### Session B27: Experimental Techniques in Biophysics

 Monday, March 15, 2010 11:15AM - 11:27AM B27.00001: Reducing the Viscosity of Blood by Pulsed Magnetic Field R. Tao , K Huang Blood viscosity is a major player in heart disease. When blood is viscous, in addition to a high blood pressure required for the blood circulation, blood vessel walls are also easy to be damaged. While this issue is very important, currently the only method to reduce the blood viscosity is to take medicine, such as aspirin. Here we report our new finding that the blood viscosity can be reduced by pulsed magnetic field. Blood is a suspension of red blood cells (erythrocytes), white blood cells (leukocytes) and platelets in plasma, a complex solution of gases, salts, proteins, carbohydrates, and lipids. The base liquid, plasma, has low viscosity. The effective viscosity of whole blood increases mainly due to the red blood cells, which have a volume fraction about 40{\%} or above. Red blood cells contain iron and are sensitive to magnetic field. Therefore, when we apply a strong magnetic field, the red cells make their diameters align in the field direction to form short chains. This change in rheology reduces the effective viscosity as high as 20-30{\%}. While this reduction is not permanent, it lasts for several hours and repeatable. The reduction rate can be controlled by selecting suitable magnetic field and duration of field application to make blood viscosity within the normal range. Monday, March 15, 2010 11:27AM - 11:39AM B27.00002: Eukaryotic cell flattening Albert Bae , Christian Westendorf , Christoph Erlenkamper , Edouard Galland , Carl Franck , Eberhard Bodenschatz , Carsten Beta Eukaryotic cell flattening is valuable for improving microscopic observations, ranging from bright field to total internal reflection fluorescence microscopy. In this talk, we will discuss traditional overlay techniques, and more modern, microfluidic based flattening, which provides a greater level of control. We demonstrate these techniques on the social amoebae Dictyostelium discoideum, comparing the advantages and disadvantages of each method. Monday, March 15, 2010 11:39AM - 11:51AM B27.00003: Studies of Molecular Excitons formed by Porphyrin Dimers in Lipid Bilayer Vesicles using Two-Dimensional Electronic Coherence Spectroscopy Geoffrey Lott , Andrew Marcus When a macromolecular complex is labeled with two or more closely spaced fluorescent chomophores, resonant dipolar coupling can give rise to delocalized exciton states whose energies and transition strengths are sensitive to site conformation. We study the exciton structure of dimers of Magnesium tetraphenylporphyrin (MgTPP), which self-assemble within phospholipid bilayer vesicles. As the concentration of membrane-bound MgTPP is increased, low-lying vibronic features of the linear absorption spectrum are observed to broaden and develop blue-shifted shoulders. We apply a fluorescence-detected phase-modulation approach to two-dimensional electronic coherence spectroscopy (2D-ECS) to measure the 2D electronic spectrum of the exciton-coupled dimers. The 2D spectra exhibit clearly resolved diagonal and off-diagonal features associated with the exciton splitting, whose shapes and relative amplitudes evolve on femtosecond time scales. Monday, March 15, 2010 11:51AM - 12:03PM B27.00004: Simultaneous multi-point laser ablation using a spatial light modulator Aroshan Jayasinghe , Shane Hutson Laser-microsurgery has emerged as a powerful technique for evaluating in vivo tissue mechanics; however, for incisions involving multiple pulses, only the very first pulse ablates tissue with unaltered mechanical stress; subsequent pulses ablate tissue that is recoiling from earlier ablations. To avoid this, we have developed a system for simultaneously ablating tissue at several points by using a single laser pulse shaped by a phase-only spatial light modulator (SLM). The ablating laser system is connected to a confocal microscope with a high-speed camera attachment. Using the high-speed camera and pulsed illumination, we have imaged the dynamics of multi-point ablation -- including the formation and interaction of multiple simultaneous plasmas and cavitation bubbles. We report preliminary results from simultaneous ablation of multiple spots and/or extended lines in aqueous solution, gels and fruit fly embryos. Monday, March 15, 2010 12:03PM - 12:15PM B27.00005: Nonlinear nonlocal infrared plasmonic arrays for