Bulletin of the American Physical Society
APS March Meeting 2022
Volume 67, Number 3
Monday–Friday, March 14–18, 2022; Chicago
Session Z07: Novel InstrumentationRecordings Available
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Sponsoring Units: DBIO Chair: Shubham Tripathi, Rice University Room: McCormick Place W-179A |
Friday, March 18, 2022 11:30AM - 11:42AM |
Z07.00001: Determining partitioning and transport coefficients for hydrophobic chemicals in polydimethylsiloxane (PDMS)-based devices Nathaniel G Hermann, Shane Hutson, Lisa McCawley, Dmitry Markov Organ-on-chip devices are an emerging technology with many possible novel applications. These devices are commonly made using polydimethylsiloxane (PDMS), a ubiquitous, versatile, and inexpensive polymer. However, partitioning of hydrophobic compounds in PDMS limits the current ability of organ-on-chip devices to control dosage of chemical solutions accurately and precisely. Here, we measure and model chemical-PDMS interactions, accounting for chemical partitioning across the PDMS interface mediated by free energy differential and internal diffusion in bulk PDMS. The model is validated via optical measurements of fluorescent dyes binding to and diffusing within PDMS. Interaction experiments then extract relevant chemical-PDMS interaction parameters, including diffusion and partition constants. This is demonstrated for nine chemicals, selected for current use in organ-on-chip studies. |
Friday, March 18, 2022 11:42AM - 11:54AM |
Z07.00002: Order-of-Magnitude Tuning of Microbial Growth Rates through Photo-Excitation Growth Spectroscopy Mehmet E Kilinc, Jacky Wan, Thomas E Kuhlman, Nathaniel M Gabor When exposed to light, microbial life shows unique and complex behaviors that are yet to be fully understood. Major questions remain about the effects of light on ATP production (e.g., photobiomodulation in mitochondria) and the near theoretical quantum limits of efficiency of photosynthetic bacteria. To unambiguously quantify such phenomena, it is necessary to develop new techniques that probe microbial growth dynamics in a highly controlled multi-parameter light environment. Here, we describe a novel method, called Photo-Excitation Growth Spectroscopy (PhotoExGS), that combines broadband supercontinuum excitation and real-time laser probes of microbial growth in highly controlled environments. Using this technique, we measured the growth of highly characterized and metabolically diverse bacteria, Rhodobacter sphaeroides and several key mutants. By shining spatially separated white light (supercontinuum laser wavelength from 400 nm to 2300 nm) with a highly characterized intensity distribution on an agarose microbial farm, we are able to generate high resolution movies of spectrally resolved dynamic growth. Strikingly, we find that the growth rate of Rhodobacter sphaeroides can be fine tuned over an order of magnitude using only light intensity and wavelength. Such tuning reveals several surprising features of growth dynamics in Rhodobacter sphaeroides, yet also indicates a powerful control for future population dynamics measurements in microbial systems. |
Friday, March 18, 2022 11:54AM - 12:06PM |
Z07.00003: Input-response function for spatiotemporally modulated input transcription factor and target gene outputs Anand P Singh, Ping Wu, Sergey Ryabichko, João Raimundo, Michael Swan, Eric F Wieschaus, Jared Toettcher, Thomas Gregor During embryonic development, cell specification is tightly regulated in space and time by specific DNA-binding proteins called transcription factors (TFs). However, the input-response functions between transcription factors and the gene outputs that will eventually give rise to distinct cell fates are largely unknown. To address this issue, we engineered embryos with no natural input sources and with a fast optogenetically tunable single-input system. Our custom-built light delivery platform allows acute temporal and spatial light patterns to instruct light-sensitive TF concentrations in living fly embryos. These tools enable us to induce and control input TF concentration on a minute-time scale and record output gene transcription in real-time. We found the target genes showing differential transcriptional activity in response to acute TF concentration perturbations in time and space. Our synthetic approach to modulate TF concentration while concurrently recording transcriptional gene output in real-time presents a powerful approach to study transcriptional dynamics in vivo. |
Friday, March 18, 2022 12:06PM - 12:18PM |
