Bulletin of the American Physical Society
APS March Meeting 2011
Volume 56, Number 1
Monday–Friday, March 21–25, 2011; Dallas, Texas
Session A7: Prize Session: Single Molecule Biophysics I: Recent Advancements in Technology and Applications |
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Sponsoring Units: DBP DPOLY DCMP Chair: Yan Mei Wang, Washington University in St. Louis Room: Ballroom C3 |
Monday, March 21, 2011 8:00AM - 8:36AM |
A7.00001: Single Fluorescent Molecules as Nano-Illuminators for Biological Structure and Function Invited Speaker: Since the first optical detection and spectroscopy of a single molecule in a solid (Phys. Rev. Lett. \textbf{62}, 2535 (1989)), much has been learned about the ability of single molecules to probe local nanoenvironments and individual behavior in biological and nonbiological materials in the absence of ensemble averaging that can obscure heterogeneity. Because each single fluorophore acts a light source roughly 1 nm in size, microscopic imaging of individual fluorophores leads naturally to superlocalization, or determination of the position of the molecule with precision beyond the optical diffraction limit, simply by digitization of the point-spread function from the single emitter. For example, the shape of single filaments in a living cell can be extracted simply by allowing a single molecule to move through the filament (PNAS \textbf{103}, 10929 (2006)). The addition of photoinduced control of single-molecule emission allows imaging beyond the diffraction limit (super-resolution) and a new array of acronyms (PALM, STORM, F-PALM etc.) and advances have appeared. We have used the native blinking and switching of a common yellow-emitting variant of green fluorescent protein (EYFP) reported more than a decade ago (Nature \textbf{388}, 355 (1997)) to achieve sub-40 nm super-resolution imaging of several protein structures in the bacterium\textit{ Caulobacter crescentus}: the quasi-helix of the actin-like protein MreB (Nat. Meth. \textbf{5}, 947 (2008)), the cellular distribution of the DNA binding protein HU (submitted), and the recently discovered division spindle composed of ParA filaments (Nat. Cell Biol. \textbf{12}, 791 (2010)). Even with these advances, better emitters would provide more photons and improved resolution, and a new photoactivatable small-molecule emitter has recently been synthesized and targeted to specific structures in living cells to provide super-resolution images (JACS \textbf{132}, 15099 (2010)). Finally, a new optical method for extracting three-dimensional position information based on a double-helix point spread function enables quantitative tracking of single mRNA particles in living yeast cells with 15 ms time resolution and 25-50 nm spatial precision (PNAS \textbf{107}, 17864 (2010)). These examples illustrate the power of single-molecule optical imaging in extracting new structural and functional information in living cells. [Preview Abstract] |
Monday, March 21, 2011 8:36AM - 9:12AM |
A7.00002: Multiple Pathways of Single-Stranded DNA Stretching Observed Using Single-Molecule Manipulation Invited Speaker: DNA has a double helix structure and contains the genetic code of life. When the information needed to be read, the DNA double helix has to be opened up to allow access to the bases that make up the DNA. During the reading process the DNA adopt a different conformation, and the energetics and mechanics of the dynamic process is important in gene regulation. We used an atomic force microscope to pull single DNA molecules and measured the force associated with the conformational changes of poly(dA), a single-stranded DNA composed of uniform A bases. We found that the DNA can be stretched in two different ways, and the DNA can hop between these two conformations. These results suggest that poly(dA) has a novel conformation when highly stretched, and the unique conformation makes poly(dA) more stable at large extensions. The unique property of poly(dA) may play a role in biological processes such as gene expression. Moreover, single molecule force measurement allows us to quantify the elastic and thermodynamic properties of single biological molecules, and may ultimately be developed into a tool for drug screening. \\[4pt] [1] W.-S. Chen, W.-H. Chen, Z. Chen, A. A. Gooding, K.-J. Lin, and C.-H. Kiang, ``Direct Observation of Multiple Pathways of Single-Stranded DNA Stretching,'' {\em Phys. Rev. Lett.} {\bf 105} (2010) 218104. \\[0pt] [2] C. P. Calderon, W.-H. Chen, K.-J. Lin, N. C. Harris, and C.-H. Kiang, ``Quantifying DNA Melting Transitions using Single-Molecule Force Spectroscopy,'' invited paper in special issue on DNA Melting, {\em J. Phys.: Condens. Matter} {\bf 21} (2009) 034114. [Preview Abstract] |
