Bulletin of the American Physical Society
2005 APS March Meeting
Monday–Friday, March 21–25, 2005; Los Angeles, CA
Session W21: Techniques in Biophysics |
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Sponsoring Units: DBP Chair: Robert Riehn, Princeton University Room: LACC 409A |
Thursday, March 24, 2005 2:30PM - 2:42PM |
W21.00001: Linear Image Restoration of Sets of Images with Residual Misalignments Philip Baldwin, Pawel Penczek In cryo-electrron microscopy, images are collected in the form of 2-D projections of 3-D macromolecular assemblies of unknown shape. These 2-D images suffer from a very low signal-to-noise- ratio. In order to proceed with reconstructing a 3-D model, individual images are aligned and then classified. Class averages are then formed by averaging the resulting aligned images. Unfortunately, it is difficult to completely eliminate alignment errors from the alignment procedures; the high- frequency components in the resulting class-averages are therefore detiorated. We propose to use (two-point) correlation functions to provide estimates for the variances in the alignment parameters. With estimates of the variances at hand, one can use a linear procedure to find better estimates of class averages, where the blurring due to the initial misalignments has been corrected for. Tests are performed on a variety of real protein data. [Preview Abstract] |
Thursday, March 24, 2005 2:42PM - 2:54PM |
W21.00002: Femtosecond laser-assisted delivery of molecules into single living cells Cheng Peng, Ingrid Wilke, Robert Palazzo The controlled delivery of membrane impermeable molecules into single living cells (micro-injection) is important for a variety of applications such genomics, proteomics or drug screening and testing. Recently, it has been demonstrated that opto-injection with femtosecond (fs) laser pulses at near-infrared (nir) wavelengths (700-1100nm) has the potential to create transient pores in single living cells with high cell survival rates and transfection efficiency. However, the physical mechanisms of fs nir opto-injection is poorly understood. Here, we present an experimental study of fs nir laser-assisted opto--injection of Bovine Aorthic Endothelial Cells (BAEC). Transient pore creation is studied experimentally by fluorescent dye uptake. Using this experimental approach we have investigated individual BAE cells for laser power levels up to 400mW. We found that the minimum laser power density for pore creation is 2.5$\times $10$^{6}$W/mm$^{2}$.We observe that fs nir opto-injection is a stable and reliable method of micro-injection and that pore creation is transient. For power levels between 50mW and 150mW we observe 100{\%} cell survival as judged by the unaltered cell morphology. [Preview Abstract] |
Thursday, March 24, 2005 2:54PM - 3:06PM |
W21.00003: Diffractive Imaging of Single Viruses Robert Coridan, John Butler, Thomas Angelini, Gerard C.L. Wong The resolution of Electron Microscopy (EM) images is limited by instrumentation lens aberration. Nanobeam electron diffraction in principle allows for diffraction-limited resolution analysis of single particles. However, images are not directly recoverable from the diffraction pattern due to the well-known phase problem. Algorithms for solving the phase problem have long been a topic of theoretical research, but only recently has diffractive imaging been experimentally actualized using coherent diffraction. We will discuss these algortihms and the potential of improving the resolution of TEM imaging of biomolecular systems through a combination of these algorithms and cryogenic sample preparation. Preliminary data on single virus diffraction data will be presented. [Preview Abstract] |
Thursday, March 24, 2005 3:06PM - 3:18PM |
W21.00004: Optical detection of changes in glass surface properties induced during wet photolithography R. Masina, J. P. Landry, X. D. Zhu, A. N. Parikh Recently Yee et al. (Adv. Mater. 2004, \textbf{16}: 1184-1189) demonstrated a wet UV-photolithographic method for patterning phospholipid bilayers into two-dimensional arrays of voids and patches on hydrophilic glass surfaces. In this method, the glass surface is chemically etched to remove organic contaminants and expose hydrophilic groups before a bilayer is formed on top of it, using the method of small unilamellar phospholipid vesicle fusion. The bilayer is subsequently illuminated with short-wavelength UV light through a photomask, creating voids in the irradiated regions. We studied the effects of the chemical etching and subsequent UV irradiation on the surfaces of commercially available microscope glass slides using an oblique-incidence optical reflectivity difference (OI-RD) microscope. We found that the UV irradiation after chemical etching further changes the properties of the surface, even in the absence of a lipid bilayer. As a result, irradiating the chemically etched surface before the UV-photolithography step prevents further UV-induced changes in surface properties. We found that the lipid bilayer formed on such an UV-irradiated surface retains its fluidic properties. [Preview Abstract] |
Thursday, March 24, 2005 3:18PM - 3:30PM |