Z07.00004: Automated detection and identification of membrane proteins in atomic force microscopy images using machine learning Creighton Lisowski, Dylan Weaver, Gavin M King, Ioan Kosztin Atomic force microscopy (AFM) is an effective single molecule technique for imaging membrane proteins in their native environment under physiological conditions. In spite of the high out of membrane (z-axis) spatial resolution (~1Å) of AFM images, due to the finite size of the AFM tip and other factors, the corresponding lateral resolution is usually more than an order of magnitude lower (>1nm). By employing a recently developed localization AFM (LAFM) approach from the Scheuring laboratory, the lateral resolution can be enhanced (typically by a factor of three), a major advance for the field. Yet, the processing and evaluation of the large number of images recorded in AFM experiments are time consuming and challenging. Here we present a workflow for the automated detection and identification of membrane proteins in a previously recorded stack of AFM images. The protein blobs are detected by standard image segmentation methods. After proper alignment and LAFM resolution enhancement, the protein blobs are identified by using a deep convolutional neural network (CNN). The CNN is trained by using (i) simulated AFM images of atomistic protein structures in the OPM database, and (ii) previously validated AFM images. The effectiveness of the proposed workflow is demonstrated for a system comprising membrane proteins from the general secretory system in a supported E. coli lipid bilayer. |
Friday, March 18, 2022 12:18PM - 12:30PM Withdrawn |
Z07.00005: Approaches to Peptide Photodissociation for Single-Molecule Protein Sequencing Derek M Stein We analyze the feasibility of using light to fragment a peptide into its constituent amino acids before identifying them by mass spectrometry (MS) for the purpose of single-molecule protein sequencing. Laser power considerations strongly favor photofragmenting peptides in solution before they leave the ion source rather than in the gas phase. %in the mass spectrometer. Ultaviolet (UV) wavelengths near 200~nm are weakly absorbed in water, and a single photon can selectively cleave the peptide bonds that link amino acids together. These properties make UV photofragmentation more promising than methods based on infrared or x-ray light. We develop a simple model of the probability of liberating an amino acid intact by cleaving the peptide bonds on either side of it before the light damages the amino acid itself. We predict 193~nm light can liberate many amino acids with probabilities ranging from 0.65-0.92; however, the aromatic amino acids and histidine, methionine, arginine, and lysine, which are relatively susceptible to photodamage, would be liberated intact with a probability in the range 0.004-0.330. These findings suggest that UV photofragmentation could reveal a significant amount of a single protein's sequence information to a mass spectrometer. |
Friday, March 18, 2022 12:30PM - 12:42PM |
Z07.00006: Biocompatible surface functionalization architecture for a diamond quantum sensor Mouzhe Xie, Xiaofei Yu, Lila Rodgers, Daohong Xu, Ignacio Chi Durán, Adrien Toros, Niels Quack, Nathalie P de Leon, Peter Maurer Diamond with nitrogen-vacancy (NV) centers is a quintessential material used in quantum sensing and other high precision measurements. One promising application of such novel NV sensors is to study single-molecule biophysics, which generally require chemical modification of the substrate to immobilize target molecules of interest and prevent nonspecific adsorption of unwanted molecular species. However, diamond surfaces are known to be difficult to chemically modify, bottlenecking the realization of single-molecule experiments on target molecules under their biologically relevant conditions. |
Friday, March 18, 2022 12:42PM - 12:54PM |
Z07.00007: Enhanced nonenzymatic glucose detection through the synergy of Cu and Ni in a co-sputtered heteroatomic thin film Brianna Barbee, Baleeswaraiah Muchharla, Wei Cao, Hani E. Elsayed-Ali, Adetayo Adedeji, Abdennaceur Karoui, Kishor Kumar Sadasivuni, Bijandra Kumar Diabetes is a chronic disease that spreads because of having too much sugar in blood. Millions of people across the globe suffer health condition due to diabetes. Continuous blood glucose monitoring is a key to detect and treat patients in early stages of diabetes. In this work, we report the synthesis and characterization of nickel (Ni) and copper (Cu) heteroatomic thin films using RF magnetron sputtering technique. As prepared Cu-Ni thin films were utilized as sensing platform for nonenzymatic glucose sensors. A comparative study with pristine Ni and Cu films evidence that Cu-Ni heteroatomic film exhibits synergistic effect for glucose detection in basic media. It also displayed applicability for glucose detection with varying concentration from 10 µM to 5 mM. The developed sensing platform with superior selectivity and enhanced stability can be used for realistic application. |
Friday, March 18, 2022 12:54PM - 1:06PM |
Z07.00008: Direct measurement of single molecule DNA bend-stiffness with nano-magnetic torque balance Isaac Shelby, Zeeshawn Kazi, Kai-Mei C Fu, Paul Wiggins The tight-bending of DNA plays a key role in many biological contexts, from nucleosome formation to DNA packaging in viral capsids. Despite its biological significance, questions remain about the mechanical properties of DNA on sub-persistence-length scales. Using magPI, a novel approach for measuring the position and orientation of magnetic nanoparticle probes, we perform a torque-balance measurement on short dsDNA molecules to directly measure the DNA bending free energy. We expect the magPI approach will be widely applicable to other biological systems where long-duration probe-orientation tracking is critical. |
Friday, March 18, 2022 1:06PM - 1:18PM |
