Bulletin of the American Physical Society
APS March Meeting 2017
Volume 62, Number 4
Monday–Friday, March 13–17, 2017; New Orleans, Louisiana
Session E4: Advances in Cellular and Multicellular ImagingFocus
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Sponsoring Units: DBIO Chair: Ralf Bundschuh, The Ohio State University Room: 263 |
Tuesday, March 14, 2017 8:00AM - 8:36AM |
E4.00001: Mechanoregulation of clathrin-mediated endocytosis in isolated cells and developing tissues Invited Speaker: Comert Kural Clathrin-coated assemblies bear the largest fraction of the endocytic load from the plasma membrane of eukaryotic cells. However, dynamics of clathrin-mediated endocytosis (CME) have not been established within tissues of multicellular organisms due to experimental and analytical bottlenecks in determining the lifespan of clathrin-coated structures. We found that clathrin coat growth rates obtained from fluorescence microscopy acquisitions can be utilized as reporters of CME dynamics. Growth rates can be assembled within time windows shorter than the average clathrin coat lifetime and, thereby, allow probing the changes in CME dynamics in real time. Furthermore, this novel approach is applicable to tissues as it is not prone to particle detection and tracking errors, which result in underestimation of the clathrin coat lifetimes. Exploiting these advantages, we detected spatial and temporal changes in CME dynamics within Drosophila amnioserosa tissues at different stages of embryo development. We also found that increased membrane tension impedes CME through inhibition of formation and dissolution of clathrin-coated structures. Therefore, the parameters defining clathrin coat dynamics (i.e., lifetime, formation density and growth rates) can be utilized to monitor the spatiotemporal gradients of the plasma membrane tension during cell migration and spreading. [Preview Abstract] |
Tuesday, March 14, 2017 8:36AM - 8:48AM |
E4.00002: Correlated Fluorescence-Atomic Force Microscopy Studies of the Clathrin Mediated Endocytosis in SKMEL Cells Steve Smith, Amy Hor, Anh Luu, Lin Kang, Brandon Scott, Elizabeth Bailey, Adam Hoppe Clathrin-mediated endocytosis is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorescent fusion proteins (actin filaments labeled with green phalloidin-antibody and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. Results from our work are compared against dynamical polarized total internal fluorescence (TIRF), super-resolution photo-activated localization microscopy (PALM) and transmission electron microscopy (TEM) to draw conclusions regarding the prominent model of vesicle formation in clathrin-mediated endocytosis. [Preview Abstract] |
Tuesday, March 14, 2017 8:48AM - 9:00AM |
E4.00003: High-Throughput Light Sheet Microscopy for the Automated Live Imaging of Larval Zebrafish Ryan Baker, Savannah Logan, Christopher Dudley, Raghuveer Parthasarathy The zebrafish is a model organism with a variety of useful properties; it is small and optically transparent, it reproduces quickly, it is a vertebrate, and there are a large variety of transgenic animals available. Because of these properties, the zebrafish is well suited to study using a variety of optical technologies including light sheet fluorescence microscopy (LSFM), which provides high-resolution three-dimensional imaging over large fields of view. Research progress, however, is often not limited by optical techniques but instead by the number of samples one can examine over the course of an experiment, which in the case of light sheet imaging has so far been severely limited. Here we present an integrated fluidic circuit and microscope which provides rapid, automated imaging of zebrafish using several imaging modes, including LSFM, Hyperspectral Imaging, and Differential Interference Contrast Microscopy. Using this system, we show that we can increase our imaging throughput by a factor of 10 compared to previous techniques. We also show preliminary results visualizing zebrafish immune response, which is sensitive to gut microbiota composition, and which shows a strong variability between individuals that highlights the utility of high throughput imaging. [Preview Abstract] |
Tuesday, March 14, 2017 9:00AM - 9:12AM |
E4.00004: Analysis of T lymphocyte activation measured by Super-Resolution Microscopy Leonard Campanello, Maria Traver, Hari Schroff, Brian Schaefer, Wolfgang Losert Tight regulatory control of the activation signal in T lymphocytes is necessary to prevent the immune response from getting too large or persisting too long. Utilizing cutting-edge super-resolution imaging technologies in combination with quantitative image analysis, we explore one aspect of this regulation in activated cells: the dynamics of the protein MALT1. Our goal is to analyze how the motion of MALT1 within the cell affects the transduction and regulation of the activation signal. A focus of our analysis is to measure anisotropies in the spatial organization of MALT1 and the shape of the related larger scale protein complex that it is a part of, the POLKADOTS Signalosome. [Preview Abstract] |
Tuesday, March 14, 2017 9:12AM - 9:24AM |
