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

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).

Light sources

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

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

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

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

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):

Mechanical synthesis of ATP

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

Molecular tracking Yildiz, Selvin Science 2003

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

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.

sources of noise in a camera Readout noise

EMCCD

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

Pixel size

lens-less microscopy

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)

Sub-wavelength localization

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

Myosin walking on actin

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 Jun 27;300(5628): Epub 2003 Jun 5.

Molecular tracking Cy3

Yildiz, Selvin Science 2004

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 Feb 1;102(5): Epub 2005 Jan 24.

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 Apr 11;103(15): Epub 2006 Apr 3.

Photoswitchable FPs

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

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

Super-resolution Reconstruction

Multicolor STORM Bates, Zhuang Science 2007

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

STED microscopy

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

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

Single molecule FRET on Ribozymes Zhuang, Chu, Science 2002

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

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

Memory effects in enzymes

Enzyme turn-over time distribution is multi-exponential.

Sanger Sequencing

Illumina Sequencing

Pyro-sequencing

Problem: how to increase speed?

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

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 Apr 15;464(7291):

Example: Single base stepping of RNA polymerase

Example: single-molecule DNA gyrase

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

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