Z07.00009: Quantifying out-of-equilibrium transcriptional bursting in living fly embryos Po-Ta Chen, Benjamin Zoller, Michal Levo, Thomas Gregor Gene expression is intrinsically dynamic. A series of molecular events, including RNA polymerase II (Pol II) loading, underlie the regulation of gene (or transcriptional) activation. However, knowledge about the dynamics of these molecular events remains elusive. Here, we present an optimized two-photon microscope to measure gene activity of individual loci in living fly embryos. We achieve single RNA detection sensitivity, and combined with statistical analysis we construct time serieses of single Pol II loading events from the transcriptional output. This leads, for the first time, to the observation of non-steady state transcriptional bursting. We quantified the kinetic parameters of Pol II loading events, such as waiting time distributions of Pol II loading, time-dependent transcription rates, and durations of transcriptional bursting. With a novel inference framework, we extract the non-stationary bursting parameters from the transcriptional output. Applying our approach to multiple genes, we found common regulatory strategies that are shared among different genes, regardless of space and time during embryo development, opening the path to universal features of out-of-equilibrium gene control. |
Friday, March 18, 2022 1:18PM - 1:30PM |
Z07.00010: Tuning cell division machinery in oocytes using light Jinghui Liu, Tzer Han Tan, S. Zachary Swartz, Nikta Fakhri The process of cell division relies on the coordination of chemical and mechanical signals in response to cell cycle cues. During early developmental processes in starfish egg cells, actomyosin contractility is patterned by Cdk1 gradients through the activation of Rho-GTP pathway, and organizes the large-scale shape deformation that can be observed in either asymmetric (meiosis) or symmetric (mitosis) divisions. To investigate the role of geometry, mechanics and biochemistry, we decouple the Rho-actomyosin pathway from pre-patterned cell cycle signals by engineering and delivering an optogenetic activator of Rho-GTP into starfish oocytes arrested at the prophase of meiosis I. Using the light activation of local Rho-GTP combined with global shape perturbations, we can induce large-scale mechanical deformations seen during symmetric and asymmetric cell division. Our results can shed light on mechanisms of mechanochemical patterns and provide a blueprint for designing programmable active mechanochemical materials. |
Friday, March 18, 2022 1:30PM - 1:42PM |
Z07.00011: FRET Based Biosensors for CBRN Threat Detection Steven M Demers, Kaitlin Lawrence, Ashlee Swindle, Wendy Kuhne, Candace Langan Advanced sensor capabilities for the simultaneous on-site detection of chemical, biological, and radiological/nuclear (CBRN) threats would significantly limit the risk of exposure to personnel and allow for the rapid, in field collection of essential scientific data. One way to accomplish this is to create a multiplexed Förster Resonance Energy Transfer (FRET) based sensor that is capable of simultaneous CBRN detection. FRET based sensors are tailorable, specific, and highly dependent on donor-acceptor distance, where the distance dependence is the key component of FRET based biosensors. The energy transfer was measured between donors (quantum dot and dyes) and acceptors (gold nanoparticles and quenchers) bound by double-stranded DNA aptamers that target specific CBRN threats. Binding of the target analyte to the DNA aptamer causes conformational changes to the DNA structure that alters the donor-acceptor distance, creating a detectable signal change in the fluorescence response. Nanometal surface energy transfer (NSET) for different plasmonic metal (gold, silver, and copper) nanoparticles was investigated to examine the effectiveness and reliability of these different metals for NSET based biosensing. Refinements to the original NSET equations with regards to particle size and composition were implemented to measure the effect of energy transfer from different metallic nanoparticles. Calculated NSET and FRET distances for Cu, Ag, and Au metal nanoparticles will be presented as well as results for the detection of a sarin metabolite (methylphosphonic acid) and anthrax protective antigen 63 using different biosensor arrangements. |
Friday, March 18, 2022 1:42PM - 1:54PM |
Z07.00012: Anatomical Study of Cells and Tissues using Helium Ion Microscopy Leila Kasaei, Antonio Merolli, Hussein Hijazi, Viacheslav Manichev, Leonard C Feldman Helium Ion Microscopy (HeIM) has advantages for the study of cells and tissues, not available with other ultra-microscopy techniques like Scanning Electron Microscopy (SEM). Imaging without an electroconductive coating of the sample, reveals more details of biological structures (an insulating material) compared to conductivity-coated SEM, making HeIM an ideal component for correlative microscopy analysis, combining nanoscale imaging with optical spectroscopy. The lack of coating allows samples to be re-imaged easily with fluorescence or optical microscopy in a reversible process, allowing multiple data sets to be acquired for the same sample. We show how a minimal sample preparation (often a simple formalin fixation) permits the analysis of a larger number of specimens in the same unit time compared to other techniques that demand multiple steps. We report our specific experience in employing HeIM based correlative microscopy on cells (Vero E6, Schwann cells and oligodendrocytes) and tissue (brain, nerve, retina, kidney). For instance, HeIM shows clearly the morphological network of microfilaments (filopodia, lamellipodia or tunneling nanotubes), after fluorescence microscopy highlighted the presence of fibrillar actin (filament forming proteins). In the case of tissue imaging, the absence of coating provided surface details of fine structure on nerve tissue devoid of shrinkage artifacts in myelinated fibers. |