pump-probe studies on protein monolayers Shyamsunder Erramilli , Ronen Adato , Alan Gabel , Ahmet Ali Yanik , Hatice Altug , Mi K. Hong Infrared spectroscopy is an exquisite bond-specific tool for studying biomolecules with characteristic vibrational normal modes that serve as a molecular fingerprint''. Intrinsic absorption cross-sections for proteins are significant ($\sim$10$^{-19}$ -10$^{-21}$ cm$^{2})$, although small compared to label-based fluorescence methods. We have shown that carefully designed plasmonic nanoantenna arrays can enhance the vibrational signatures by $\sim$ 10$^{5}$ (Adato et al, \textit{Proc Natl Acad Sci USA}, 2009). Theoretical modeling combined with polarized FTIR-microscopy show that enhancement is due both to localized effects and nonlocal collective effects, governed by the dielectric properties of silicon and gold nanoantennae, coupled to protein molecules. The resonance properties can be modulated by photoinduced excitation of charge carriers and excitons, causing both a shift in the resonance frequency and a change in the enhancement factor. An ultrafast visible pump laser can then be used to extend visible pump-infrared probe studies to protein molecules even when the molecules lack a chromophore. This provides a toolkit for biophysical studies in which the nonlinear, nonlocal interaction between a 35-fs visible or near-infrared laser and the designed plasmonic nanoantenna arrays are used to study dynamics of protein molecules. Monday, March 15, 2010 12:15PM - 12:27PM B27.00006: \textit{In situ} and \textit{in vitro} characterization of biointerfaces using linear and non-linear optical spectroscopy Patrick Koelsch The actively emerging field of biointerfaces requires novel experimental techniques which allow us to deduce molecular-level based information under normal conditions and, in terms of biological relevance, in aqueous environments. In this contribution we demonstrate in several examples the feasibility of spectroscopic techniques to analyze complex structures at interfaces \textit{ex situ}, \textit{in situ} and \textit{in vitro}: (i) DNA Films: We studied the chemical integrity, packing density, orientation, and ordering in monomolecular films of single-stranded DNA on Au by a combination of X-ray and IR spectroscopy. In addition, \textit{in situ} broadband sum-frequency-generation (SFG) spectroscopy has been employed to determine the conformation and orientation of various homo-oligonucleotides in aqueous environments and to follow hybridization processes \textit{in situ} on a molecular scale. (ii) Cell adhesion: We probed various cell types adhered on solid surfaces via \textit{in vitro} broadband SFG spectroscopy to demonstrate the feasibility of using SFG spectroscopy as an experimental tool to characterize the extracellular matrix (ECM) layer adjacent to a solid substrate beneath a layer of cells. (iii) Membrane Proteins: Transmembrane proteins in Xenopus laevis oocytes and Zebrafish embryos have been studied by attenuated total reflectance infrared spectroscopy. Monday, March 15, 2010 12:27PM - 12:39PM B27.00007: Multi-photon Fluorescence Recovery After Photobleaching Applied to Systems Confined in One, Two or Three Dimensions Kelley D. Sullivan , Edward B. Brown Multi-photon fluorescence recovery after photobleaching is a microscopy technique used to measure the diffusion coefficient of macromolecules in biological systems. As multi-photon fluorescence recovery after photobleaching is introduced into more systems in vivo, the need arises to adapt the technique for application to a wider range of physiological situations. In this talk, we present our findings into measuring diffusion coefficients in systems with boundary conditions on the order of the size of the focal volume. Using Monte Carlo methods, we model both diffusion and the fluorescence recovery process in systems where diffusion is limited in one, two and finally all three dimensions. We compare our calculations with experiments where diffusion is limited in one dimension by one and two boundaries. From our results, we can define where boundaries are sufficiently far away to apply multi-photon fluorescence recovery after photobleaching with the conventional analysis; where boundaries are too close to use the technique at all; and an intermediate range of boundary locations, where measurements of the diffusion coefficient are possible with minor modifications to the fluorescence recovery analysis. Monday, March 