1. Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012

2. Single Molecule detection of GFP R. M. Dickson, A. B Cubitt, R. Y. Tsien, and W. E. Moerner, "On/Off Blinking and Switching Behavior of Single Green Fluorescent Proteins," Nature, 388, 355 (1997).

3. Light sources

4. SM counting and stochiometry Isacoff EY, Subunit counting in membrane-bound proteins. Nat Method 4(4):319-21 2007

5. measuring binding kinetics M.P. Elenko, J.W. Szostak and A.M. van Oijen, J Am Chem Soc, 131 (2009), pp. 9866–9867.

6. Fluorescence Correlation Spectroscopy (FCS) Variants: image FCS, FRET-FCS, TIRF-FCS

7. F1-ATPase rotation Kinoshita, Direct observation of the rotation of F1-ATPase. Nature 386(6622):299-302. 1997

8. 52ATP/s -> 17 rps. ATP = 80pN nm, friction load is estimated at 80pN nm. Kinoshita, Direct observation of the rotation of F1-ATPase. Nature 386(6622):299-302. 1997

9. Mechanical synthesis of ATP

10. Yoshida et al, F 1 -ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits. Nature Structural & Molecular Biology 14, 841 - 846 (2007)

11. Molecular tracking Yildiz, Selvin Science 2003

12. Cameras Front-illuminated Back-illuminated Compare to 2% in photographic film

13. sources of noise in a camera Dark current 25 o C 2 o C Problematic at long exposure times, but we keep our camera cooled to -70 o C.

14. sources of noise in a camera Readout noise

15. EMCCD

16. Cmos cameras Parallel and fast readout (no need for amplification) Smaller pixel size (1-2um) Not quite as sensitive yet (~60% QE) iMicroscopes

17. Pixel size

18. lens-less microscopy

19. iPhone flow-cytometer A. Ozcan, S.O. Isikman, O. Mudanyali, D. Tseng, I. Sencan, “Lensfree on- chip holography facilitates novel microscopy applications” SPIE Newsroom, (2010)

20. Sub-wavelength localization

21. Sub-diffraction localization Precise nanometer localization analysis for individual fluorescent probes. Thompson REThompson RE, Larson DR, Webb WW. Biophys J. 2002 May;82(5):2775-83.Larson DRWebb WW Biophys J.

22. Myosin walking on actin

23. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Yildiz AYildiz A, Forkey JN, McKinney SA, Ha T, Goldman YE, Selvin PR.Forkey JNMcKinney SAHa TGoldman YESelvin PR Science.Science. 2003 Jun 27;300(5628):2061-5. Epub 2003 Jun 5.

25. Molecular tracking Cy3

26. Yildiz, Selvin Science 2004

27. single-molecule high-resolution colocalization (SHREC) Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Churchman LSChurchman LS, Okten Z, Rock RS, Dawson JF, Spudich JA.Okten ZRock RSDawson JF Spudich JA Proc Natl Acad Sci U S A.Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1419-23. Epub 2005 Jan 24.

28. A base-excision DNA-repair protein finds intrahelical lesion bases by fast sliding in contact with DNA. Blainey PCBlainey PC, van Oijen AM, Banerjee A, Verdine GL, Xie XS.van Oijen AMBanerjee AVerdine GLXie XS Proc Natl Acad Sci U S A.Proc Natl Acad Sci U S A. 2006 Apr 11;103(15):5752-7. Epub 2006 Apr 3.

29. Photoswitchable FPs

30. PALM and STORM Betzig E, et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science. 2006 Sep 15;313(5793):1642-5. Rust MJ, Bates M, Zhuang X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods. 2006 Oct;3(10):793-5. Hess ST, Girirajan TP, Mason MD. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J. 2006 Dec 1;91(11):4258-72.

31. 5’ 3’ STORM with 1 color probes Cy3-Cy5 GFP mRNA GFP mRNA

32. Super-resolution Reconstruction

33. Multicolor STORM Bates, Zhuang Science 2007

35. Breaking the diffraction barrier Super-resolution microscopy (~10 nm resolution)

36. STED microscopy

37. Saturated structured illumination microscopy (SSIM) Nonlinear structured-illumination microscopy: Wide- field fluorescence imaging with theoretically unlimited resolution. Mats Gustafsson, PNAS 2005

38. Z. Liu et al., Science 315, 5818 (March 23, 2007).

39. Single molecule FRET on Ribozymes Zhuang, Chu, Science 2002

41. Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein. Nature 2007

42. Single molecule enzymology BP English et al. Nature Chemical Biology 2, 87 - 94 (2006)

43. Memory effects in enzymes

44. Enzyme turn-over time distribution is multi-exponential.

45. Sanger Sequencing

46. Illumina Sequencing

47. Pyro-sequencing

48. Problem: how to increase speed?

49. Pac biosciences sequencer Real-Time DNA Sequencing from Single Polymerase Molecules, Science 2008

51. Real-time tRNA transit on single translating ribosomes at codon resolution Uemura S, Aitken CE, Korlach J, Flusberg BA, Turner SW, Puglisi JD. Real-time tRNA transit on single translating ribosomes at codon resolution. Nature. 2010 Apr 15;464(7291):1012-7.

53. Example: Single base stepping of RNA polymerase

54. Example: single-molecule DNA gyrase

55. DNA motor proteins Van Oijen, AM Science 2003 Single-Molecule Kinetics of λ Exonuclease Reveal Base Dependence and Dynamic Disorder

56. Trombone model of DNA replication Van Oijen, AM Nature 2006 DNA primase acts as a molecular brake in DNA replication