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| Open AccessAutomatic deep learning-driven label-free image-guided patch clamp system
Patch clamp recording of neurons is slow and labor-intensive. Here the authors present a method for automated deep learning driven label-free image guided patch clamp physiology to perform measurements on hundreds of human and rodent neurons.
- Krisztian Koos
- , Gáspár Oláh
- & Peter Horvath
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Article
| Open AccessNonlinear mechanics of lamin filaments and the meshwork topology build an emergent nuclear lamina
Mechanical strength of in situ assembled nuclear lamin filaments arranged in a 3D meshwork is unclear. Here, using mechanical, structural and simulation tools, the authors report the hierarchical organization of the lamin meshwork that imparts strength and toughness to lamin filaments at par with silk and Kevlar®
- K. Tanuj Sapra
- , Zhao Qin
- & Ohad Medalia
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Article
| Open AccessSound generation in zebrafish with Bio-Opto-Acoustics
Existing tools to study hearing are limited. Here the authors report Bio-OptoAcoustic (BOA) stimulation wherein they use optical forces to generate localised sound and activate the auditory system of zebrafish larvae.
- Itia A. Favre-Bulle
- , Michael A. Taylor
- & Ethan K. Scott
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Article
| Open AccessThe hierarchical assembly of septins revealed by high-speed AFM
Septins are GTP-binding proteins involved in diverse cellular processes including division, polarity maintenance and membrane remodeling. Here authors use high-speed atomic force microscopy to show that assembly of septin filaments is a diffusion-driven process, while septin assembly into higher-order involves septin self-templating
- Fang Jiao
- , Kevin S. Cannon
- & Simon Scheuring
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Article
| Open AccessMillisecond dynamics of an unlabeled amino acid transporter
Excitatory amino acid transporters (EAATs) are crucial for the removal of excitatory amino acids from the synaptic cleft. Here authors combined high-speed atomic force microscopy line-scanning with automated state assignment for the determination of transport dynamics of GltPh, a prokaryotic EAAT homologue, with millisecond temporal resolution.
- Tina R. Matin
- , George R. Heath
- & Simon Scheuring
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Article
| Open AccessMapping cellular-scale internal mechanics in 3D tissues with thermally responsive hydrogel probes
Local mechanical properties are important to cellular function; but conventional measurement techniques are limited in intact, living, 3D tissues. Here, the authors report on swellable hydrogel microparticles to monitor mechanical properties in situ via a temperature change.
- Stephanie Mok
- , Sara Al Habyan
- & Christopher Moraes
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| Open AccessMultiplex flow magnetic tweezers reveal rare enzymatic events with single molecule precision
Single molecule force measurements have shed light on dynamic biological events, but rare events escape notice owing to low throughput of the methods. Here, the authors combine an array of magnetic tweezers with lateral flow to increase throughput 100-fold, and detect rare DNA breaks induced by gyrase.
- Rohit Agarwal
- & Karl E. Duderstadt
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Article
| Open AccessFully organic compliant dry electrodes self-adhesive to skin for long-term motion-robust epidermal biopotential monitoring
Reported wearable dry electrodes have limited long-term use due to their imperfect skin compliance and high motion artifacts. Here, the authors report an intrinsically conductive, stretchable polymer dry electrode with excellent self-adhesiveness for long-term high-quality biopotential detection.
- Lei Zhang
- , Kirthika Senthil Kumar
- & Jianyong Ouyang
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Article
| Open AccessModulating mechanical stability of heterodimerization between engineered orthogonal helical domains
Mechanically stable specific heterodimerization formed with reversible bonds are used as a molecular anchorage in single-molecule force spectroscopy studies with unique mechanical properties. Here authors develop a variety of heterodimerization molecular systems with a range of mechanical stability from a set of recently engineered helix-heterotetramers.
- Miao Yu
- , Zhihai Zhao
- & Jie Yan
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Article
| Open AccessCFTR trafficking mutations disrupt cotranslational protein folding by targeting biosynthetic intermediates
Cystic fibrosis (CF) is a lethal genetic disease that is primarily caused by misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR). Here authors show that disease-causing mutations located within the first nucleotide binding domain of CFTR have distinct effects on nascent polypeptides.
