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| Open AccessSingle molecule delivery into living cells
Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here, the authors describe a platform to deliver defined numbers of macromolecules into cultured cell lines at single molecule resolution.
- Chalmers C. Chau
- , Christopher M. Maffeo
- & Paolo Actis
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Article
| Open AccessMicrofibril-associated glycoprotein 4 forms octamers that mediate interactions with elastogenic proteins and cells
Microfibrillar-associated protein 4 (MFAP4) is involved in fibrotic and cardiovascular diseases. Wozny et al. reveal structural aspects mediating MFAP4 octamer formation critical for its interaction with elastogenic proteins and cells.
- Michael R. Wozny
- , Valentin Nelea
- & Dieter P. Reinhardt
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Article
| Open AccessAnalysis of early intermediate states of the nitrogenase reaction by regularization of EPR spectra
Here, the authors characterize selenium and sulphur incorporated FeMo cofactors of the catalytic MoFe protein component from Azotobacter vinelandii under turnover conditions using EPR.
- Lorenz Heidinger
- , Kathryn Perez
- & Erik Schleicher
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Article
| Open AccessGiant organelle vesicles to uncover intracellular membrane mechanics and plasticity
The biophysical properties of cellular organelles are difficult to study directly. Here, the authors generate and characterize osmotically-expanded giant vesicles of several organelles, which maintain some of their functional properties.
- Alexandre Santinho
- , Maxime Carpentier
- & Abdou Rachid Thiam
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Article
| Open AccessHydrophobic mismatch drives self-organization of designer proteins into synthetic membranes
The organization of membrane proteins is critical to cellular function. Here the authors explore how computational protein design, MD simulation, and cell-free systems can be combined to elucidate how membrane-protein hydrophobic mismatch affects protein folding and organization in synthetic lipid membranes.
- Justin A. Peruzzi
- , Jan Steinkühler
- & Neha P. Kamat
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Article
| Open AccessMeasuring sub-nanometer undulations at microsecond temporal resolution with metal- and graphene-induced energy transfer spectroscopy
Studying the fluctuations of biological membranes with high resolution is challenging. Here, the authors combine metal- and graphene-induced energy transfer (MIET/GIET) with fluorescence correlation spectroscopy (FCS) to monitor such fluctuations with nanometer and microsecond resolution.
- Tao Chen
- , Narain Karedla
- & Jörg Enderlein
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Article
| Open AccessMassively parallel profiling of RNA-targeting CRISPR-Cas13d
Systematic understanding of CRISPR enzyme RNA binding specificity and cleavage is lacking. Here the authors report RNA chip-hybridised association-mapping platform (RNA-CHAMP), a workflow that repurposes next generation DNA sequencing chips to measure the binding affinity for RNA targets.
- Hung-Che Kuo
- , Joshua Prupes
- & Ilya J. Finkelstein
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Article
| Open AccessQuantification of ligand and mutation-induced bias in EGFR phosphorylation in direct response to ligand binding
The authors show that the oncogenic L834R EGFR mutation induces bias in EGFR signal transduction across the plasma membrane. While EGFR signalling is biased toward Y1068 phosphorylation, the mutation switches the preference to Y1173 phosphorylation.
- Daniel Wirth
- , Ece Özdemir
- & Kalina Hristova
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Article
| Open AccessIn situ quantification of osmotic pressure within living embryonic tissues
Osmotic pressure is thought to play a key role in many cellular and developmental processes, but remains challenging to measure it in cells and tissues. Here, the authors present a sensor based on double emulsion droplets that allows quantification of osmotic pressure in situ and in vivo.
- Antoine Vian
- , Marie Pochitaloff
- & Otger Campàs
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Article
| Open AccessMapping mechanical stress in curved epithelia of designed size and shape
Marin-Llaurado and colleagues engineer curved epithelial monolayers of controlled geometry and develop a new technique to map their state of stress. They show that pronounced stress anisotropies influence cell alignment.
- Ariadna Marín-Llauradó
- , Sohan Kale
- & Xavier Trepat
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Article
| Open AccessDynamic interplay between target search and recognition for a Type I CRISPR-Cas system
The details of CRISPR-Cas target search are unresolved. Here the authors analyse the target search process of the Type I CRISPR-Cas complex Cascade: they show that target search and target recognition are tightly linked, and DNA supercoiling and limited 1D diffusion play a role.
