Session H3: Biophysics, Fluids and Fields

Nanapore Sequencing with MSPA

Nanopore sequencing is the simplest concept of converting the sequence of a single DNA molecule directly into an electronic signal. We introduced the protein pore MspA. derived from Mycobacterium smegmatis, to nanpore sequencing [1]. MspA has a single, narrow (-1.2nm) and short ($<$1nm) constriction, ideal to identify single nucleotides. Compared to solid state devices, MspA is reproducible with sub-nanometer precision and is engineerable using genetic mutations. DNA moves through the pore at rates exceeding 1nt/microsec. too fast to observe the passage of each nucleotide. However, when DNA is held with double stranded DNA sections or an avidin anchor, single nucleotides resident in MspA's constriction can be identified with highly resolved current differences. We have provided proof of principle of a nanopore sequencing method [2] in which we use DNA modified by inserting double stranded DNA-sections between every nucleotide. The double stranded sections are designed to halt translocation for long enough to sequentially read the sequence of the original DNA molecule. Prospects and developments to sequence unmodified native DNA using MspA will be discussed.\\ [4pt] [1] T.Z. Butler, et al, PNAS 105 20647 (2008)\\[0pt] [2] I.M. Derrington, et al, PNAS 107 16060 (2010).   

Probing Chromosome Structure and Dynamics: The Physics of a Long Tangledd Polymer?

The stochasticity of chromosome organization, dynamices, and micro-structure are investigated in live cells. The E. coli chromosome is precisely organized into a nucleoid filament with a linear order. Loci in the body of the nucleoid show a precision of inter-locus distance of genomically-proximate loci was better than 4\% of the cell length. The measured dependence of the precision of inter-locus distance on genomic distance singles out intra -nucleoid interactions as the mechanism responsible for chromosome organization. We probe the nature of this structure using super-resolution fluorescence microscopy and investigate the processivity of segregation of newly replicated genetic loci and their condensation into the nucleoid.   

Mitigating radiation damage in x-ray macromolecular crystallography at synchrotron sources

A new strategy is presented to reduce primary x-ray damage in macromolecular crystallography using synchrotron radiation. The basic principle was to separate as much as possible the x-ray irradiated region, where the diffracted signal originates, from the region where damage accumulates. This is possible since, by far, most of the damage is caused by the photo-electrons (PE's) excited as an x-ray photon is absorbed, and distributes its damage typically over several $\mu $m's. The optimum method to accomplish this was to focus the x-rays to a vertical line of sub-$\mu $m width. Diffraction experiments performed recently at the Advanced Photon Source, with a 0.83x60$\mu $m vertical line focus, allowed us to directly measure the spatial dependence of radiation damage in lysozyme crystals. The PE's caused radiation damage to accumulate predominatly outside the irradiateded region of the crystal exposed with line focused beam leading to a 4.5 factor (450{\%}) decease of radiation damage in the focused diffraction signal.   

Probing Lipid Membrane Rafts (Microdomains) with Fluorescent Phospholipids

Membrane rafts are enriched in sphingolipids and cholesterol, they exist in a more ordered state (the liquid-ordered phase; lo) than the bulk membrane (the liquid-disordered phase; ld). Ternary mixtures of palmitoyl-oleoyl-phosphocholine (POPC; 16:0,18:1 PC), sphingomyelin (SPM), and cholesterol (Chol) form membrane rafts over a wide range of molar ratios. We are examining the ability of two fluorescent probes, NBD linked to di-16:0 PE which partitions into the lo phase, and NBD linked to di-18:1 PE which partitions into the ld phase, to detect these two phases. We are also examining the effect of the highly polyunsaturated phospholipid stearoyl-docosahexanoyl-phosphocholine (SDPC; 18:0, 22:6 PC) on the size and stability of POPC/SPM/Chol membrane rafts. We report on the fluorescence lifetime and anisotropy decay dynamics of two fluorescent probes. Data were acquired via frequency-domain measurements from 5 to 250 MHz.   

Acoustic cavitation of individual ultrasound contrast agent microbubbles confined in capillaries

Ultrasound targeted therapies mainly rely on the inertial cavitation of ultrasound contrast agent (UCA) microbubbles. Our objective is to determine the cavitation acoustic pressure threshold for the destruction of UCA microbubbles inside cellulose capillaries. Acoustic emission from individual Optison{\texttrademark} microbubbles confined inside a 200-$\mu $m diameter capillary was detected using a passive cavitation detection system. Excitation signals from a 2.25 MHz transmitter were applied to the microbubbles while their acoustic emission was detected by a broadband 15 MHz receiver. Time traces were recorded (100 MHz sampling, 12- bit), and frequency-domain analysis of the received signals was performed to characterize microbubble cavitation. The cavitation acoustic pressure threshold was found to be 1 MPa inside the capillary in comparison with $\sim $0.7 MPa previously reported for unconfined UCA microbubbles. This work provides a clearer understanding of the role of ultrasound contrast agent dynamics inside a capillary.   

Capillary Action may Cool Systems and Precisely balance Chemical Reactions

It is well known that it takes no work for Water to rise in a Capillary tube against the force of Gravity. There is a precise balance in this system that resembles Robert Millikan's ``Oil Drop'' experiment, where mass was balanced against the electrostatic force. If at the top of the capillary tube there is evaporation, one can see that the system is cooled as another water molecule has room to move up the column. Furthermore, if the evaporation process can be controlled one photon at a time, a precise balance is created between a photon, and the height/mass of the column. If other molecules are place in the column, they can be moved up and down the column, in a chromatograph way, in a fairly precise manner, by controlling evaporation and molecular weight. If in addition to all of this, the interface of the solution against the walls of the column have Fermi levels, it can be seen as a very precise Electrochemical Device. In the situation of nanotubes, as opposed to trees and plants, these properties can be used to create measure environmental properties and to Balance Chemical Reactions. Forests, and Plants may cool themselves and their environment using this process, and using this process coupled with more energetic photons through photosynthesis.   

Scaling of Magnetic Particle Imaging for Sub-micrometer Resolution

In this work, we discuss the potential for scaling magnetic particle imaging (MPI) systems to resolve intracellular distributions of magnetic marker particles in biological samples, and present preliminary experimental results. MPI is a tomographic method developed for \textit{in vivo} imaging [1]. It utilizes the nonlinear response of magnetic particles. A magnetic field is established within the scanning volume such that it is zero at only one point. Only particles within this field free point respond to a small ac magnetic field and can be detected inductively. An image is formed by moving the field free point relative to the sample. Previous work has achieved a spatial resolution of 1 mm [2]. We will present a theoretical analysis showing that sub-micrometer spatial resolution can, in principle, be achieved. Our first laboratory implementation has demonstrated a spatial resolution of 1 mm. We continue to scale and optimize the system for a first-ever demonstration of tomographic imaging of sub-cellular structures and processes in real-time. \\[4pt] [1] B. Gleich and J. Weizenecker, Nature, v. 435, p. 1214, 2005 \\[0pt] [2] B. Gleich, et al., Phy. Med. Bio., v. 54, p.L1, 2009