W21.00005: Comparison of sensitivities of two label-free optical detection techniques: SPR vs. oblique-incidence reflectivity difference (OI-RD) X. D. Zhu, J. P. Landry Surface plasmon resonance (SPR) and recently developed oblique- incidence optical reflectivity difference or OI-RD (a special form of nulling ellipsometry) are two label-free techniques for detecting biochemical reactions and other surface processes on a solid support. In SPR, one measures the change in surface plasmon resonance angle, $\Delta \theta _{SP}$, as a result of changes in thickness and dielectric response of a molecular adlayer on the solid support coated with gold. In OI- RD, one measures the difference in fractional reflectivity change between p-polarized and s-polarized light defined as (r$_{p}$-r$_{p0}) $/r$_{p0}$ - (r$_{s}$-r$_{s0})$/r$_{s0} \quad \equiv \quad \Delta _{p}- \Delta _{s}$, again in response to changes in thickness and dielectric response of a molecular adlayer on the solid support. I show that the two techniques measure the same physical quantities of the molecular adlayer, up to a constant that depends on the incidence angle in OI-RD. And since $\Delta _{p}-\Delta _{s}$ is enhanced near the Brewster angle, the OI- RD technique is at least as sensitive as and more versatile than the SPR technique for label-free detection of biochemical reactions and other processes on solid surfaces. [Preview Abstract] |
Thursday, March 24, 2005 3:30PM - 3:42PM |
W21.00006: A New Class of Quantum Imaging Materials:50 nm Diameter Infrared Upconverting Phosphors. Shuang Fang Lim, Robert Riehn, Chih-kuan Tung, Robert H Austin, Alex McDonald, David Tank Upconversion phosphors (UCPs) convert infrared (IR) photons into visible light using stepwise excitation levels, so that inexpensive CW IR diode lasers can be used for multi-photon imaging. These phosphors are ideal candidates as biological markers, due to their high quantum yield, very rich spectral features, absence of photobleaching, and tunable emission from the red through the blue with IR excitation. The multiphoton intensity dependence gives rise to high spatial resolution confocal imaging possibilities. There is a complete lack of background fluorescence excitation in biological tissue with CW IR excitation, greatly enhancing sensitivity. We have succeeded in synthesizing spherical nanoparticles of 50 nm. We have also succeeded in modifying the surface of the UCP nanoparticles with a biotin-streptavidin coupling scheme, have demonstrated end-labeling of lambda DNA to the upconversion phosphors. We have also shown that electron beam excitation of these UPCs gives rise to complex emission patterns related to the optical patterns, so that both 2-photon confocal imaging and much higher resolution SEM imaging using optical detection is possible. [Preview Abstract] |
Thursday, March 24, 2005 3:42PM - 3:54PM |
W21.00007: Differential Adhesion of Amino Acids to Inorganic Surfaces Robert Willett, Ken West, Loren Pfeiffer Interactions at the interface of biological molecules and inorganic materials are an open question in materials science; understanding these hybrid interfaces at the molecular level can have extensive basic and practical implications. In an extensive set of measurements we have systematically examined the adhesion of amino acids to a series of inorganic surfaces used in semiconductor devices. Peptides comprised of each of the twenty amino acids were exposed in solution to surfaces including metals, insulators, and semiconductors. Significant differential adhesion to the various surfaces is observed over the complement of amino acids, with adhesion determined largely by the amino acid side-chain charge. Mapping of adhesion findings for the amino acids versus materials in multiple solutions has been accomplished, in addition to examination of concentration and pH dependence. These results provide an empirical basis for building peptide to inorganic surface structures. In this vein, we have designed inorganic nano-structures that are shown to selectively bind to prescribed primary peptide sequences. [Preview Abstract] |
Thursday, March 24, 2005 3:54PM - 4:06PM |
W21.00008: A novel approach to measurement of the adhesion strength of a single cell on a substrate Marie-Josee Colbert, Kari Dalnoki-Veress, Cecile Fradin The fundamental study of the adhesion of cells on solid surfaces is crucial to the characterization and development of materials suitable for use in biological environments (i.e. implants). We will present our work on the adhesion of a single vesicle on a substrate. A vesicle is held at the end of a micropipette mounted on a micromanipulator and put into contact with a surface. Adhesion is measured by pulling the vesicle from the substrate. Rather than using suction to infer the adhesion strength, we take advantage of the spring constant of an L-shaped micropipette to directly measure the adhesion force. The effect of surface roughness on the adhesion strength of vesicles will be discussed. [Preview Abstract] |
Thursday, March 24, 2005 4:06PM - 4:18PM |