- Pierre Aldag
- , Marius Rutkauskas
- & Ralf Seidel
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Article
| Open AccessIn-vivo programmable acoustic manipulation of genetically engineered bacteria
In vivo manipulation of cells has applications in cell-based therapy, tissue engineering and targeted drug delivery. Here the authors demonstrate in vivo programmable acoustic manipulation of genetically engineered bacteria using holographic acoustic tweezers.
- Ye Yang
- , Yaozhang Yang
- & Hairong Zheng
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Article
| Open AccessEmergent collective organization of bone cells in complex curvature fields
It remains unclear how cells respond to complex extracellular geometries at the mesoscale. Here, the authors study the organization of bone cells in landscapes with varying curvatures, observing a preference for local concavities, multicellular bridging, and collective stress fiber orientation.
- Sebastien J. P. Callens
- , Daniel Fan
- & Amir A. Zadpoor
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Article
| Open AccessHedgehog is relayed through dynamic heparan sulfate interactions to shape its gradient
The Hedgehog morphogen creates gradients during development, but diffusion alone cannot explain its spatiotemporal dynamics. Hedgehog transport requires binding heparan sulfate sugar chains, and the authors now show that Hedgehogs can spread by interacting with sequential heparan molecules.
- Fabian Gude
- , Jurij Froese
- & Kay Grobe
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Article
| Open AccessTuning phenylalanine fluorination to assess aromatic contributions to protein function and stability in cells
Aromatic amino acids in proteins support ligand binding and protein stability. To parse the physiocochemical roles of aromatic interactions, here Galles, Infield and co-authors identify pyrrolysine-based aminoacyl-tRNA synthetases that enable the encoding of fluorinated phenylalanine amino acids.
- Grace D. Galles
- , Daniel T. Infield
- & Christopher A. Ahern
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Article
| Open AccessPICH acts as a force-dependent nucleosome remodeler
In anaphase, any unresolved DNA entanglements between the segregating sister chromatids can give rise to chromatin bridges. Here, the authors present an in vitro single-molecule assay that mimics chromatin under tension, to show that PICH is a tension- and ATP-dependent nucleosome remodeler.
- Dian Spakman
- , Tinka V. M. Clement
- & Gijs J. L. Wuite
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Perspective
| Open AccessBottom-up assembly of viral replication cycles
Synthetic biology encompasses engineering approaches to either redesign biological organisms with new molecular abilities or build up organism from the bottom to improve our understanding of life. Here, the authors summarize current approaches that can be applied for the synthetic engineering of viral replication cycles and discuss the potential DURC risk associated with such bottom-up approaches in infectious disease.
- Oskar Staufer
- , Gösta Gantner
- & Joachim P. Spatz
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Article
| Open AccessBinary-FRET reveals transient excited-state structure associated with activity-dependent CaMKII - NR2B binding and adaptation
FRET can be used to study conformational changes and protein-protein interactions. Here the authors report Binary-FRET for monitoring two FRET reactions, one encoded in the fluorescence lifetime of the donor, another encoded in its anisotropy, and monitor the dynamics of CaMKII and its interaction with NR2B.
- Tuan A. Nguyen
- , Henry L. Puhl III
- & Steven S. Vogel
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Article
| Open AccessAntibiotic polymyxin arranges lipopolysaccharide into crystalline structures to solidify the bacterial membrane
Manioglu et al use high-resolution atomic force microscopy to resolve how polymyxins interact with the bacterial membrane. Polymyxins arrange the bacterial lipids into regular hexagonal structures that stiffen the membrane and lead to rupture.
- Selen Manioglu
- , Seyed Majed Modaresi
- & Sebastian Hiller
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Article
| Open AccessMultistep orthophosphate release tunes actomyosin energy transduction
Release of the ATP hydrolysis product orthophosphate (Pi) from the myosin active site is central in force generation but is poorly understood. Here, Moretto et al. present evidence for multistep Pi-release reconciling apparently contradictory results.
