Bulletin of the American Physical Society
APS March Meeting 2011
Volume 56, Number 1
Monday–Friday, March 21–25, 2011; Dallas, Texas
Session W39: Experimental Techniques in Biophysics |
Hide Abstracts |
Sponsoring Units: DBP Chair: Mark Reeves, George Washington University Room: A124/127 |
Thursday, March 24, 2011 11:15AM - 11:27AM |
W39.00001: ABSTRACT WITHDRAWN |
Thursday, March 24, 2011 11:27AM - 11:39AM |
W39.00002: Spatially resolved absorption spectroscopy of bio - assemblies on a micron scale Silki Arora, Jennifer Mauser, Debopam Chakrabarti, Alfons Schulte We have developed a novel approach to measure optical absorption spectra with spatial resolution at the micron scale. The setup employs a confocal microscope with a broadband white light excitation beam in transmission geometry. An aperture controls the amount of illuminating light and localizes the area of excitation. The setup is employed to measure the absorption spectrum of single red blood cells ($\sim $ 7 microns diameter) under solution conditions. The spatial resolution in the lateral direction is found to be better than three microns. Through measurements of the transmitted intensity in met- myoglobin and calcein dye nanoliter solutions at fixed path lengths, we establish that the absorbance varies linearly with concentration over the range from 0.1 to 2 mM. Our instrument enables measurements of spatial variations in the optical density of small samples and may find application in monitoring biological assemblies at the single cell level. [Preview Abstract] |
Thursday, March 24, 2011 11:39AM - 11:51AM |
W39.00003: Stochastic super resolution imaging by diffusive probes Thomas Dorn, John Ewalt, Francisco Marquez, George Shubeita Optical microscopy is a powerful tool for the imaging of cells and bio-materials, however the resolution is limited by diffraction and thus objects closer than a few hundred nanometers cannot be individually resolved. We report a novel stochastic super-resolution technique which relies on diffusing probes in which the resolution is determined by probe size and Forster radius of energy transfer. By recording a time-series of images similar to other super-resolution techniques, the centers of bright spots can be determined with sub-pixel accuracy by fitting to the point spread function. The centroids can then be used to reconstruct a super-resolution image. [Preview Abstract] |
Thursday, March 24, 2011 11:51AM - 12:03PM |
W39.00004: Near-field Approaches to Subcellular Tissue Abalation Deepa Raghu, Joan Hoffmann, Benjamin Gamari, Andrew Gomella, Mark Reeves We report on the development of a near-field approach to MALDI (Matrix-assisted laser desorption and Ionization). In this technique analytes embedded in an energy- absorbing matrix are ablated from the surface of a sample. In the infrared region, the matrix can be water by exciting the 3-micron vibrational mode of the water molecule. We use a 3-micron wavelength lasers, coupled with a near-field scanning microscope to ablate material from cells of different membrane stiffness. We have been able to reproducibly ablate features as small as 1 micron in diameter in cell and have characterized the power-dependence of the ablation process. We will review our findings and describe demonstrations of tissue modification by this approach at length scales smaller than a single cell. This approach has the potential to allow the identification and mapping of proteins expressed in intact cells and tissues, which is of great interest as protein expression connects genomic information with the functioning of an organism. [Preview Abstract] |
Thursday, March 24, 2011 12:03PM - 12:15PM |
W39.00005: Rotational Diffusion of Plasmon-Resonant Gold Nanorods for Depth-Resolved Microrheology Using Optical Coherence Tomography Amy Oldenburg, Raghav Chhetri, Krystian Kozek, Aaron Johnston-Peck, Joseph Tracy The ability to perform microrheology in optically thick samples would enable analysis of bulk tissues. Optical coherence tomography (OCT) provides imaging several mean free scattering path lengths into tissue. In this study we report the use of plasmon-resonant gold nanorods as microrheological sensors in OCT. Nanorods exhibit a longitudinal mode that is excited when they are oriented parallel to the polarization of the incident light, which is favorable for passive microrheology using polarized light to monitor their rotational diffusion. We demonstrate measurements of the rotational diffusion of unconfined, colloidal gold nanorods using polarization-sensitive OCT, and validate the Stokes-Einstein relationship for the nanorods in simple fluids of varying viscosity. We then show that OCT provides depth-resolved imaging of fluid viscosity through measurements of the rotational diffusion rate of the nanorods. [Preview Abstract] |
Thursday, March 24, 2011 12:15PM - 12:27PM |
W39.00006: Monte-Carlo Study of Binding Kinetics in Surface Plasmon Resonance Systems Matthew Raum, Uwe T\"auber, Kimberly Forsten-Williams Surface plasmon resonance (SPR) has become a standard tool for studying ligand-receptor binding reactions in real-time. Ideally the data obtained with this technique allows measurement of kinetic reaction rates (rather than merely the equilibrium constant for the reaction). In typical experimental configurations one species is immobilized near the active surface while its binding partner is initially suspended in solution, flowing across the active surface. It is generally appreciated that reaction rates observed in SPR experiments are affected by mass transport if the time scales for reaction and transport in the system are comparable. The issue of ensuing effective reaction rates has been treated through different approaches in the literature. The goal of this research is a quantitative study of how faithfully intrinsic binding rates can be measured in SPR devices. We employ a lattice Monte Carlo method to simulate SPR experiments in order to test the efficacy of common SPR analytical techniques. We point out where existing analytical techniques succeed or fail in measuring binding and dissociation rates, and investigate the influence of secondary parameters in the system (such as the flow rate) on experimental data. [Preview Abstract] |
