Lecture 2: in vitro Single molecule and
Super-resolution Microscopy
Long Cai
Bi/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
Compare to 2% in photographic film
Back-illuminated
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 onchip 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.
STORM with 1 color probes
GFP mRNA
Cy3-Cy5
5’
3’
Super-resolution Reconstruction
150
100
50
150
100
50
50
50
100
150
100
150
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: Widefield 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 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