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
Cmos cameras Parallel and fast readout (no need for amplification) Smaller pixel size (1-2um) Not quite as sensitive yet (~60% QE) iMicroscopes
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-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.
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
Multicolor STORM Bates, Zhuang Science 2007
Breaking the diffraction barrier Super-resolution microscopy (~10 nm resolution)