Thursday, March 24, 2011 12:27PM - 12:39PM |
W39.00007: Fluorescence Correlation Spectroscopy of Tryptophan-containing Proteins in Sugar Solutions using Two Photon Excitation David Sidebottom, Nathan Holman, Yuli Wang, Michael Nichols Simple sugars are often incorporated in cryopreserving media to aid in the preservation of biomaterials and functional proteins. However, the mechanism by which sugars provide protection is still openly debated. As part of a project to investigate the behavior of proteins in sugar solutions, we are developing Fluorescence Correlation Spectroscopy (FCS), using a novel two photon excitation at 532 nm, as a selective probe of protein dynamics for tryptophan-containing proteins. Our goal is to monitor possible alterations in the protein's hydrodynamic radius caused by preferential binding of sugars to its surface. [Preview Abstract] |
Thursday, March 24, 2011 12:39PM - 12:51PM |
W39.00008: Continuous Nano-Particle Transport in a Standing Wave Optical Line Trap Vassili Demergis, Ernst-Ludwig Florin Since the introduction of the single beam optical trap (SBT) by Ashkin et. al. in 1986, trapping and manipulation of micron-sized particles by optical forces has become instrumental in many areas of research. However, controlled transport of large numbers of particles is difficult using a SBT. Here we introduce a technique for controlled transport that we call an Optical Capillary (OC), named for its ability to strongly confine and continuously transport nanometer-sized particles. The OC, generated by an optical standing wave pattern, is especially strong along the optical axis due to the compensation of the axial scattering force. We utilize the lateral scattering forces to control the transport of particles along a line perpendicular to the optical axis. The measured velocity profiles of single particles in the OC agree with our model predictions. [Preview Abstract] |
Thursday, March 24, 2011 12:51PM - 1:03PM |
W39.00009: Laser- based Insect Tracker (LIT) Leonardo Mesquita, Shiva Sinha, Rob de Ruyter Van Steveninck Insects are excellent model systems for studying learning and behavior, and the potential for genetic manipulation makes the fruitfly especially attractive. Many aspects of fruitfly behavior have been studied through video based tracking methods. However, to our knowledge no current system incorporates signals for behavioral conditioning in freely moving flies. We introduce a non-video based method that enables tracking of single insects over large volumes ($>$8000cm3) at high spatial ($<$1mm) and temporal ($<$1ms) resolution for extended periods ($>$1 hour). The system uses a set of moveable mirrors that steer a tracking laser beam. Tracking is based on feedback from a four-quadrant sensor, sampling the beam after it bounces back from a retro reflector. Through the same mirrors we couple a high speed camera for flight dynamics analysis and an IR laser for aversive heat conditioning. Such heat shocks, combined with visual stimuli projected on a screen surrounding the flight arena, enable studies of learning and memory. By sampling the long term statistics of behavior, the system augments quantitative studies of behavioral phenotypes. Preliminary results of such studies will be presented. [Preview Abstract] |
Thursday, March 24, 2011 1:03PM - 1:15PM |
W39.00010: Power dependent study of kinetics of TRF2 recruitment in cells due to DNA damage caused by ultrafast near-IR Laser Manas Bhalerao, Nazmul Huda, David Gilley, Samarendra Mohanty Ultrafast laser microbeam is finding widespread applications in eliciting highly localized damage to cellular components allowing study of in-situ repair machinery. While the high peak power density that exists in ultrafast laser can cause various types of DNA damage including double strand breaks (DSB), tuning the power of these laser microbeams may cause specific type of DNA damage. Here, we report wavelength and dose dependent parametric study of kinetics of TRF2 recruitment in cells due to DNA damage caused by ultrafast near-IR Laser. A tunable Ti: Sapphire laser beam was coupled via laser port of an inverted microscope. Spot and line laser micro-irradiation pattern in nuclear sites of HT1080 cells expressing YFP-tagged TRF2 was achieved by piezo-scanning mechanism. The recruitment of TRF2-YFP was found to depend highly on the peak irradiance of the near-IR laser microbeam, the required threshold irradiance being much higher than that observed for DSB. Further, recruitment kinetics revealed that the time constant for TRF2 recruitment depends on the laser irradiance parameters. The time required for TRF2 recruitment was found to decrease with increased peak irradiance. We will present these results and also elucidate on physical mechanism of DNA damage caused by ultrafast laser microbeam. [Preview Abstract] |
Thursday, March 24, 2011 1:15PM - 1:27PM |