15, 2010 12:39PM - 12:51PM B27.00008: Origin of Different Color Hues in Fluorescent Proteins with the Same Chromophore Aleksander Rebane , Mikhail Drobizhev , Shane Tillo , Nikolay Makarov , Thomas Hughes Fluorescent proteins (FPs) exhibit broad varietyof absorption and emission colors even though some mutants share the same chromophore structure. We demonstrate that in red FPs including DsRed, mRFP, and mFruits (absorption peak 540 to 590 nm), as well as in green FPs including EGFP, TagGFP, mWasabi, GX variants, and mTFP variants (absorption peak 450 to 500 nm) the colors are caused by internal Stark effect. We use quantitative two-photon absorption spectroscopy to show that the colors changes can be explained by quadratic Stark shifts due to variations of the strong local electric field within the beta barrel. This allows us, for the first time tour knowledge, to directly measure the internal electric field inside a protein. The obtained maximum values up to 10 to 100 MV/cm in the mFruits series are rather large, however, these field strengths are still 1 -2 orders less than those required to ionize the chromophore. These measured values also correspond well with previous theoretical estimates for different proteins. Our finding suggests a new way to sense electrical fields in biological systems, while it also bring order to a bewildering diversity of FP properties Monday, March 15, 2010 12:51PM - 1:03PM B27.00009: Initial stage of the phase separation, observed with fluorescent excitation transfer technique Alexei Goun , Michael Fayer The use of the fluorescent resonant excitation transfer (FRET) to study the phase transition kinetics is demonstrated. The laser temperature jump is applied to the water/2,6-lutidine mixture and causes the demixing of the mixture components. Coumarin 480 and hydroxypyrene laser dyes form excitation transfer pair once they are in the uniform phase of the mixture. Due to the differential solubility of these dyes in the componens of the mixture, the excitation transfer ceases once the phase separation occurs. The increase of the donor fluorescence indicates the extent of the phase separation. The spatial resolution of the method is determined by the Forster distance of the excitation transfer pair, and in this case is equal to 3 nm. The phase separation is completed within 1 microsecond. The rising edge of the fluorescence is consistent with polynomial growth of the phase separated domains, and not with Cahn-Hilliard fixed length instability. Monday, March 15, 2010 1:03PM - 1:15PM B27.00010: Fabrication of Magnetic Particles to Directly Measure Torque and Force on Single DNA Molecules Paula Vivas , Adam Hauser , Marek Simon , Fengyuan Yang , Ezekiel Johnston-Halperin , Michael Poirier A common approach for manipulating single biomolecules is to attach them to a super-paramagnetic bead and manipulate the bead with a magnetic trap [1]. An advantage of this approach is that it is straightforward to apply forces and twists to single DNA molecules. However, \textbf{\textit{torque}} measurements have remained elusive because of the complication in determining the direction of the spherical bead's magnetic moment. To overcome this difficulty, we fabricated anisotropic, elongated micron-to-nanometer sized magnetic particles. We grow ferromagnetic films using ultra high vacuum (UHV) sputtering and photolithography patterning to create magnetic particles with elongated shapes. The particles are coated with gold and functionalized for single DNA molecule attachment. Preliminaries results on magnetic particle preparation, functionalization and manipulation will be presented. \\[4pt] [1] \textit{Kapanidis AN, Strick T. Biology, one molecule at a time. Trends Biochem Sci. 2009 May;34(5):234-43.} Monday, March 15, 2010 1:15PM - 1:27PM B27.00011: Mobile magnetic traps for manipulation of magnetically labeled and unlabeled cells Thomas Henighan , Aaron Chen , Greg Vieira , Adam Hauser , Fengyuan Yang , Jeffrey Chalmers , Ratnasingham Sooryakumar Magnetic forces are frequently used for the manipulation of biological cells because magnetic fields are typically easier to use and have fewer effects on the cells than optical or electrical fields. While magnetic forces are typically used for bulk separation, it is considerably harder to magnetically manipulate a single cell, or a small number of cells. In this study we employ reprogrammable magnetization profiles created through lithographically