Bulletin of the American Physical Society
2009 APS March Meeting
Volume 54, Number 1
Monday–Friday, March 16–20, 2009; Pittsburgh, Pennsylvania
Session Z7: Nanoprobes of Molecules and Cells |
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Sponsoring Units: DBP Chair: Adam Cohen, Harvard University Room: 407 |
Friday, March 20, 2009 11:15AM - 11:51AM |
Z7.00001: Real time imaging of mRNA expression dynamics in live cells using protein complementation methods Invited Speaker: Traditional methods for mRNA quantification in cells, such as northern blots, quantitative PCR or microarrays assays, require cell lysis and therefore do not preserve its dynamics. These methods cannot be used to probe the spatio-temporal localization of mRNA in cells, which provide useful information for a wide range biomolecular process, including RNA metabolizim, expression kinetics and RNA interference. To probe mRNA dynamics in live prokaryotic and eukaryotic cells, we develop a method, which exploit the strong affinity of the eukaryotic initiation factor 4A (eIF4A) to specific RNA aptamers. Two parts of the eIF4A are fused to a split Green Fluorescence Protein (GFP), and are expressed in the cells at high abundance. However, only when the RNA apatmer is also present, the two protein parts complement and become fluorescent. Thus, the fluorescent background remains low, allowing us to directly image the expression of mRNA molecules in live \textit{e-coli} cells from its early onset, over hours. We find that the expression kinetics can be classified in one out of at least three forms, which also display distinct spatial distributions. I will discuss the possible biological origin for these distributions and their time evolution. [Preview Abstract] |
Friday, March 20, 2009 11:51AM - 12:27PM |
Z7.00002: Nanopores and nanofluidics for single DNA studies Invited Speaker: Lab-on-a-chip fluidic technology takes inspiration from electronic integrated circuits, from which its name, its fabrication methods, and its ``smaller, cheaper, faster'' paradigm are derived. For silicon-based electronics, miniaturization eventually gave rise to qualitatively different behavior, as quantum mechanical phenomena grew increasingly important. As we shrink fluidic devices down to the nanoscale to probe samples as minute as a single molecule, what physical phenomena will dominate in this new regime, and how might we take advantage of them? This talk will focus on our studies of single DNA molecules using nanofluidic devices and solid-state nanopores. We are studying how nanofluidic structures, whose critical dimensions are tens to hundreds of nanometers, can manipulate long DNA molecules by a variety of nanoscale phenomena, including electrokinetics, hydrodynamics, Coulomb interactions, and the statistical properties of polymers. Our work also focuses on solid-state nanopores, single-nanometer-scale devices that can not only manipulate single molecules, but also detect them electronically. The basic principle behind this is that when DNA is electrophoretically driven through a nanopore, it blocks a measureable fraction of the ionic current that is transmitted through the pore. Thanks to its size, the nanopore also forces each base along the DNA to pass through in sequence, suggesting intriguing possibilities for genetic analysis. [Preview Abstract] |
Friday, March 20, 2009 12:27PM - 1:03PM |
Z7.00003: Toward Single-Molecule Nanomechanical Mass Spectrometry Invited Speaker: Mass spectrometry (MS) has become a preeminent methodology of proteomics since it provides rapid and quantitative identification of protein species with relatively low sample consumption. Yet with the trend toward biological analysis at increasingly smaller scales, ultimately down to the volume of an individual cell, MS with few-to-single molecule resolution will be required. We report the first realization of MS based on single-biological-molecule detection with nanoelectromechanical systems (NEMS). NEMS provide unparalleled mass resolution, now sufficient for detection of individual molecular species in real time. However, high sensitivity is only one of several components required for MS. We demonstrate a first complete prototype NEMS-MS system for single-molecule mass spectrometry providing proof-of-principle for this new technique. Nanoparticles and protein species are introduced by electrospray injection from the fluid phase in ambient conditions into vacuum and subsequently delivered to the NEMS detector by hexapole ion optics . Mass measurements are then recorded in real-time as analytes adsorb, one-by-one, onto a phase-locked, ultrahigh frequency (UHF) NEMS resonator. These first NEMS-MS spectra, obtained with modest resolution from only several hundred mass adsorption events, presage the future capabilities of this methodology. We outline the substantial improvements feasible in near term, through recent advances and technological avenues that are unique to NEMS-MS. [Preview Abstract] |
Friday, March 20, 2009 1:03PM - 1:39PM |
Z7.00004: Using Photoactivation Light Microscopy (PALM) to construct comprehensive, nanometer precision atlases of signaling complexes Invited Speaker: The E. coli chemotaxis network is a model system for biological signal processing. In E. coli, transmembrane receptors responsible for signal transduction assemble into large clusters containing several thousand proteins. These sensory clusters have been observed at cell poles and future division sites. Despite extensive study, it remains unclear how chemotaxis clusters form, what controls cluster size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells. Here we use photoactivated localization microscopy (PALM) to map the cellular locations of three proteins central to bacterial chemotaxis (the Tar receptor, CheY, and CheW) with a precision of 15 nanometers. We find that cluster sizes are approximately exponentially distributed, with no characteristic cluster size. One third of Tar receptors are part of smaller lateral clusters and not the large polar clusters. Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly. The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport. [Preview Abstract] |
Friday, March 20, 2009 1:39PM - 2:15PM |
Z7.00005: Single-molecule dynamics in nanofabricated traps Invited Speaker: The Anti-Brownian Electrokinetic trap (ABEL trap) provides a means to immobilize a single fluorescent molecule in solution, without surface attachment chemistry. The ABEL trap works by tracking the Brownian motion of a single molecule, and applying feedback electric fields to induce an electrokinetic motion that approximately cancels the Brownian motion. We present a new design for the ABEL trap that allows smaller molecules to be trapped and more information to be extracted from the dynamics of a single molecule than was previously possible. In particular, we present strategies for extracting dynamically fluctuating mobilities and diffusion coefficients, as a means to probe dynamic changes in molecular charge and shape. If one trapped molecule is good, many trapped molecules are better. An array of single molecules in solution, each immobilized without surface attachment chemistry, provides an ideal test-bed for single-molecule analyses of intramolecular dynamics and intermolecular interactions. We present a technology for creating such an array, using a fused silica plate with nanofabricated dimples and a removable cover for sealing single molecules within the dimples. With this device one can watch the shape fluctuations of single molecules of DNA or study cooperative interactions in weakly associating protein complexes. [Preview Abstract] |
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