Monday, March 21, 2011 9:12AM - 9:48AM |
A7.00003: Max Delbruck Prize in Biological Physics Talk: Zoom into life at the nanoscale with STORM Invited Speaker: Powered by its molecule-specific contrast and live-cell compatibility, fluorescence microscopy is one of the most widely used imaging methods in biological research. The resolution of fluorescence microscopy is classically limited by the diffraction of light to several hundred nanometers. This resolution limit is substantially larger than the typical molecular length scales in cells, preventing detailed characterization of most sub-cellular structures. Here, I describe a new imaging method, stochastic optical reconstruction microscopy (STORM), which breaks the diffraction limit and allows for super-resolution imaging. STORM uses single-molecule imaging and photo-switchable fluorescent probes to temporally separate the spatially overlapping images of individual molecules, thereby allowing each molecule to be localized with high precision and a super-resolution image to be reconstructed from the numerous measured positions of the molecules. Using this approach, we have imaged cellular structures with nanometer-scale resolution. In this talk, I will discuss the general concept, recent technical advances, and various biological applications of STORM. [Preview Abstract] |
Monday, March 21, 2011 9:48AM - 10:24AM |
A7.00004: DNA overstretching transition and the biophysical properties of S-DNA Invited Speaker: DNA double helix undergoes an ``overstretching'' transition in a narrow tensile force range slightly above 60 pN. Overstretched DNA is about 1.7 times longer than B-DNA. Despite numerous studies the basic question of whether the strands are separated or not remains controversial. Our recent experiments show that two distinct transitions are involved in DNA overstretching: a slow hysteretic strand-unpeeling transition to strand separation from free DNA ends or nicks, and a fast, non-hysteretic B-to-S transition to an elongated double helix called ``S-DNA''. We find that the relative fraction of these two overstretched forms is sensitive to factors that affect DNA base pair stability. Under conditions when S-DNA is stable, we characterize its force-extension curve and compare it with that of single-stranded DNA. We find that the S-DNA is 0.01 - 0.02 nm/bp shorter than that of a nucleotide of single-stranded DNA in the force range 75 - 110 pN. Under conditions when S-DNA is less stable than single-stranded DNA, a slow force increase leads to direct strand separation from B-DNA, while a quick force jump to greater than 70 pN leads to a quick formation of the S-DNA first, followed by a slow secondary transition which is a strand separation from S-DNA. From the secondary transition, the extension difference between S-DNA and single-stranded DNA can be directly calculated, which is found in perfect agreement with that computed from the force-extension curves. Finally, we show that DNA in between a pair of small GC-rich segments is biased toward B-to-S transition. This result also demonstrates that in the absence of nicks and free ends, torsion-unconstrained DNA still undergoes the overstretching transition but only through the B-S transition pathway. [Preview Abstract] |
Monday, March 21, 2011 10:24AM - 11:00AM |
A7.00005: Ultra-high resolution optical trap with single fluorophore sensitivity Invited Speaker: We present a new single-molecule instrument that combines ultra- high resolution optical tweezers with single-fluorophore fluorescence microscopy. The new instrument will enable the simultaneous measurement of angstrom-scale mechanical motion of individual DNA-binding proteins (e.g., single base-pair stepping of DNA translocases) along with the detection of fluorescently labeled protein properties (e.g., internal configuration). The optical tweezers portion of the instrument is based on a timeshared dual optical trap design and is interlaced with a confocal fluorescence microscope. In a demonstration experiment, individual single-fluorophore labeled DNA oligonucleotides can be observed to bind and unbind to complementary DNA suspended between two trapped beads. Simultaneous with the single-fluorophore detection, coincident angstrom-scale changes in tether extension can be clearly observed. [Preview Abstract] |
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