Bulletin of the American Physical Society
APS March Meeting 2021
Volume 66, Number 1
Monday–Friday, March 15–19, 2021; Virtual; Time Zone: Central Daylight Time, USA
Session V12: Instrumentation and TechniquesLive
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Sponsoring Units: DBIO Chair: Lauren Lastra, University of California Riverside; Panayotis Benetatos, Kyungpook Natl Univ |
Thursday, March 18, 2021 3:00PM - 3:12PM Live |
V12.00001: Multi-modal Single-molecule Imaging with Continuously Controlled Spectral-resolution (CoCoS) Microscopy Jonathan Jeffet, Yael Michaeli, Dmitry Torchinsky, Ifat Israel-Elgali, Noam Shomron, Timothy David Craggs, Yuval Ebenstein The ever-growing need to acquire high-throughput, dynamic data from multicolor species is driving the development of optical schemes that optimize the achievable spectral, temporal, and spatial resolution needed in order to follow biological, chemical and physical processes. Here we introduce Continuously Controlled Spectral-resolution (CoCoS) microscopy, an imaging scheme that encodes color into spatial read-out in the image plane, with continuous control over the spectral resolution. The concept enables single-frame acquisition of multiple color channels, allowing simultaneous, single-molecule colocalization for barcoding and Förster resonance energy transfer (FRET) experiments. The simple control over the spectral dispersion allows switching between imaging modalities at a click of a button. We demonstrate the utility of CoCoS for multicolor localization microscopy of microRNA barcodes in clinical samples, single-molecule FRET measurements, and single-molecule spectroscopy. CoCoS may be integrated as a simple add-on to existing microscopes and will find use in applications that aim to record dynamic, multicolor localization events such as in multiplex FRET and tracking of multi-component, interacting complexes. |
Thursday, March 18, 2021 3:12PM - 3:24PM Live |
V12.00002: Optimal Signal Transduction with Silicon Transistors Enable Therapeutic Enzyme Regulation Arvind Balijepalli, Son Le, Michelle Morris, Harish Pant, Curt Richter We used commercially sourced n-channel silicon field-effect transistors (nFETs) operating under PID control to demonstrate pH measurements with a resolution of (7.2+/-0.3)x10^-3 at 10 Hz. The results represent a 3-fold improvement over open loop operation of the nFETs and over ion sensitive field-effective transistors (ISFETs). The improved performance was realized when the pH sensing membrane was separated from the nFETs and connected electrically to the transistor gate. The technique leverages key operating procedures from our previous work with dual-gate 2D field-effect transistors (dg2DFET) fabricated with 2D semi-conducting MoS2 channels. The devices were used to measure the function of the enzyme Cdk5, which facilitates signaling within cells by modifying proteins via the hydrolysis of adenosine triphosphate (ATP), and thereby changing the pH of the surrounding solution by a very small amount. By measuring this subtle change in pH, we quantified the activity of Cdk5, which has been previously implicated in Alzheimer’s disease, under physiological conditions. Finally, we demonstrated the effectiveness of a custom polypeptide, p5, as a therapeutic agent in restoring the function of the pathological form of Cdk5. |
Thursday, March 18, 2021 3:24PM - 3:36PM Live |
V12.00003: Characterizing the Simulated Anomalous Diffusion of Proteins in Relation to the Nanoporous Structure of Extracellular Matrix-Relevant Hydrogels Shawn Yoshida, William Schmid, Nam Vo, Lydia Kisley Local drug delivery requires therapeutics to diffuse through the nanoporous structure of the extracellular matrix. To enable the efficient delivery of drugs, both the structure of this hydrogel environment and the diffusion of the drug must be understood. We simulated fluorescence microscopy data of BSA diffusing in binarized images of polyacrylamide at various concentrations. Conventional methods are unable to quantify both nanoscale structure and diffusion, but we overcame these limitations with a technique known as "fluorescence correlation spectroscopy super-resolution optical fluctuation imaging" (fcsSOFI), which can quantify local anomaleity and diffusion dynamics, along with the size, shape, and frequency of nanopore structures. Delauney triangulation was applied to calculate pore sizes, and showed agreement with the ground truth. Combining the characterizations of pore sizes and local anomaleity allowed us to relate the subdiffusivity of simulated proteins with pore size. These findings can help inform drug-delivery applications where nanoparticle therapeutics must diffuse through the extracellular matrix. |
Thursday, March 18, 2021 3:36PM - 3:48PM Live |
V12.00004: Distinguishing Catalytic and Noncatalytic Motions of Individual Taq Polymerase Molecules Jeffrey Taulbee, Mackenzie W Turvey, Calvin J Lau, Wonbae Lee, Kristin Nichelle Gabriel, Cynthia Chen, Rebecca Vargas, Gregory A Weiss, Philip G Collins Single-molecule field-effect transistors (smFETs) provide a powerful way to probe biochemical activity with a level of detail that is elusive to ensemble techniques. For example, smFETs have been used to record bond-by-bond catalysis by enzymes acting upon a variety of substrates [1], including the duplication of DNA by DNA polymerases [2]. With the temperature raised to 72 °C, the smFET method has now been applied to study Taq polymerase and key catalytic steps in the polymerase chain reaction (PCR). During PCR, Taq incorporates nucleotides into complementary Watson-Crick base pairs while rejecting noncomplementary mismatches. smFETs resolved both processes, even though the rejection events had durations averaging only 20 µs. This presentation will summarize the Taq immobilization orientations and analysis methods that best recorded and differentiated between catalytic and noncatalytic motions, producing detailed single-molecule kinetics and energy landscapes for the two processes. |