patterned ferromagnetic disks as a template for producing highly localized trapping fields. The resulting magnetic field gradients can be modulated by an external magnetic field enabling directed forces to be applied on, (a) single, or a small number of immunomagnetically labeled biological cells and, (b) magnetic microspheres that act as magnetically actuated force transmitting probes to navigate fluid-borne unlabeled cells with micrometer precision. We demonstrate the mobile traps by remotely transporting and arranging, with programmed routines (\textit{a la} joystick), T-lymphocyte and leukemia cells on the platform. Without producing damage, the forces transport the cells with speeds up to 20 microns/sec across a silicon platform to predetermined sites. Monday, March 15, 2010 1:27PM - 1:39PM B27.00012: Picosecond Time-Resolved Small- and Wide-Angle X-ray Scattering of Proteins in Solution: A New Method for Tracking Protein Structural Changes Naranbaatar Dashdorj , Hyun Sun Cho , Friedrich Schotte , Philip Anfinrud A detailed mechanistic understanding of protein function requires knowledge of structural change on ultrafast time scales. Small- and wide-angle X-ray scattering (SAXS/WAXS) of proteins in solution exhibit a radial intensity distribution that is sensitive to protein size, shape, and structure. When acquired in a time-resolved fashion, these scattering patterns unveil conformational changes that occur as a protein executes its designed function. We recently developed the infrastructure required to record SAXS/WAXS scattering snapshots with 100-ps time resolution on the BioCARS beamline at the Advanced Photon Source in Argonne, IL. This methodology was used to probe structural changes in several proteins following laser excitation. In all systems studied thus far, the time-resolved scattering fingerprints reveal an unresolvably fast ($<$100 ps) structure change that presumably corresponds to tertiary motions. Subsequent structure changes occur on time scales that can span more than 8 decades. By characterizing the nature of protein structural changes in solution over a broad range of time scales, we aim to assess their biomechanics and probe the origins of the forces that drive allosteric structure transitions in multi-subunit proteins. Monday, March 15, 2010 1:39PM - 1:51PM B27.00013: Neutron Spin Echo Studies of Protein Dynamics Jyotsana Lal , Peter Fouquet , Marco Maccarini , Lee Makowski Neutron spin-echo (NSE) spectroscopy was used to study structural fluctuations that occur in hemoglobin (Hb) and myglobin (Mb) in solution. Using NSE in conjunction with Wide Angle X-ray Scattering (WAXS) to very high momentum transfer, q (up to 0.62 inverse Angstroms), the internal dynamics of these proteins were characterized at the level of the dynamical pair correlation function and self-correlation function in the time range of several picoseconds to a few nanoseconds. Comparison of data from the two homologous proteins collected at different temperatures and protein concentrations was used to assess the contributions to the data made by translational and rotational diffusion and internal modes of motion. Monday, March 15, 2010 1:51PM - 2:03PM B27.00014: Residence-time analysis of \textit{D. discoideum}-nanoelectrode contacts Bret Flanders , Prem Thapa Electrode-cellular interfaces are critical features of electrophysiological devices. The present effort investigates the effect of small negative voltages applied to on-chip, polythiophene nanoelectrodes on the attachment of \textit{D. discoideum} cells to the nanoelectrodes. The recently developed \textit{directed electrochemical nanowire assembly} method has been used to grow polyethylene dioxythiophene (PEDOT) nanoelectrodes on microscopic electrode arrays. \textit{D. discoideum} that are cultured on the arrays forage randomly in chemically and electrically isotropic environments and occasionally contact the polymeric nanoelectrodes. The distribution of residence times during which single cells were in contact with unbiased nanoelectrodes was measured. The statistics are Poisson with an average residence time of 250 s. The residence time-distribution for cells in contact with a -50 mV biased electrode is not Poisson, and the probability of observing a residence time greater that 250 s is significant. This effect will be discussed in light of recent evidence for the coupling of the seemingly disparate processes, cell-substrate adhesion and voltage-gated ion flux.