- Luisa Moretto
- , Marko Ušaj
- & Alf Månsson
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Article
| Open AccessSubstrate binding in the mitochondrial ADP/ATP carrier is a step-wise process guiding the structural changes in the transport cycle
The mitochondrial ADP/ATP carrier transports adenine nucleotides. Here, authors identify the residues involved in substrate binding. One set forms the central substrate binding site and two asparagine/arginine pairs help to guide the substrates during the transport cycle.
- Vasiliki Mavridou
- , Martin S. King
- & Edmund R. S. Kunji
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Article
| Open AccessMapping the conformational energy landscape of Abl kinase using ClyA nanopore tweezers
Quantitative characterization of kinase conformational dynamics remains challenging. Here, the authors show that protein nanopore tweezers allow analyzing the conformational energy landscape and ligand binding of the Abl kinase domain.
- Fanjun Li
- , Monifa A. Fahie
- & Min Chen
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Article
| Open AccessLight-sheet photonic force optical coherence elastography for high-throughput quantitative 3D micromechanical imaging
The authors introduce photonic force optical coherence elastography that uses a light-sheet for parallelised and localised mechanical loading. They demonstrate the combination of 3D imaging of extracellular matrix mechanics with cellular-scale resolution and dynamic monitoring of cell-mediated changes.
- Yuechuan Lin
- , Nichaluk Leartprapun
- & Steven G. Adie
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Article
| Open AccessEmbryo-scale epithelial buckling forms a propagating furrow that initiates gastrulation
Drosophila mesoderm invagination begins with the formation of a furrow. Here they show that a long-range mechanism, powered by actomyosin contraction between the embryo polar caps, works like a ‘cheese-cutter wire’ indenting the tissue surface and folding it into a propagating furrow.
- Julien Fierling
- , Alphy John
- & Matteo Rauzi
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Article
| Open AccessInhibition mechanism of the chloride channel TMEM16A by the pore blocker 1PBC
TMEM16A, a calcium-activated chloride channel involved in multiple cellular processes, is implicated in various diseases, but its pharmacology remains poorly understood. Here, the authors combine cryo-EM and electrophysiology to elucidate the mechanism of TMEM16A inhibition by the pore blocker 1PBC.
- Andy K. M. Lam
- , Sonja Rutz
- & Raimund Dutzler
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Article
| Open AccessTemplated folding of the RTX domain of the bacterial toxin adenylate cyclase revealed by single molecule force spectroscopy
The authors use optical tweezers to show that the folding of repeats-in-toxin (RTX) block-iv in adenylate cyclase is templated by the folded RTX block-v. The findings suggest a possible mechanism for transmitting the folding signal in the RTX domain.
- Han Wang
- , Guojun Chen
- & Hongbin Li
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Article
| Open AccessGasdermin-A3 pore formation propagates along variable pathways
Gasdermin-A3 pore formation propagates along diverse pathways. It begins with membrane attachment and oligomeric pre-assembly. Once inserted in the membrane, the oligomers re-assemble into various shapes and sizes, which open their lytic pores.
- Stefania A. Mari
- , Kristyna Pluhackova
- & Daniel J. Müller
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Article
| Open AccessAuxiliary ATP binding sites support DNA unwinding by RecBCD
RecBCD is a remarkably fast DNA helicase. Using a battery of biophysical methods, Zananiri et. al reveal additional, non-catalytic ATP binding sites that increase the ATP flux to the catalytic sites that allows fast unwinding when ATP is scarce.
- Rani Zananiri
- , Sivasubramanyan Mangapuram Venkata
- & Arnon Henn
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Article
| Open AccessMembrane thickness, lipid phase and sterol type are determining factors in the permeability of membranes to small solutes
Membrane permeability of small molecules depends on the composition of the lipid bilayer. Here, authors compare permeability measured on membranes in different physical states and conclude that the yeast membrane exists in a highly ordered phase.
- Jacopo Frallicciardi
- , Josef Melcr
- & Bert Poolman
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Article
| Open Accessα-catenin switches between a slip and an asymmetric catch bond with F-actin to cooperatively regulate cell junction fluidity
By using laser tweezers, the authors show that a single α-catenin molecule does not resist force on F-actin. However, clustering of multiple molecules and force applied toward F-actin pointed end engage a molecular switch in α-catenin, which unfolds and strongly binds F-actin.
