exposure time (Originally 0.3 mM ; 500 msec exp. time) [ATP] (16.6x higher), 125x faster acq. pixel size is 160nm 2 x real time

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

13. Dynein Kinesin We have great x-y accuracy in vitro with fluorescent dyes and quantum dots… Can we get this accuracy in vivo? Yes…in Drosophilia cells, individual kinesin & dynein moving cooperatively(Kural, Science, 2005) dr = 1.5 nm dt = 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) TIR- (q > qc) Exponential decay Total Internal Reflection (TIR) Microscopy dp=(l/4p)[n12sin2i) - n22]-1/2 For glass (n=1.5), water (n=1.33): TIR angle = >57° Penetration depth = dp = 58 nm With dp = 58 nm , can excite sample and not much background.

15. 132.9 ± 0.93 nm 72.1 ± 3.5 nm 8.7 ± 1.4 nm Super-Resolution: Nanometer Distances between two (or more) dyes SHRImP Super High Resolution IMaging with Photobleaching 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) In vitro

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

17. 1 mm 1 mm 1 mm PhotoActivation Localization Microscopy (F)PALM (Photoactivatable GFP) TIRF PALM TEM Mitochondrial targeting sequence tagged with mEOS 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.