Thursday, March 18, 2021 3:48PM - 4:24PM Live |
V12.00005: Synthetic Electrophysiology: Manipulating and measuring bioelectric pattern formation with light Invited Speaker: Harold M McNamara
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Thursday, March 18, 2021 4:24PM - 4:36PM Live |
V12.00006: DNA Flossing in a Dual Pore Device An Vuong, Xavier Capaldi, Swarnadeep Seth, Philip Zimny, Roland Nagel, Aniket Bhattacharya, William Dunbar, Walter Reisner Solid-state nanopores are a leading technology for label-free single-molecule sensing of DNA. However, fundamental challenges hinder exploitation of solid-state pore devices: the need to (1) ensure molecule linearization (eliminate folding), (2) reduce effect of molecular fluctuations that introduce random error and (3) perform accurate genomic distance calibration. Two-pore devices with active control have potential to address these challenges. We have developed a feedback-driven dynamic control approach based on Field Programmable Gate Arrays that can adjust the opposing forces during translocation so as to scan the DNA molecule back and forth (“DNA flossing” [1-3]) This multi-scanning capability reduces random error by enabling averaging over repeated scans of linearized molecules. We demonstrate ability to perform 100’s of multi-scan cycles of a DNA molecule labeled with sequence specific tags and present simulation motivated analysis protocols for performing optimal time to sequence domain calibration of extracted barcodes. |
Thursday, March 18, 2021 4:36PM - 4:48PM On Demand |
V12.00007: The Origin of Conductive-Pulse Sensing Inside a Nanopore and the Role of Electro-Hydrodynamics Lauren Lastra, Kevin Freedman Despite the highly negatively charged backbone of DNA, electroosmotic flow (EOF) within a nanopore can lead to DNA travelling opposite to electrophoretic force at low ionic strengths. However, EOF-pumping and its role in producing current-enhancing events is ambiguous due to the complicated interactions between nanopore walls, DNA grooves, ion mobility, and counterion clouds. Here, we discuss how current-enhancing DNA events could be the result of a flux imbalance between anions and cations. The contributing factors for driving a flux imbalance within a nanopore include pore size, voltage bias, and type of alkali chloride electrolyte. Once the mechanism behind conductive events is established, the physics of transducing a DNA translocation into an electrical signal can be further exploited for improving DNA sequencing and, more broadly, bio-sensing. |
Thursday, March 18, 2021 4:48PM - 5:00PM On Demand |
V12.00008: Parameter estimation for correlated Ornstein-Uhlenbeck processes Helmut Strey In many fields of science, we observe time-series of a fluctuating quantity: local density fluctuations of a liquid, or fluctuations of neural activity as measured by fMRI. Here, we are exploring how to characterize correlations between two such time-series taking into account that the time series exhibit a relaxation time (memory). In particular, we will restrict ourselves to the simplest random process that results in a fluctuating time series with a characteristic relaxation time: the Ornstein-Uhlenbeck (OU) process. Here we show that by coupling two OU processes, we can create correlated time series with Pearson correlation coefficients from -1 to 1. We express the likelihood probability of an equally spaced time-series and can numerically solve for the maximum likelihood with respect to the parameters. From the Hessian of log(p) we can estimate the variance of the estimated parameters. To test our approach, we simulated correlated time-series of different lengths and degree of correlation and validated our maximum likelihood against Markov-Chain Monte-Carlo methods (pymc3). Our method is better suited to estimate correlations in time-series with memory than the often-used Pearson correlation. |
Thursday, March 18, 2021 5:00PM - 5:12PM On Demand |
V12.00009: Electroosmotic Flow and Pressure Influences DNA Configuration in Nanopores Lauren Lastra Nanopores have a promising ability of DNA sequencing. One disadvantage with sequencing using solid-state nanopores is that the molecules translocate through the pore far too quickly for detection of each nucleotide. Nanopore research has pushed for the discovery of new techniques to slow down translocating molecules, such as: changing the electrolyte solution, modifying the nanopore surface, and modulating fluid flow. An example of fluid flow modulation is incorporating a trans-pore pressure bias, which has been shown to ‘slow-down’ translocating molecules in high salt conditions1. Here, we use low salt conditions (i.e. electro-osmotic flow, EOF, dominated) to translocate Lambda phage DNA (λ-DNA) through glass nanopores. When a negative voltage is applied, λ-DNA translocates the pore, resulting in current enhancing (conductive) spikes. Under EOF dominated conditions, pressure affects not only the λ-DNA dwell time, but also the configuration. We describe how λ-DNA configurations can fluctuate depending on pore size, voltage applied, and method of translocation (i.e. EOF or electrophoretically). This work reveals the optimal environment slowing down linearly translocating DNA in preparation for using solid-state nanopores to sequence DNA. |
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