Biophysics

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    Protocols in Current Issue
    Spatiotemporal Quantification of Cytosolic pH in Arabidopsis Pollen Tubes
    Authors:  Maria Teresa Portes, Daniel S.C. Damineli and José A. Feijó, date: 07/20/2021, view: 231, Q&A: 0

    Ion-specific probes and fluorescent indicators have been key in establishing the role of ion signaling, namely calcium, protons, and anions, in plant development, providing a robust approach for monitoring spatiotemporal changes in intracellular ion

    ...
    Method for Rapid Enzymatic Cleaning for Reuse of Patch Clamp Pipettes: Increasing Throughput by Eliminating Manual Pipette Replacement between Patch Clamp Attempts

    The whole-cell patch-clamp method is a gold standard for single-cell analysis of electrical activity, cellular morphology, and gene expression. Prior to our discovery that patch-clamp pipettes could be cleaned and reused, experimental throughput and

    ...
    A Multi-color Bicistronic Biosensor to Compare the Translation Dynamics of Different Open Reading Frames at Single-molecule Resolution in Live Cells
    Authors:  Amanda L. Koch, Tatsuya Morisaki and Timothy J. Stasevich, date: 07/20/2021, view: 344, Q&A: 0

    Here, we describe how to image and quantitate the translation dynamics of a bicistronic biosensor that we recently created to fairly compare cap-dependent and IRES-mediated translation at single-molecule resolution in live human cells. This

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    Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom
    Authors:  Benjamin Boyer, Benoist Laurent, Charles H. Robert and Chantal Prévost, date: 07/20/2021, view: 450, Q&A: 0

    Protein filaments are dynamic entities that respond to external stimuli by slightly or substantially modifying the internal binding geometries between successive protomers. This results in overall changes in the filament architecture, which are

    ...
    Spatiotemporal Quantification of Cytosolic pH in Arabidopsis Pollen Tubes
    Authors:  Maria Teresa Portes, Daniel S.C. Damineli and José A. Feijó, date: 07/20/2021, view: 231, Q&A: 0
    [Abstract]

    Ion-specific probes and fluorescent indicators have been key in establishing the role of ion signaling, namely calcium, protons, and anions, in plant development, providing a robust approach for monitoring spatiotemporal changes in intracellular ion dynamics. The integration of protons/pH in signaling mechanisms is especially important as reports

    ...
    Method for Rapid Enzymatic Cleaning for Reuse of Patch Clamp Pipettes: Increasing Throughput by Eliminating Manual Pipette Replacement between Patch Clamp Attempts
    [Abstract]

    The whole-cell patch-clamp method is a gold standard for single-cell analysis of electrical activity, cellular morphology, and gene expression. Prior to our discovery that patch-clamp pipettes could be cleaned and reused, experimental throughput and automation were limited by the need to replace pipettes manually after each experiment. This

    ...
    A Multi-color Bicistronic Biosensor to Compare the Translation Dynamics of Different Open Reading Frames at Single-molecule Resolution in Live Cells
    Authors:  Amanda L. Koch, Tatsuya Morisaki and Timothy J. Stasevich, date: 07/20/2021, view: 344, Q&A: 0
    [Abstract]

    Here, we describe how to image and quantitate the translation dynamics of a bicistronic biosensor that we recently created to fairly compare cap-dependent and IRES-mediated translation at single-molecule resolution in live human cells. This technique employs a pair of complementary intrabodies loaded into living cells that co-translationally bind

    ...
    Modeling Perturbations in Protein Filaments at the Micro and Meso Scale Using NAMD and PTools/Heligeom
    Authors:  Benjamin Boyer, Benoist Laurent, Charles H. Robert and Chantal Prévost, date: 07/20/2021, view: 450, Q&A: 0
    [Abstract]

    Protein filaments are dynamic entities that respond to external stimuli by slightly or substantially modifying the internal binding geometries between successive protomers. This results in overall changes in the filament architecture, which are difficult to model due to the helical character of the system. Here, we describe how distortions in RecA

    ...
    Single-molecule Fluorescence Technique to Monitor the Co-transcriptional Formation of G-quadruplex and R-loop Structures
    Authors:  Gunhyoung Lim and Sungchul Hohng, date: 07/05/2021, view: 1306, Q&A: 0
    [Abstract]

    G-quadruplexes (GQ) and R-loops are non-canonical nucleic acid structures related to gene regulation and genome instability that can be formed during transcription; however, their formation mechanisms remain elusive. To address this question, we developed a single-molecule fluorescence technique to monitor the formation of G-quadruplex and R-loop

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    OrganoPlate Micro-fluidic Microvessel Culture and Analysis
    Authors:  Abidemi Junaid and Thomas Hankemeier, date: 07/05/2021, view: 1190, Q&A: 0
    [Abstract]

    The endothelial cells from the microvasculature are key drivers and targets of inflammatory and thrombotic processes in microvascular diseases. The study of bioactive lipids in inflammatory processes has been largely based on two-dimensional endothelial cell cultures. Three-dimensional microvessels-on-a-chip provides an opportunity to monitor the

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    Building a Total Internal Reflection Microscope (TIRF) with Active Stabilization (Feedback SMLM)
    Authors:  Simao Coelho, Jongho Baek, J. Justin Gooding and Katharina Gaus, date: 07/05/2021, view: 1515, Q&A: 0
    [Abstract]

    The data quality of high-resolution imaging can be markedly improved with active stabilization, which is based on feedback loops within the microscope that maintain the sample in the same location throughout the experiment. The purpose is to provide a highly accurate focus lock, therefore eliminating drift and improving localization precision.

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    Electron Tomography to Study the Three-dimensional Structure of the Reovirus Egress Pathway in Mammalian Cells
    [Abstract]

    Mammalian orthoreoviruses (reoviruses) are nonenveloped, double-stranded RNA viruses that replicate and assemble in cytoplasmic membranous organelles called viral inclusions (VIs). To define the cellular compartments involved in nonlytic reovirus egress, we imaged viral egress in infected, nonpolarized human brain microvascular endothelial cells

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    Electrophysiological Properties of Neurons: Current-Clamp Recordings in Mouse Brain Slices and Firing-Pattern Analysis
    Authors:  Elina Nagaeva, Ivan Zubarev and Esa R. Korpi, date: 06/20/2021, view: 1157, Q&A: 0
    [Abstract]

    Characterization of an electrically active cell, such as a neuron, demands measurement of its electrical properties. Due to differences in gene activation, location, innervation patterns, and functions, the millions of neurons in the mammalian brain are tremendously diverse in their membrane characteristics and abilities to generate action

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    Preparation of Doublet Microtubule Fraction for Single Particle Cryo-electron Microscopy
    Authors:  Corbin Black, Daniel Chen Dai, Katya Peri, Muneyoshi Ichikawa and Khanh Huy Bui, date: 06/05/2021, view: 1562, Q&A: 0
    [Abstract]

    Over the years, studying the ultrastructure of the eukaryotic cilia/flagella using electron microscopy (EM) has contributed significantly toward our understanding of ciliary function. Major complexes in the cilia, such as inner and outer dynein arms, radial spokes, and dynein regulatory complexes, were originally discovered by EM. Classical

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