W21.00009: Observation of magnetic field-induced contraction of fission yeast cells using optical projection microscopy Xi Yang, A.W. Beckwith The charges in live cells interact with or produce electric fields, which results in enormous dielectric responses, flexoelectricity, and related phenomena. Here we report on a contraction of \textbf{\textit{Schizosaccharomyces pombe}} \textbf{(fission yeast)} cells induced by \textbf{\textit{magnetic}} \textbf{fields}, as observed using a phase-sensitive projection imaging technique. Unlike electric fields, magnetic fields only act on moving charges. The observed behavior is therefore quite remarkable, and may result from a contractile Lorentz force acting on diamagnetic screening currents. This would indicate extremely high intracellular charge mobilities. Besides, we observed a large electro-optic response from fission yeast cells. [Preview Abstract] |
Thursday, March 24, 2005 4:18PM - 4:30PM |
W21.00010: Nonlinear Microrheology of Dilute Aqueous Polymer and Biopolymer Solutions J.N. Wilking, T.G. Mason We have developed an optically driven non-linear microrheometer using birefringent microdisks. With it, we measure the nonlinear rheological properties of dilute aqueous polyethylene oxide (PEO) solutions. As a demonstration, we explore the yield stress with respect to polymer concentration for a range of PEO concentrations. Discotic microparticles are trapped and rotationally driven with a laser tweezer, and LabVIEW is used to track the angular displacement of the disk from its backscattered laser light streak. Results are compared with macroscopic rheological measurements. Using this general nonlinear rheological technique, we also investigate concentrated biopolymer solutions of double stranded DNA. [Preview Abstract] |
Thursday, March 24, 2005 4:30PM - 4:42PM |
W21.00011: Dielectric Properties of Live Yeast Cells Expressed with the Motor Protein Prestin John Miller, Dharmakeerthi Nawarathna, David Warmflash, Fred Pereira, William Brownell We report on the linear and nonlinear dielectric properties of budding yeast (S. cerevisiae) cells, one strain of which has been genetically modified to express prestin. This motor protein plays a crucial role in the large electromotility exhibited by the outer hair cells (OHCs) of mammalian inner ears. Live cell suspensions exhibit enormous dielectric responses, which can be used to probe metabolic activity, membrane potential, and other properties. We observe a broad peak, centered around 20 kHz, in the normalized difference in dielectric responses between the two strains, and also observe substantial differences in nonlinear harmonic responses. Our data appears to correlate with measurements showing piezoelectric resonances in OHCs. These results suggest that dielectric probes can be used to study the electrical properties of prestin and other proteins, either expressed in live cells or (from our other recent studies on tubulin) as pure protein suspensions. [Preview Abstract] |
Thursday, March 24, 2005 4:42PM - 4:54PM |
W21.00012: A Microchannel Device for the Alignment of Self-Assembled Filamentous Protein Systems Linda S. Hirst, E. Parker, Z. Abu Samah, Y. Li, N.C. MacDonald, C. R. Safinya, R. Pynn We report a technique for the alignment of filamentous self-assembled protein systems, such as F-actin bundles and microtubules, in a surface modified titanium or silicon micro-fluidic device. Such Protein systems are delicate and typically difficult to align as they must be formed under carefully controlled solution conditions. Narrow channels allow the protein system to assemble in-situ in a confined geometry, producing an aligned sample. The device surfaces are modified via self-assembled monolayers to resist protein adsorption. Biomolecular self-assembly can be investigated in a controlled fashion under different molecular concentration gradients and conditions along the channel. This technique produces highly aligned samples for structural studies carried out via x-ray scattering and is also useful for mechanical studies of different filamentous assemblies. Use of a micro-fabricated device for protein studies is perfect for microscopy and also will allow for the incorporation of MEMS features into the device. [Preview Abstract] |
Thursday, March 24, 2005 4:54PM - 5:06PM |
W21.00013: Developing a Microfluidic Diffusional Mixer for Time-resolved Infrared Microscope Spectroscopic Studies of Protein Structural Dynamics Peter Galajda, Robert Austin, Jarmila Guijarro, Aihua Xie Information regarding protein structural dynamics is essential for understanding the structure-function relationships of proteins. Time-resolved Fourier transform infrared spectroscopy is a powerful technique for probing and characterizing the structures and structural dynamics of functional intermediate states of proteins. A key step is to trigger a synchronized biological process of proteins. We have designed and tested a microfluidic diffusional mixer for time-resolved infrared microscope spectroscopic studies of structural dynamics of chemically activated proteins. The design of the chip with micro-channels ensures rapid mixing. The microfluidic devices were fabricated in silicon using photolithography and deep reactive ion etching. An infrared microscope is employed so that the sample size can be as small as 50 microns in diameter. A syringe-pump was used to establish stable flow in the chip. A computer-controlled step-motor is used to move the micro-mixer chip for sampling different parts of the sample in the mixing channel. [Preview Abstract] |
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