- Hideki Shishido
- , Jae Seok Yoon
- & William R. Skach
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Article
| Open AccessDirected manipulation of membrane proteins by fluorescent magnetic nanoparticles
Membrane proteins are embedded in the lipid bilayer of the plasma membrane and their function in this context is often linked to their specific location and dynamics within the membrane. Here authors report the use of fluorescent magnetic nanoparticles to track membrane molecules and to manipulate their movement and pull membrane components laterally through the membrane with femtonewton-range forces.
- Jia Hui Li
- , Paula Santos-Otte
- & Helge Ewers
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Article
| Open AccessPrecise capture and dynamic relocation of nanoparticulate biomolecules through dielectrophoretic enhancement by vertical nanogap architectures
Label-free trapping of nanoparticles via dielectophoretic forces is traditionally done with electrodes in a horizontal gap layout. Here, the authors present a vertical nanogap architecture, which allows for precise capture and spatiotemporal manipulation of nanoparticles and molecular assemblies.
- Eui-Sang Yu
- , Hyojin Lee
- & Yong-Sang Ryu
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Article
| Open AccessChemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing
Chemical modification of proteins can be used to decipher function or use that function for therapeutic purposes. Here, the authors insert synthetic peptides via tandem protein trans-splicing to add post-translational modifications or non-canonical amino acids.
- K. K. Khoo
- , I. Galleano
- & S. A. Pless
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Article
| Open AccessHigh-throughput cell and spheroid mechanics in virtual fluidic channels
High-throughput rheological measurements of cells and cell clusters by microfluidics is limited by fixed channel dimensions. Here the authors create virtual fluidic channels inside the cuvette of commercial flow cytometers to dynamically tune channel cross section to enable rheological measurements from cells and cell clusters.
- Muzaffar H. Panhwar
- , Fabian Czerwinski
- & Oliver Otto
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Article
| Open AccessSingle molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search
It is not fully understood how XPA interacts with a DNA lesion during nucleotide excision repair. Here, the authors use single molecule analysis to study XPA–DNA interactions, including the DNA bend angle, protein stoichiometry, and diffusive properties during damage search.
- Emily C. Beckwitt
- , Sunbok Jang
- & Bennett Van Houten
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Article
| Open AccessMechanical properties measured by atomic force microscopy define health biomarkers in ageing C. elegans
The development of reliable measures of health in ageing organisms is a need in ageing research. Using atomic force microscopy, here, the authors assess whole body stiffness of worms and show that it reflects organismal fitness.
- Clara L. Essmann
- , Daniel Martinez-Martinez
- & Filipe Cabreiro
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Article
| Open AccessTissue mechanics drives regeneration of a mucociliated epidermis on the surface of Xenopus embryonic aggregates
The role of tissue mechanics in the regeneration of mucociliated epithelium in Xenopus is unclear. Here, the authors show that Xenopus ectoderm aggregates undergo epithelial-like phenotypic transition prior to differentiation of mucus-secreting goblet cells to enable regeneration.
- Hye Young Kim
- , Timothy R. Jackson
- & Lance A. Davidson
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Article
| Open AccessBright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy
Photoconvertible proteins occupy two color channels thereby limiting multicolour localisation microscopy applications. Here the authors present UnaG, a new green-to-dark photoswitching fluorescent protein for super-resolution imaging, whose activation is based on a noncovalent binding with bilirubin.
- Jiwoong Kwon
- , Jong-Seok Park
- & Sang-Hee Shim
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Article
| Open AccessAnnexin-V stabilizes membrane defects by inducing lipid phase transition
Annexins are cytoplasmic proteins, which bind to membranes exposing negatively charged phospholipids in a Ca2+-dependent manner. Here the authors use high-speed atomic force microscopy and other techniques to show that annexin-V self-assembles into highly structured lattices that lead to a membrane phase transition on PS-rich membranes.
- Yi-Chih Lin
- , Christophe Chipot
- & Simon Scheuring
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| Open AccessForces during cellular uptake of viruses and nanoparticles at the ventral side
Many intracellular pathogens mimic extracellular matrix motifs to specifically interact with the host membrane which may influences virus particle uptake. Here authors use single molecule tension sensors to reveal the minimal forces exerted on single virus particles and demonstrate that the uptake forces scale with the adhesion energy.