W39.00011: Background Elimination and Noise Reduction by Mechanical Modulation Raman Spectroscopy Kathleen Hinko, Chieze Ibeneche, Andrea Keidel, Tobias Bartsch, Ernst-Ludwig Florin Raman spectroscopy is widely used by biophysicists for the molecular identification of cellular substructures. However, there are high levels of background and noise associated with Raman spectra from other molecules in the microscopic detection volume. We present two methods of mechanical modulation for background subtraction and noise reduction in a Raman microscope: (1) a three-axis stage modulation for fixed objects and (2) a separate optical trap modulation for objects in solution. With our technique, we completely eliminate the background in our spectra and improve the signal-to-noise ratio by two orders of magnitude. We applied this technique to lipid vesicles and fission yeast cells in solution. Additionally, we obtained mechanical modulation Raman spectra of fission yeast in three dimensions and observed spatial differences in the molecular composition for different metabolic states of a single yeast cell. [Preview Abstract] |
Thursday, March 24, 2011 1:27PM - 1:39PM |
W39.00012: Ultraweak bioluminescence dynamics and singlet oxygen correlations during injury repair in sweet potato Marius Hossu, Lun Ma, Wei Chen Ultraweak bioluminescence at the level of hundreds of photons per second per square centimeter after cutting injury of sweet potato was investigated. A small emission peak immediate after cutting and a later and higher peak were observed. Selective singlet oxygen inhibitors and sensors have been use to study the contribution of singlet oxygen during the curing process, demonstrating increased presence of singlet oxygen during and after the late bioemission peak. It was confirmed that singlet oxygen has direct contribution to ultraweak bioluminescence but also induces the formation of other exited luminescent species that are responsible for the recorded bioluminescence. [Preview Abstract] |
Thursday, March 24, 2011 1:39PM - 1:51PM |
W39.00013: Obtaining optical properties using Representative Layer Theory Neema Razavi, Brain Yust, Dhiraj Sardar Reliable and minimally invasive methods for diagnosis of toxicity and onset of disease are important for advances in clinical practices. This is commonly achieved through the optical properties, such as a change in the absorption or scattering strength of the diseased tissue. Thus, being able to quantitatively characterize these changes is important to advancements in medical diagnostic methods. By adapting the Representative Layer Theory to the integrating sphere technique, very thin biological samples may be optically characterized, yielding a quick and easy method for monitoring optical changes as a function of disease progression. Samples, consisting of cells, dyes, and nanoparticles of known concentrations were optically characterized at multiple wavelengths. Optical properties obtained by the Representative Layer Theory are compared to those obtained through other methods, such as Kubelka-Munk and Inverse Adding Doubling which are known to have sample thickness limitations. [Preview Abstract] |
Thursday, March 24, 2011 1:51PM - 2:03PM |
W39.00014: An \textit{in vitro} approach to understanding intracellular motor-based cargo transport Rafael Longoria Casasa, Hayley Manning, George Shubeita Microtubule-based molecular motors are responsible for the long range transport of intracellular cargoes. Most cargoes move bidirectionally yet reach their destination in the cell. The mechanism by which the seemingly random bidirectional motion of cargoes is regulated by the cell to produce directed transport remains unclear. Two distinct models have been proposed: coordination via a tug-of-war, the dynamics of which depend only on the properties of the motors; and coordination via non-motor proteins. However, no direct evidence for either one has been found yet. We present an experimental method that can address the different predictions of these models. We reconstitute \textit{in vitro} transport of endogenous motor-driven lipid droplets purified from \textit{Drosophila} embryos. Global transport dynamics are observed under varied medium conditions by DIC microscopy. Combined with stall force measurements using an optical trap, these investigations relate the global dynamics to local changes in force production of the motors which give us a direct handle to differentiate between the different models of transport. [Preview Abstract] |
Thursday, March 24, 2011 2:03PM - 2:15PM |
W39.00015: Nonlinear optical microscopy in biology: Combining second-harmonic generation and two-photon fluorescence imaging Koen Clays Optical microscopy has been since long a truly enabling visualization technique in the biological and biomedical sciences. Linear optical microscopy relies on simple linear optical effects. Nonlinear optical microscopy relies on the nonlinear optical properties of endogenous or exogenous chromophores to produce a better image. Two-photon fluorescence (TPF), a third-order nonlinear optical effect and observed at the focal spot only due to the quadratic intensity dependence, results in inherently higher resolution than possible for one-photon fluorescence, observed over the complete Rayleigh range. Second-harmonic generation (SHG) is a second-order nonlinear optical effect only observed for non-centrosymmetric arrangements of non-centrosymmetric chromophores. While this does put a restriction on the chromophores that can be used, it also results in structural information about symmetry when used in combination with TPF. TPF, being a third-order nonlinear process, is not restricted by any symmetry consideration. We will review the molecular design criteria for exogenous probes for combined SHG and TPF nonlinear microscopy, provide examples of optimized chromophores and show microscopy images demonstrating the use of such chromophores in nonlinear microscopy. [Preview Abstract] |
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