Biology subjects

Tibbs, J.

Publications and source records attributed to Tibbs, J..

4 recordsLinked to original sources

bioRxiv2022

A Photonic Resonator Interferometric Scattering Microscope for Label-free Detection of Nanometer-Scale Objects with Digital Precision in Point-of-Use Environments

Label-free detection and digital counting of nanometer-scaled objects such as nanoparticles, viruses, extracellular vesicles, and protein molecules enable a wide range of applications in cancer diagnostics, pathogen detection, and life science research. The contrast of interferometric scattering microscopy is amplified through a photonic crystal surface, upon which scattered light from an object combines with illumination from a monochromatic plane wave source. The use of a photonic crystal substrate for interference scattering microscopy results in reduced requirements for high-intensity lasers or oil-immersion objectives, thus opening a pathway toward instruments that are more suitable for environments outside the optics laboratory. Here, we report the design, implementation, and characterization of a compact Photonic Resonator Interferometric Scattering Microscope (PRISM) designed for point-of-use environments and applications. The instrument incorporates two innovative elements that facilitate operation on a desktop in ordinary laboratory environments by users that do not have optics expertise. First, because scattering microscopes are extremely sensitive to vibration, we incorporated an inexpensive but effective solution of suspending the instruments main components from a rigid metal framework using elastic bands, resulting in an average of 28.7 dBV reduction in vibration amplitude compared to an office desk. Second, an automated focusing module based on the principle of total internal reflection maintains the stability of image contrast over time and spatial position, facilitating automated data collection. In this work, we characterize the systems performance by measuring the contrast from gold nanoparticles with diameters in the 10-40 nm range and by observing various biological analytes, including HIV virus, SARS-CoV-2 virus, exosomes, and ferritin protein.

bioengineering↗

bioRxiv2022

Photonic-Plasmonic Coupling Enhanced Fluorescence Enabling Digital-Resolution Ultrasensitive Protein Detection

Assays utilizing molecular fluorophores are common throughout life science research and diagnostic testing, although detection limits are generally limited by weak emission intensity, thus requiring many labeled target molecules to combine their output to achieve signal-to-noise greater than the background. Here, we describe how the synergistic coupling of plasmonic and photonic resonance modes can significantly boost the emission from fluorescent dye molecules without increasing the illumination intensity while utilizing a microscopy approach with a broad field of view. By optimally matching the resonant modes of a plasmonic fluor (PF) nanoparticle and a photonic crystal (PC) surface with the absorption and emission spectrum of the PFs fluorescent dye, we observe a 52-fold improvement in signal intensity, enabling individual PFs to be observed and digitally counted, using an approach in which one PF tag represents detection of one target molecule. The photonic amplification from the PF can be attributed to the strong near-field enhancement due to the cavity-induced activation of the PF, PC band structure-mediated improvement in collection efficiency of emitted photons, and increased rate of spontaneous emission. We demonstrate the applicability of the method by dose-response characterization of a sandwich immunoassay for human interleukin-6, a biomarker commonly used to assist diagnosis of cancer, inflammation, sepsis, and autoimmune disease. We achieve a limit of detection of 10 fg/ml, representing a capability three orders of magnitude lower than standard immunoassays.

bioengineering↗

bioRxiv2021

KERA: Analysis Tool for Multi-Process, Multi-State Single-Molecule Data

Molecular machines within cells dynamically assemble, disassemble, and reorganize. Molecular interactions between their components can be observed at the single-molecule level and quantified using colocalization single-molecule spectroscopy (CoSMoS), in which individual labeled molecules are seen transiently associating with a surface-tethered partner, or other total internal reflection fluorescence microscopy (TIRFM) approaches in which the interactions elicit changes in fluorescence in the labeled surface-tethered partner. When multiple interacting partners can form ternary, quaternary and higher order complexes, the types of spatial and temporal organization of these complexes can be deduced from the order of appearance and reorganization of the components. Time evolution of complex architectures can be followed by changes in the fluorescence behavior in multiple channels. Here, we describe the kinetic event resolving algorithm (KERA), a software tool for organizing and sorting the discretized fluorescent trajectories from a range of single-molecule experiments. KERA organizes the data in groups by transition patterns, and displays exhaustive dwell-time data for each interaction sequence. Enumerating and quantifying sequences of molecular interactions provides important information regarding the underlying mechanism of the assembly, dynamics and architecture of the macromolecular complexes. We demonstrate KERAs utility by analyzing conformational dynamics of two DNA binding proteins: RPA and XPD helicase.

biophysics↗

bioRxiv2018

Dynamics and Selective Remodeling of the DNA Binding Domains of RPA

Replication protein A (RPA) coordinates important DNA metabolic events by stabilizing single-strand DNA (ssDNA) intermediates, activating the DNA damage response, and handing off ssDNA to appropriate downstream players. Six DNA binding domains (DBDs) in RPA promote high affinity binding to ssDNA, but also allow RPA displacement by lower affinity proteins. We have made fluorescent versions of RPA and visualized the conformational dynamics of individual DBDs in the context of the full-length protein. We show that both DBD-A and DBD-D rapidly bind to and dissociate from ssDNA, while RPA as a whole remains bound to ssDNA. The recombination mediator protein Rad52 selectively modulates the dynamics of DBD-D. This demonstrates how RPA interacting proteins, with lower ssDNA binding affinity, can access the occluded ssDNA and remodel individual DBDs to replace RPA.\n\nOne Sentence SummaryThe choreography of binding and rearrangement of the individual domains of RPA during homologous recombination is revealed.

biochemistry↗