- Tina Wiegand
- , Marta Fratini
- & Joachim P. Spatz
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| Open AccessIntegrating electric field modeling and neuroimaging to explain inter-individual variability of tACS effects
Electrical stimulation of the brain can have variable effects, perhaps because of individual differences in brain structure which produce differences in the electric fields. Here, the authors show that using functional and structural brain imaging along with electric field modeling can predict the effectiveness of stimulation.
- Florian H. Kasten
- , Katharina Duecker
- & Christoph S. Herrmann
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Article
| Open AccessDynamic constriction and fission of endoplasmic reticulum membranes by reticulon
The endoplasmic reticulum (ER) is an intracellular network characterized by highly dynamic behavior whose control mechanisms are unclear. Here, the authors show that the ER-membrane protein Reticulon (Rtnl1) can constrict ER bilayers and lead to ER fission.
- Javier Espadas
- , Diana Pendin
- & Andrea Daga
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| Open AccessSingle-molecule sensing of peptides and nucleic acids by engineered aerolysin nanopores
Aerolysin pores have potential to improve the accuracy of DNA sequencing and single-molecule proteomics. Here, the authors rationally design a set of mutated pores to achieve a more accurate detection of peptides and nucleic acids.
- Chan Cao
- , Nuria Cirauqui
- & Matteo Dal Peraro
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Article
| Open AccessAn alternative framework for fluorescence correlation spectroscopy
Fluorescence correlation spectroscopy is widely used for in vivo and in vitro applications, yet extracting information from experiments still requires long acquisition times. Here, the authors exploit Bayesian non-parametrics to directly analyze the output of confocal fluorescence experiments thereby probing physical processes on much faster timescales.
- Sina Jazani
- , Ioannis Sgouralis
- & Steve Pressé
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Article
| Open AccessPolarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity
Many developing tissues require Par-driven polarization, but its role in mammalian tissue maintenance is unclear. Here, the authors show that in mouse epidermis, Par3 governs tissue homeostasis not via orientation of cell division but by coupling cell mechanics with mitotic accuracy and genome integrity.
- Martim Dias Gomes
- , Soriba Letzian
- & Sandra Iden
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Article
| Open AccessDesign and fabrication of flexible DNA polymer cocoons to encapsulate live cells
The ability to encapsulate living cells could lead to many applications. Here, the authors present a flexible method to graft DNA polymers onto bacteria, yeast and mammalian cells, polymerize them into DNA cocoons and use these to manipulate and select cells based on the encoded polymer sequences on DNA cocoons.
- Tao Gao
- , Tianshu Chen
- & Genxi Li
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Article
| Open AccessA binding cooperativity switch driven by synergistic structural swelling of an osmo-regulatory protein pair
The bacterial protein Cnu together with the transcription repressor H-NS regulate expression of virulence factors in an osmo-sensitive manner. Here authors show that the structure of Cnu swells with decreasing ionic strength driving the oligomerization of H-NS and regulating osmo-sensory response.
- Abhishek Narayan
- , Soundhararajan Gopi
- & Athi N. Naganathan
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Article
| Open AccessHigh-throughput single-cell rheology in complex samples by dynamic real-time deformability cytometry
Real-time deformability cytometry (RT-DC) is used for mechanical cell phenotyping but is limited to a single snapshot per cell and can only measure elastic modulus. Here the authors introduce dynamic RT-DC which can measure elasticity and viscosity in single cells by following dynamic cell shape changes.
- Bob Fregin
- , Fabian Czerwinski
- & Oliver Otto
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Article
| Open AccessDispersible hydrogel force sensors reveal patterns of solid mechanical stress in multicellular spheroid cultures
Understanding how forces orchestrate tissue formation requires technologies to map internal tissue stress at cellular length scales. Here, authors develop ultrasoft sensors that visibly deform under cell-generated stress to capture patterns of internal stress development during multicellular spheroid formation.
- Wontae Lee
- , Nikita Kalashnikov
- & Christopher Moraes
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| Open AccessLong-term functional and structural preservation of precision-cut human myocardium under continuous electromechanical stimulation in vitro
Myocardial tissue undergoes steady functional decline when cultured in vitro. Here, the authors report a protocol for culture of human cardiac slices that allows maintenance of contractility for up to four months, and show that the model is suitable for evaluation of drug safety, as exemplified for drugs interfering with cardiomyocyte repolarization.