E4.00005: Optical Ptychographic Microscope for Quantitative Bio-Mechanical Imaging Nicholas Anthony, Guido Cadenazzi, Keith Nugent, Brian Abbey The role that mechanical forces play in biological processes such as cell movement and death is becoming of significant interest to further develop our understanding of the inner workings of cells. The most common method used to obtain stress information is photoelasticity which maps a samples birefringence, or its direction dependent refractive indices, using polarized light. However this method only provides qualitative data and for stress information to be useful quantitative data is required. \newline Ptychography is a method for quantitatively determining the phase of a samples’ complex transmission function. The technique relies upon the collection of multiple overlapping coherent diffraction patterns from laterally displaced points on the sample. The overlap of measurement points provides complementary information that significantly aids in the reconstruction of the complex wavefield exiting the sample and allows for quantitative imaging of weakly interacting specimens\footnote{T. Godden et al., \textbf{Opt. Ex.} 2014}. \newline Here we describe recent advances at La Trobe University Melbourne on achieving quantitative birefringence mapping using polarized light ptychography with applications in cell mechanics\footnote{N. Anthony et al., \textbf{Nat. Sci. Rep.} 2016}.. [Preview Abstract] |
Tuesday, March 14, 2017 9:24AM - 9:36AM |
E4.00006: Exploring Membrane Dynamics during Electric Pulse Exposure with Second Harmonic Generation Erick Moen, Bennett Ibey, Hope Beier, Andrea Armani Optical second harmonic generation (SHG) is a powerful tool for investigating the nanostructure of symmetry-breaking materials and interfacial layers. Recently, we developed an imaging technique based on SHG for quantifying and localizing nanoporation in the plasma membrane of living cells. Nanosecond pulsed electric fields (nsPEF) were used to controllably disrupt the membrane, and the observed changes were validated against an extensible cell circuit model. In this talk, I will discuss the development of this method and its application to various cell types and stimuli, with a specific focus on bipolar (BP) nsPEF. BP nsPEF hold special interest as a cellular insult because they allow for a unique exposition of transmembrane potential and membrane charging/relaxation. Using this approach, we examine the structural response of the membrane as the temporal spacing between pulse phases was varied over several orders of magnitude and compare these results to the response when the cell is exposed to a monopolar (MP) nsPEF. Disagreement of the experimental results with the model demonstrates that biological processes may play a larger role than previously thought. These findings could lead to a greater understanding of the fundamental processes essential to all electroporation. [Preview Abstract] |
Tuesday, March 14, 2017 9:36AM - 9:48AM |
E4.00007: The 2-Body Cytoskeleton Problem: Studying Cell-Cell Fusion Mechanics in Osteoclasts with Multiscale Imaging Jesse Silverberg, Pei Ying Ng, Roland Baron, Peng Yin Most research on \textit{in vivo }cytoskeletal mechanics focuses on what happens in a single cell context. This foundational work has opened up new avenues to study higher-order problems, such as what happens when cells physically interact. For example, osteoclasts, one of the cell types responsible for maintaining healthy skeletal structure, are formed when \textasciitilde 10 or more mononuclear cells fuse into a multinuclear behemoth. But how does the cytoskeleton of two or more cells fuse? And what is the role of mechanics in understanding the resulting cytoskeletal organization? In this work, we use the multiscale multiplexed Molecular Atlas Platform to image and study the cytoskeletal mechanics of cell-cell fusion. Our work documents the processes involved and uses observed structures to infer mechanical events during these interactions. Broadly this work takes a technology-driven approach to perform fundamental exploratory work, and uses current state-of-the-art cytoskeletal mechanical modeling to interpret our observations. [Preview Abstract] |
Tuesday, March 14, 2017 9:48AM - 10:00AM |
E4.00008: Forward convolution approach to reconstructing 3D bacterial cell shape reveals MreB localizes based on geometric cues Benjamin Bratton, Randy Morgenstein, Zemer Gitai, Joshua Shaevitz Over the past few years we have developed an image-processing framework that allows us to extract precise 3D shapes of bacterial cells from fluorescence microscopy data. This approach, using active meshes, minimizes the difference between an observed Z-stack and model shapes that have been convolved with the experimental point spread function. From these xyz coordinates, we calculate geometric parameters such as local curvatures, surface areas, and the relative enrichment of fluorescent signals. This method generates reconstructions for a variety of bacterial sizes and shapes including rods, vibriods and spiral bacteria. As one example case for a particular fluorescent signal, we have been studying the localization of the bacterial actin homolog, MreB. Along with our previous work on the cell shape role of MreB polymers [Ouzounov et al., BiophysJ 2016] and MreB rotation [Morgenstein et al., PNAS 2015], here we show in straight and curved rod bacteria that MreB localizes based on local Gaussian curvature. This curvature localizing mechanism helps ensure rod-like growth of the cell and helps prevent branching. Gaussian curvature is the product of the two principal curvatures, something which can only be measured using a fully three dimensional notion of cell shape. Using MreB point mutants that have altered curvature sensitivities, we are testing the hypothesis that cells straighten deformations by patterning growth at the proper geometry. [Preview Abstract] |
Tuesday, March 14, 2017 10:00AM - 10:12AM |