Friday, March 18, 2022 1:54PM - 2:06PM |
Z07.00013: Using Neutron and X-ray Reflectivity to Investigate the Interfacial Structure of Cell Monolayers Kathleen Cao, Ann Junghans, Tanvi Subramanian, Anna Gaffney, Adam Lam, Wei Bu, Binhua Lin, Mark L Schlossman, Jaroslaw Majewski, Ka Yee C Lee, Luka Pocivavsek Decades worth of scattering studies on model membrane systems, including lipid monolayers at the air-water interface and supported lipid bilayers on solid substrates, is motivated by their connection to the cell membrane. However, cell biology does not readily translate to physiology. Most biological interfaces are multi-cellular monolayers and monolayer biology is very different than single cell systems. Depending on the biological situation, cellular adhesion to interfaces is desirable or undesirable. In order to optimize material properties of biomedical devices, knowledge of whether cells stick or not stick to these materials is crucially important. Our prior studies utilized neutron reflectometry to study the cell membrane-substrate separation in cell monolayers, where the separation length is indicative of adhesion strength. We extend our work to x-rays because they provide a rich set of tools to probe the structure and interactions of cellular monolayers. X-rays can also overcome the low intensity, long collection times, and narrow Qz range limitations of neutrons. Applying x-ray scattering techniques to a true living cellular system will help us gain insight to parameters important for the improved design of medical devices. |
Friday, March 18, 2022 2:06PM - 2:18PM |
Z07.00014: 3D Tracking of Continuous Nanoscale Objects in Complex Environments Peter T Brown, Douglas P Shepherd Understanding 3D behavior of nanoscale biological systems commonly relies on 2D optical imaging combined with computational analyses to extract 3D motion. For example, point-spread function (PSF) engineering approaches infer axial position from the PSF shape but are restricted to sparse objects. To relax the sparsity requirement, rapidly acquired 2D planes can approximate a 3D image as in single-objective light-sheet fluorescence microscopy. Ultimately the brightness and density of emitters limit the speed and accuracy of fluorescence-based 3D tracking. A promising alternative is optical diffraction tomography (ODT), an interferometric technique that infers the 3D refractive index by combining different views of a sample under coherent illumination. Because ODT does not depend on fluorescence it provides high signal at ~1000 volumes per second. Here we describe our recent development of a multi-modal structured illumination (SIM) and ODT microscope based on a digital micromirror device. Using binary patterns to generate SIM and ODT patterns improves speed and reliability compared to previous approaches. We demonstrate our approach by tracking continuous nanoscale objects such as isolated cilia, and study hydrodynamic interactions of diffusing colloidal particles with a boundary. |
Friday, March 18, 2022 2:18PM - 2:30PM |
Z07.00015: Onset of lipid-nanoparticle cooperative association in biomembranes: role of nanoparticle charge and membrane stiffness Anurag Chaudhury, Wei Bu, Maximilian Skoda, Koushik Debnath, Nikhil Jana, Jaydeep K Basu Nanoparticles are of great interest in Biophysics because they have been considered as next-generation tools for nanoscale targeted drug delivery. In nanoparticle-cell interactions, the first encounter occurs with the cell membrane, whose properties along with the type of nanoparticles determine the binding behaviour and changes brought upon to the cell membrane. Our recent study with multi-component phase-segregated Floating Lipid Monolayers as a model using X-ray scattering reveals that the Cationic nanoparticles have a preferential binding to one of the phases. The structural disordering and penetration depth is determined by the stiffness and charge density of the membrane1. The recent extension of this work using Neutron Reflectivity studies reveals that the nature of the nanoparticle surface charge distributions affects their penetration and coverage over the membrane. Interestingly, it has been observed that the type of charge of the nanoparticles cause them to form lipid-nanoparticle complex by extracting lipids out of the membrane. This is mediated by particle-particle interactions and is found to occur at a critical concentration. |
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