- C. Arbore
- , M. Sergides
- & M. Capitanio
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Article
| Open AccessSonogenetic control of mammalian cells using exogenous Transient Receptor Potential A1 channels
Ultrasound can be used to non-invasively control neuronal functions. Here the authors report the use of human Transient receptor potential ankyrin 1 (hsTRPA1) to achieve ultrasound sensitivity in mammalian cells, and show that it can be used to manipulate neurons in the mammalian brain.
- Marc Duque
- , Corinne A. Lee-Kubli
- & Sreekanth H. Chalasani
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Article
| Open AccessDuplex DNA and BLM regulate gate opening by the human TopoIIIα-RMI1-RMI2 complex
Here the authors probe the cleavage and gate opening of single-stranded DNA by the human topoisomerase TRR using a unique single-molecule strategy to reveal structural plasticity in response to both double-stranded DNA and the helicase BLM.
- Julia A. M. Bakx
- , Andreas S. Biebricher
- & Erwin J. G. Peterman
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Article
| Open AccessMicroskeletal stiffness promotes aortic aneurysm by sustaining pathological vascular smooth muscle cell mechanosensation via Piezo1
Mechanobiological signals have been reported to contribute to abdominal aortic aneurysm (AAA) development. Here the authors report that the microskeletal stiffness and a Piezo1-mediated mechanism influence vascular smooth muscle cell mechanosensation and AAA disease development.
- Weiyi Qian
- , Tarik Hadi
- & Weiqiang Chen
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Article
| Open AccessFocused ultrasound excites cortical neurons via mechanosensitive calcium accumulation and ion channel amplification
The mechanisms underlying the effects of ultrasonic modulation of neural activity are unclear. Here, the authors show that focused ultrasound excites cultured primary murine cortical neurons via calcium accumulation through specific mechanosensitive ion channels.
- Sangjin Yoo
- , David R. Mittelstein
- & Mikhail G. Shapiro
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Article
| Open AccessResonator nanophotonic standing-wave array trap for single-molecule manipulation and measurement
Applications of nanophotonic tweezers have been limited by the low trapping force. Here, the authors present enhanced force generation in a nanophotonic standing-wave array trap by integrating a critically-coupled resonator design and demonstrate common single-molecule experiments.
- Fan Ye
- , James T. Inman
- & Michelle D. Wang
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Article
| Open AccessMonitoring the binding and insertion of a single transmembrane protein by an insertase
The insertion and folding nascent or fully synthesized polypeptides into membranes is assisted by insertases. Here, the authors use a range of biophysical approaches to provide molecular details of how the transmembrane insertase YidC facilitates the insertion a protein into a phospholipid membrane.
- Pawel R. Laskowski
- , Kristyna Pluhackova
- & Daniel J. Müller
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Article
| Open AccessKinetic and structural mechanism for DNA unwinding by a non-hexameric helicase
UvrD is a model helicase from the non-hexameric Superfamily 1. Here, the authors use optical tweezers to measure directly the stepwise translocation of UvrD along a DNA hairpin, and propose a mechanism in which UvrD moves one base pair at a time, but sequesters the nascent single strands, releasing them after a variable number of ATP hydrolysis cycles.
- Sean P. Carney
- , Wen Ma
- & Yann R. Chemla
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Article
| Open AccessMolecular insights into receptor binding energetics and neutralization of SARS-CoV-2 variants
Here, the authors combine single-molecule atomic force spectroscopy measurements and molecular dynamics simulations to investigate the binding of spike proteins from four SARS-CoV-2 variants of concern (VoC) to the human ACE2 receptor. They observe an increase in the RBD-ACE2 complex stability for several of the VoCs and derive how the mutations affect the kinetic, thermodynamic and structural properties of complex formation.
- Melanie Koehler
- , Ankita Ray
- & David Alsteens
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Article
| Open AccessMeasuring kinetics and metastatic propensity of CTCs by blood exchange between mice
Current methods for acquiring dissemination kinetics of rare circulating tumor cells (CTCs) that form metastases have several limitations. Here, the authors show an approach for measuring endogenous CTC kinetics by continuously exchanging CTC-containing blood between un-anesthetized, tumor-bearing mice and healthy, tumor-free counterparts.
- Bashar Hamza
- , Alex B. Miller
- & Scott R. Manalis
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Article
| Open AccessFunctional basis of electron transport within photosynthetic complex I
Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. Here the authors determine the reduction potentials of the iron-sulphur clusters of PS-CI and thus the bioenergetics of the electron transfer relay.
