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

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

Long CaiBi/Ch 132

Oct 9th, 2012

Page 2: 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).

Page 3: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Light sources

Page 4: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

SM counting and stochiometry

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

Page 5: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

measuring binding kinetics

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

Page 6: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Fluorescence Correlation Spectroscopy (FCS)

Variants: image FCS, FRET-FCS, TIRF-FCS

Page 7: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

F1-ATPase rotation

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

Page 8: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

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

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

Mechanical synthesis of ATP

Page 10: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Yoshida et al, F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits. Nature Structural & Molecular Biology 14, 841 - 846 (2007)

Page 11: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Molecular tracking

Yildiz, Selvin Science 2003

Page 12: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Cameras

Front-illuminated

Back-illuminated

Compare to 2% in photographic film

Page 13: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

sources of noise in a camera• Dark current

25oC 2oC

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

Page 14: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

sources of noise in a camera• Readout noise

Page 15: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

EMCCD

Page 16: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Cmos cameras

• Parallel and fast readout (no need for amplification)

• Smaller pixel size (1-2um)• Not quite as sensitive yet (~60% QE)• iMicroscopes

Page 17: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Pixel size

Page 18: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

lens-less microscopy

Page 19: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

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)

Page 20: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Sub-wavelength localization

Page 22: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Myosin walking on actin

Page 24: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.
Page 25: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Molecular tracking

Cy3

Page 26: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Yildiz, Selvin Science 2004

Page 29: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Photoswitchable FPs

Page 30: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

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.

Page 31: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

5’ 3’

STORM with 1 color probes

Cy3-Cy5 GFP mRNA

Page 32: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

150 100 50 50 100 150

150

100

50

50

100

150

Super-resolution Reconstruction

Page 33: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Multicolor STORM

Bates, Zhuang Science 2007

Page 34: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.
Page 35: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Breaking the diffraction barrier

Super-resolution microscopy (~10 nm resolution)

Page 36: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

STED microscopy

Page 37: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Saturated structured illumination microscopy (SSIM)

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

Page 38: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

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

Page 39: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Single molecule FRET on Ribozymes

Zhuang, Chu, Science 2002

Page 40: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.
Page 41: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

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

Page 42: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Single molecule enzymology

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

Page 43: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Memory effects in enzymes

Page 44: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Enzyme turn-over time distribution is multi-exponential.

Page 45: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Sanger Sequencing

Page 46: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Illumina Sequencing

Page 47: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Pyro-sequencing

Page 48: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Problem: how to increase speed?

Page 49: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Pac biosciences sequencer

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

Page 50: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.
Page 51: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

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.

Page 52: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.
Page 53: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Example: Single base stepping of RNA polymerase

Page 54: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Example: single-molecule DNA gyrase

Page 55: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

DNA motor proteins

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

Page 56: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

Trombone model of DNA replication

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

Page 57: Lecture 2: in vitro Single molecule and Super-resolution Microscopy Long Cai Bi/Ch 132 Oct 9 th, 2012.

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