Lecture 2: in vitro Single molecule and Super-resolution Microscopy

Long CaiBi/Ch 132

Oct 9th, 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):319-21 2007

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):299-302. 1997

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

Mechanical synthesis of ATP

Yoshida et al, F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits. Nature Structural & Molecular Biology 14, 841 - 846 (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

25oC 2oC

Problematic at long exposure times, but we keep our camera cooled to -70oC.

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 RE, Larson DR, Webb WW.Biophys J. 2002 May;82(5):2775-83.

Myosin walking on actin

Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization.Yildiz A, Forkey JN, McKinney SA, Ha T, Goldman YE, Selvin PR.Science. 2003 Jun 27;300(5628):2061-5. 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 LS, Okten Z, Rock RS, Dawson JF, Spudich JA.Proc Natl Acad Sci U S A. 2005 Feb 1;102(5):1419-23. Epub 2005 Jan 24.

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

Photoswitchable FPs

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.

5’ 3’

STORM with 1 color probes

Cy3-Cy5 GFP mRNA

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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, 87 - 94 (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. 2010 Apr 15;464(7291):1012-7.

Example: Single base stepping of RNA polymerase

Example: single-molecule DNA gyrase

DNA motor proteins

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

Trombone model of DNA replication

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