- Katherine H. Richardson
- , John J. Wright
- & Maxie M. Roessler
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Article
| Open AccessReconfigurable multi-component micromachines driven by optoelectronic tweezers
Light-driven micromotors can convert energy to motion in sub-millimeter dimensions. Here, the authors extend this concept and introduce reconfigurable micromachines with multiple components, driven by optoelectronic tweezers, and demonstrate new functionalities.
- Shuailong Zhang
- , Mohamed Elsayed
- & Aaron R. Wheeler
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Article
| Open AccessCorrelation of membrane protein conformational and functional dynamics
High-speed atomic force microscopy height spectroscopy and single channel electrophysiology recordings are used to correlate conformational and functional dynamics of the model membrane protein, outer membrane protein G (OmpG). These techniques show that both states coexist and rapidly interchange in all conditions supported by molecular dynamics simulations.
- Raghavendar Reddy Sanganna Gari
- , Joel José Montalvo‐Acosta
- & Simon Scheuring
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Article
| Open AccessActuation enhances patterning in human neural tube organoids
Mechanical forces, along with gene regulatory networks and cell-cell signalling, play an important role in the complex organization of tissues. Here the authors describe devices that actively apply mechanical force to developing neural tube, demonstrating that mechanical forces increase growth and enhance patterning.
- Abdel Rahman Abdel Fattah
- , Brian Daza
- & Adrian Ranga
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Article
| Open AccessRheology of rounded mammalian cells over continuous high-frequencies
While rheology studies have contributed to the understanding of the viscoelastic properties of living cells, the use of higher frequencies promises elucidate the link between cellular and molecular properties. Here authors introduce a rheological assay that measures the cell mechanical response across a continuous frequency range ≈ 1 – 40 kHz.
- Gotthold Fläschner
- , Cosmin I. Roman
- & Daniel J. Müller
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Article
| Open AccessIncreased excitatory to inhibitory synaptic ratio in parietal cortex samples from individuals with Alzheimer’s disease
Synaptic loss may disturb the excitatory to inhibitory balance (E/I ratio) in circuits vulnerable in Alzheimer’s disease (AD). The authors find reduced synaptic levels of PSD-95 and gephyrin and show that individuals with AD exhibit a pro-excitatory shift of postsynaptic densities and the electrophysiological synaptic E/I ratio in the parietal cortex.
- Julie C. Lauterborn
- , Pietro Scaduto
- & Agenor Limon
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Article
| Open AccessTwo-dimensional TIRF-SIM–traction force microscopy (2D TIRF-SIM-TFM)
Quantifying rapid and small cellular forces is a major challenge in mechanobiology. Here, the authors show a >2-fold spatially and >10-fold temporally force sampling improvement combining traction force microscopy with total internal reflection fluorescence super-resolution structured illumination microscopy.
- Liliana Barbieri
- , Huw Colin-York
- & Marco Fritzsche
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Article
| Open AccessAstigmatic traction force microscopy (aTFM)
Quantifying rapidly progressing three-dimensional forces generated by cells remains a major challenge in mechanobiology. Here, the authors show that combining traction force microscopy with astigmatic imaging permits sensitive out-of-plane force estimation on the second timescale.
- Di Li
- , Huw Colin-York
- & Marco Fritzsche
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Article
| Open AccessMechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism
The GntR superfamily is one of the largest families of transcription factors in prokaryotes. Here the authors combine biophysical analysis and structural biology to dissect the mechanism by which NanR — a GntR-family regulator — binds to its promoter to repress the transcription of genes necessary for sialic acid metabolism.
- Christopher R. Horne
- , Hariprasad Venugopal
- & Renwick C. J. Dobson
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Article
| Open AccessA noninvasive fluorescence imaging-based platform measures 3D anisotropic extracellular diffusion
It is challenging to quantify anisotropic diffusion in biological systems. Here the authors report light-sheet imaging-based Fourier transform fluorescence recovery after photobleaching (LiFT-FRAP) to noninvasively determine 3D diffusion tensors of various biomolecules at physiological diffusivity.
- Peng Chen
- , Xun Chen
- & Hai Yao