- Carola Fischer
- , Hendrik Milting
- & Andreas Dendorfer
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Article
| Open AccessAn electrogenic redox loop in sulfate reduction reveals a likely widespread mechanism of energy conservation
The bacterial complex QrcABCD plays a key role in the bioenergetics of sulfate respiration. Here, Duarte et al. show that this complex is electrogenic, with protons and electrons required for quinone reduction being extracted from opposite sides of the membrane.
- Américo G. Duarte
- , Teresa Catarino
- & Inês A. C. Pereira
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Article
| Open AccessMechanical loading of desmosomes depends on the magnitude and orientation of external stress
Desmosomes are intercellular adhesion complexes that connect the intermediate filament cytoskeletons of neighboring cells but direct evidence for their load-bearing nature is lacking. Here the authors develop FRET-based tension sensors to measure the forces experienced by desmoplakin and infer that desmosomes become mechanically loaded when cells are exposed to external mechanical stresses.
- Andrew J. Price
- , Anna-Lena Cost
- & Carsten Grashoff
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Article
| Open AccessDynamic and non-contact 3D sample rotation for microscopy
Sample orientation is crucial to ensure optimal image quality in light microscopy. Here the authors enable multi-axis orientation of fixed mouse embryos and shrimp, and live zebrafish embryos and larvae by introducing magnetic beads and rotating the sample with a magnetic field in a microscope.
- Frederic Berndt
- , Gopi Shah
- & Jan Huisken
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Article
| Open AccessHigh-speed AFM height spectroscopy reveals µs-dynamics of unlabeled biomolecules
The dynamics of biomolecules can occur over a wide range of time and length scales. Here the authors develop a high-speed AFM height spectroscopy method to directly detect the motion of unlabeled molecules at Angstrom spatial and microsecond temporal resolution.
- George R. Heath
- & Simon Scheuring
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| Open AccessThe fluid membrane determines mechanics of erythrocyte extracellular vesicles and is softened in hereditary spherocytosis
Red blood cell disorders are often accompanied by increased release of extracellular vesicles (EVs), but their structural and mechanical properties are not fully understood. Here, the authors show that red blood cell EVs show liposome-like mechanical features and are softened in blood disorder patients.
- Daan Vorselen
- , Susan M. van Dommelen
- & Wouter H. Roos
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| Open AccessMechanochemical feedback control of dynamin independent endocytosis modulates membrane tension in adherent cells
Plasma membrane tension is an important factor that regulates many key cellular processes. Here authors show that a specific dynamin-independent endocytic pathway is modulated by changes in tension via the mechano-transducer vinculin.
- Joseph Jose Thottacherry
- , Anita Joanna Kosmalska
- & Satyajit Mayor
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| Open AccessFocused clamping of a single neuronal SNARE complex by complexin under high mechanical tension
The SNARE complex enables the fusion of synaptic vesicles with presynaptic membrane via a zippering process that is modulated by the protein complexin, though the precise mechanism remains unclear. Here, the authors used magnetic tweezers to show how complexin prepares a SNARE complex for fusion under mechanical tension.
- Min Ju Shon
- , Haesoo Kim
- & Tae-Young Yoon
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Article
| Open AccessAtomic force microscopy methodology and AFMech Suite software for nanomechanics on heterogeneous soft materials
Atomic force microscopy is an indispensable method in characterizing soft materials but the complexity of biological samples makes reproducible measurements difficult. Here the authors use a 3-step method to investigate biological specimens in which vertical and lateral heterogeneity hinders a precise quantitative characterization.
- Massimiliano Galluzzi
- , Guanlin Tang
- & Florian J. Stadler
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Article
| Open AccessRevealing circadian mechanisms of integration and resilience by visualizing clock proteins working in real time
The circadian clock proteins KaiA, KaiB, and KaiC reconstitute a circa-24 h oscillation of KaiC phosphorylation in vitro. Here the authors use high-speed atomic force microscopy to visualize in real time and quantify the dynamic interactions of KaiA with KaiC on the sub-second timescale to discover mechanisms of oscillatory resilience.
