lecture 2: in vitro single molecule and super-resolution microscopy long cai bi/ch 132 oct 9 th,...
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Lecture 2: in vitro Single molecule and Super-resolution Microscopy
Long CaiBi/Ch 132
Oct 9th, 2012
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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).
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Light sources
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SM counting and stochiometry
Isacoff EY, Subunit counting in membrane-bound proteins.Nat Method 4(4):319-21 2007
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measuring binding kinetics
M.P. Elenko, J.W. Szostak and A.M. van Oijen, J Am Chem Soc, 131 (2009), pp. 9866–9867.
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Fluorescence Correlation Spectroscopy (FCS)
Variants: image FCS, FRET-FCS, TIRF-FCS
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F1-ATPase rotation
Kinoshita, Direct observation of the rotation of F1-ATPase. Nature 386(6622):299-302. 1997
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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
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Mechanical synthesis of ATP
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Yoshida et al, F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits. Nature Structural & Molecular Biology 14, 841 - 846 (2007)
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Molecular tracking
Yildiz, Selvin Science 2003
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Cameras
Front-illuminated
Back-illuminated
Compare to 2% in photographic film
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sources of noise in a camera• Dark current
25oC 2oC
Problematic at long exposure times, but we keep our camera cooled to -70oC.
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sources of noise in a camera• Readout noise
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EMCCD
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Cmos cameras
• Parallel and fast readout (no need for amplification)
• Smaller pixel size (1-2um)• Not quite as sensitive yet (~60% QE)• iMicroscopes
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Pixel size
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lens-less microscopy
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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)
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Sub-wavelength localization
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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.
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Myosin walking on actin
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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.
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Molecular tracking
Cy3
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Yildiz, Selvin Science 2004
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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.
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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.
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Photoswitchable FPs
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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.
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5’ 3’
STORM with 1 color probes
Cy3-Cy5 GFP mRNA
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Super-resolution Reconstruction
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Multicolor STORM
Bates, Zhuang Science 2007
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Breaking the diffraction barrier
Super-resolution microscopy (~10 nm resolution)
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STED microscopy
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Saturated structured illumination microscopy (SSIM)
Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. Mats Gustafsson, PNAS 2005
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Z. Liu et al., Science 315, 5818 (March 23, 2007).
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Single molecule FRET on Ribozymes
Zhuang, Chu, Science 2002
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Stepwise protein-mediated RNA folding directs assembly of telomerase ribonucleoprotein. Nature 2007
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Single molecule enzymology
BP English et al. Nature Chemical Biology 2, 87 - 94 (2006)
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Memory effects in enzymes
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Enzyme turn-over time distribution is multi-exponential.
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Sanger Sequencing
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Illumina Sequencing
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Pyro-sequencing
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Problem: how to increase speed?
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Pac biosciences sequencer
Real-Time DNA Sequencing from Single Polymerase Molecules, Science 2008
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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.
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Example: Single base stepping of RNA polymerase
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Example: single-molecule DNA gyrase
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DNA motor proteins
Van Oijen, AM Science 2003Single-Molecule Kinetics of λ Exonuclease Reveal Base Dependence and Dynamic Disorder
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Trombone model of DNA replication
Van Oijen, AM Nature 2006DNA primase acts as a molecular brake in DNA replication
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