E4.00009: Subcellular Nanoparticle Distribution from Light Transmission Spectroscopy Alison Deatsch, Nan Sun, Jeffrey Johnson, Sharon Stack, Carol Tanner, Steven Ruggiero We have measured the particle-size distribution (PSD) of subcellular structures in plant and animal cells. We have employed a new technique developed by our group, Light Transmission Spectroscopy---combined with cell fractionation---to accurately measure PSDs over a wide size range: from \textasciitilde 10 nm to 3000nm, which includes objects from the size of individual proteins to organelles. To date our experiments have included cultured human oral cells and spinach cells. These results show a power-law dependence of particle density with particle diameter, implying a universality of the packing distribution. We discuss modeling the cell as a self-similar (fractal) body comprised of spheres on all size scales. This goal of this work is to obtain a better understanding of the fundamental nature of particle packing within cells in order to enrich our knowledge of the structure, function, and interactions of sub-cellular nanostructures across cell types. [Preview Abstract] |
Tuesday, March 14, 2017 10:12AM - 10:24AM |
E4.00010: How to measure separations and angles between intra-molecular fluorescent markers Henrik Flyvbjerg, KIm I. Mortensen, Jongmin Sung, James A. Spudich We demonstrate a novel, yet simple tool for the study of structure and function of biomolecules by extending two-colour co-localization microscopy to fluorescent molecules with fixed orientations and in intra-molecular proximity. From each color-separated microscope image in a time-lapse movie and using only simple means, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space with accuracy and precision. The positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA molecules internally labelled with two fixed fluorophores, we demonstrate the accuracy and precision of our method using the known structure of double-stranded DNA as a benchmark, resolve 10-base-pair differences in fluorophore separations, and determine the unique 3D orientation of each DNA molecule, thereby establishing short, double-labelled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly. [Preview Abstract] |
Tuesday, March 14, 2017 10:24AM - 10:36AM |
E4.00011: Probing 4D chromatin dynamics in live cells Yoon Jung, Kuan-Chung Su, Iain Cheeseman, Nikta Fakhri The cell nucleus is a structurally complex architecture whose spatial organization and dynamics play a major role in gene regulation. Here, we introduce a new experimental technique combined with data analysis algorithms to generate live single cell 4D (space-time) chromatin dynamic maps. The technique builds upon the unique near-infrared photoluminescence properties of single-walled carbon nanotubes (SWNTs). We develop a biochemical platform to introduce the SWNTs into the nucleus of HeLa cells and covalently attach them to specific loci. We track the position of individual SWNTs in the nucleus with high temporal and spatial resolution to create dynamic correlation maps of interactions between different loci and uncover patterns of individual and collective motions and organization. [Preview Abstract] |
Tuesday, March 14, 2017 10:36AM - 10:48AM |
E4.00012: NaGdF$_{\mathrm{4}}$:Nd$^{\mathrm{3+}}$/Yb$^{\mathrm{3+}}$ Nanoparticles as Multimodal Imaging Agents Francisco Pedraza, Chris Rightsell, GA Kumar, Jason Giuliani, Car Monton, Dhiraj Sardar Medical imaging is a fundamental tool used for the diagnosis of numerous ailments. Each imaging modality has unique advantages; however, they possess intrinsic limitations. Some of which include low spatial resolution, sensitivity, penetration depth, and radiation damage. To circumvent this problem, the combination of imaging modalities, or multimodal imaging, has been proposed, such as Near Infrared Fluorescence imaging (NIRF) and Magnetic Resonance Imaging (MRI). Combining individual advantages, specificity and selectivity of NIRF with the deep penetration and high spatial resolution of MRI, it is possible to circumvent their shortcomings for a more robust imaging technique. In addition, both imaging modalities are very safe and minimally invasive. Fluorescent nanoparticles, such as NaGdF4:Nd3$+$/Yb3$+$, are excellent candidates for NIRF/MRI multimodal imaging. The dopants, Nd and Yb, absorb and emit within the biological window; where near infrared light is less attenuated by soft tissue. This results in less tissue damage and deeper tissue penetration making it a viable candidate in biological imaging. In addition, the inclusion of Gd results in paramagnetic properties, allowing their use as contrast agents in multimodal imaging. The work presented will include crystallographic results, as well as full optical and magnetic characterization to determine the nanoparticle's viability in multimodal imaging. [Preview Abstract] |
Tuesday, March 14, 2017 10:48AM - 11:00AM |
E4.00013: Nanoplasmonic Upconverting Nanoparticles as Orientation Sensors for Single Particle Microscopy. Shuang Fang Lim We show that the anisotropic disk shape of nanoplasmonic upconverting nanoparticles (NP-UCNPs) create changes in fluorescence intensity in the event of rotational motion. We determine the orientation by a three-fold change in fluorescence intensity, and further show a strong dependence of the luminescence intensity on the particle orientation and polarization of the excitation light. The luminescence intensity shows a three fold difference between flat and on edge orientation. The intensity also varies sinusoidally with the polarization of the incident light, with the ratio, I$_{\mathrm{max}}$/I$_{\mathrm{min}}$ of up to 2.02. Both the orientation dependence and I$_{\mathrm{max}}$/I$_{\mathrm{min}}$ is dependent on the presence of the gold shell on the UCNP. This orientation dependence of the fluorescence will enable the detection of the rotational motion of biomolecules coupled to the nanoparticle. Finally, we demonstrate tracking of the real-time rotational motion of a single NP-UCNP in a microfluidic channel.. [Preview Abstract] |
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