- Tetsuya Mori
- , Shogo Sugiyama
- & Toshio Ando
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Article
| Open AccessLong-term deep-supercooling of large-volume water and red cell suspensions via surface sealing with immiscible liquids
Supercooled water is susceptible to spontaneous freezing, and preventing this process is a challenge. Here, the authors use surface sealing with immiscible liquids to eliminate primary ice nucleation at the water/air interface, enabling deep supercooling of large volumes of water and red cell suspensions for long time periods.
- Haishui Huang
- , Martin L. Yarmush
- & O. Berk Usta
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Article
| Open AccessDissecting myosin-5B mechanosensitivity and calcium regulation at the single molecule level
Myosin-5B is an actin-based motor important for endosome recycling, but the molecular mechanism underlying its motility remains unknown. Here authors use single molecule imaging and high-speed laser tweezers to dissect the mechanoenzymatic properties of myosin-5B, which shows processive motility with peculiar mechanosensitivity.
- Lucia Gardini
- , Sarah M. Heissler
- & Marco Capitanio
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Article
| Open AccessQuantifying compressive forces between living cell layers and within tissues using elastic round microgels
Increasing importance is placed upon the effect of mechanical forces on cell regulation, fate and disease states. Here, the authors describe a deformable fluorescent nanoparticle labeled elastic microsphere which can be used to calculate strain and traction forces in vitro and in vivo.
- Erfan Mohagheghian
- , Junyu Luo
- & Ning Wang
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Article
| Open AccessTrue equilibrium measurement of transcription factor-DNA binding affinities using automated polarization microscopy
Methods to measure selective transcription factor-DNA binding often lack sensitivity and are not performed in solution. Here the authors develop a method to perform fluorescence anisotropy measurements of transcription factor-DNA binding energies with high sensitivity and throughput.
- Christophe Jung
- , Peter Bandilla
- & Ulrike Gaul
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Article
| Open AccessQuantitative diffusion measurements using the open-source software PyFRAP
FRAP analysis often relies on simplified assumptions that can affect measurement accuracy. Here the authors present a Python-based FRAP analysis software using simulations instead of simplified theoretical models to fit the data, which accounts for complex sample geometries and bleach conditions.
- Alexander Bläßle
- , Gary Soh
- & Patrick Müller
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Article
| Open AccessRapid measurement of inhibitor binding kinetics by isothermal titration calorimetry
There is growing evidence that the kinetics of interactions between inhibitors and their targets can strongly impact therapeutic efficacy. Here the authors describe an isothermal titration calorimetry-based method that can rapidly quantify inhibition kinetics and measure sub-nM binding affinities.
- Justin M. Di Trani
- , Stephane De Cesco
- & Anthony K. Mittermaier
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Article
| Open AccessBri2 BRICHOS client specificity and chaperone activity are governed by assembly state
The BRICHOS domain is a chaperone that can act against amyloid-β peptide fibril formation and non-fibrillar protein aggregation. Here the authors use a multidisciplinary approach and show that the Bri2 BRICHOS domain has qualitatively different chaperone activities depending on its quaternary structure.
- Gefei Chen
- , Axel Abelein
- & Jan Johansson
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Article
| Open AccessRhythmic potassium transport regulates the circadian clock in human red blood cells
Circadian rhythms usually rely on cyclic variations in gene expression. Red blood cells, however, display circadian rhythms while being devoid of nuclear DNA. Here, Henslee and colleagues show that circadian rhythms in isolated human red blood cells are dependent on rhythmic transport of K+ ions.
- Erin A. Henslee
- , Priya Crosby
- & Fatima H. Labeed
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Article
| Open AccessA very large-scale microelectrode array for cellular-resolution electrophysiology
Large electronics limit low-noise, non-invasive electrophysiological measurements to a thousand simultaneously recording channels. Here the authors build an array of 65k simultaneously recording and stimulating electrodes and use it to sort and classify single neurons across the entire mouse retina.
- David Tsai
- , Daniel Sawyer
- & Kenneth L. Shepard
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Article
| Open AccessOptical trapping of otoliths drives vestibular behaviours in larval zebrafish
The neural circuits of the vestibular system, which detects gravity and motion, remain incompletely characterised. Here the authors use an optical trap to manipulate otoliths (ear stones) in zebrafish larvae, and elicit corrective tail movements and eye rolling, thus establishing a method for mapping vestibular processing.
- Itia A. Favre-Bulle
- , Alexander B. Stilgoe
- & Ethan K. Scott