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Article
| Open AccessWaffle Method: A general and flexible approach for improving throughput in FIB-milling
Here the authors describe the Waffle Method, aimed at increasing the throughput of and solves several challenges present in cryo-FIB/SEM sample preparation for cryo-ET analysis — the highest-resolution method for obtaining 3D views of native biological specimens in-situ.
- Kotaro Kelley
- , Ashleigh M. Raczkowski
- & Alex J. Noble
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Article
| Open Access100 Hz ROCS microscopy correlated with fluorescence reveals cellular dynamics on different spatiotemporal scales
In live-cell microscopy, motion blur limits resolution and contrast. Here the authors use 100 Hz super-resolving Rotating Coherent Scattering (ROCS) microscopy on various dynamic biological systems, and time-window analysis to understand biological effects.
- Felix Jünger
- , Dominic Ruh
- & Alexander Rohrbach
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Article
| Open AccessEffects of cryo-EM cooling on structural ensembles
The rapid temperature drop during plunge-freezing affects the structural ensembles obtained by cryo-EM. To quantify the extent of perturbation, Bock and Grubmüller combined continuum calculations, MD simulations, and kinetic models.
- Lars V. Bock
- & Helmut Grubmüller
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Article
| Open AccessDeep-learning two-photon fiberscopy for video-rate brain imaging in freely-behaving mice
The acquisition speed of two-photon fiberscopes is currently suboptimal. Here the authors report advances, including a high-speed scanner and down-sampling scheme as well as a two-stage deep learning (DL) algorithm, to allow high-speed, high-resolution imaging in freely moving mice.
- Honghua Guan
- , Dawei Li
- & Xingde Li
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Article
| Open AccessA deep-learning approach for online cell identification and trace extraction in functional two-photon calcium imaging
Processing of two-photon calcium imaging data is generally time-consuming, especially for large fields of view. Here, the authors present CITE-On, a tool based on a convolutional neural network, enabling online automatic cell identification, segmentation, identity tracking, and trace extraction.
- Luca Sità
- , Marco Brondi
- & Tommaso Fellin
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Article
| Open AccessLarge field-of-view non-invasive imaging through scattering layers using fluctuating random illumination
The authors demonstrate non-invasive fluorescence imaging behind scattering layers beyond the optical memory effect. They achieve this by demixing speckle patterns emitted by a fluorescent object under variable unknown random illumination, using matrix factorization and a fingerprint-based reconstruction.
- Lei Zhu
- , Fernando Soldevila
- & Sylvain Gigan
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Article
| Open AccessDirect imaging of the disconnection climb mediated point defects absorption by a grain boundary
Grain boundaries can improve the radiation resistance of a material by annihilating point defects formed under irradiation, however the atomistic mechanism is still unclear. Here the authors demonstrate grain boundaries absorb point defects through the climb motion of disconnections.
- Jiake Wei
- , Bin Feng
- & Yuichi Ikuhara
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Article
| Open AccessBinary-state scanning probe microscopy for parallel imaging
High-throughput imaging has generally been challenging for scanning probe microscopy techniques. Here, the authors introduce binary-state scanning probe microscopy, which uses a cantilever-free elastomeric probes and a hierarchical measurement architecture for parallel topography imaging.
- Gwangmook Kim
- , Eoh Jin Kim
- & Wooyoung Shim
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Article
| Open AccessMeasuring mechanical anisotropy of the cornea with Brillouin microscopy
Here, Brillouin optical microscopy noninvasively visualizes microscale anisotropy of the porcine cornea owing to its lamellar fiber structure and quantifies the longitudinal moduli of the bulk tissue. Anisotropy is also detected in angle-resolved measurement of the human cornea in vivo.
- Amira M. Eltony
- , Peng Shao
- & Seok-Hyun Yun
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Article
| Open AccessUltrafast infrared nano-imaging of far-from-equilibrium carrier and vibrational dynamics
Ultrafast infrared nano-imaging has enabled the study of nanoscale dynamics, but has been limited to probing short-lived carrier lifetimes. Here, the authors present pump-probe nano-spectroscopy with enhanced sensitivity to image both carrier and vibrational dynamics associated with long-lived excitations.
- Jun Nishida
- , Samuel C. Johnson
- & Markus B. Raschke
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Article
| Open AccessOsteoblast-derived vesicles induce a switch from bone-formation to bone-resorption in vivo
Bone remodeling involves a switch between bone formation and resorption, but the mechanisms is unclear. Here, the authors show that intercellular communication via extracellular vesicles secreted by mature osteoblasts is a key factor for the switching, via a microRNA-mediated mechanism.
- Maki Uenaka
- , Erika Yamashita
- & Masaru Ishii
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Article
| Open AccessDual clathrin and integrin signaling systems regulate growth factor receptor activation
The integration and organization of growth factor signaling, adhesion, and endocytosis is poorly understood. Here the authors use light and electron microscopy to shed light on the role of flat clathrin lattices and cell adhesion in growth factor signaling.
- Marco A. Alfonzo-Méndez
- , Kem A. Sochacki
- & Justin W. Taraska
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Article
| Open AccessLive-dead assay on unlabeled cells using phase imaging with computational specificity
Common methods for characterising cell viability involve cell staining with chemical reagents. Here the authors report a method for cell viability assessment that does not require labelling; this uses quantitative phase imaging combined with deep learning.
- Chenfei Hu
- , Shenghua He
- & Gabriel Popescu
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Article
| Open AccessDirect-laser writing for subnanometer focusing and single-molecule imaging
Focus-locking improves localization precision in single-molecule microscopy, but fiducials are often deposited at random and provide limited 3D compensation. Here, the authors fabricate 3D optical fiducials with nanometer accuracy by two-photon direct laser writing, and demonstrate isotropic 3D focus locking.
- Simao Coelho
- , Jongho Baek
- & Katharina Gaus
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Article
| Open AccessCircularly polarised luminescence laser scanning confocal microscopy to study live cell chiral molecular interactions
Here, the authors introduce a live-cell imaging system using chiroptical contrast, enabling the study of chiral interactions. They demonstrate simultaneous imaging of enantiomeric pairs of molecular probes emitting circularly polarised light, using both single and two-photon excitation.
- Patrycja Stachelek
- , Lewis MacKenzie
- & Robert Pal
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Article
| Open AccessQuantification of bone marrow interstitial pH and calcium concentration by intravital ratiometric imaging
The fate of hematopoietic stem cells can be controlled by factors such as calcium ion concentration. Here the authors report an intravital ratiometric analysis method to measure extracellular calcium ion concentrations and absolute pH in mouse bone marrow.
- S-C. A. Yeh
- , J. Hou
- & C. P. Lin
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Article
| Open Access4polar-STORM polarized super-resolution imaging of actin filament organization in cells
Single-molecule localisation microscopy does not give orientation information. Here the authors combine Stochastic Optical Reconstruction Microscopy (STORM) with single molecule orientation and wobbling measurements using four-polarisation image splitting, 4polar-STORM.
- Caio Vaz Rimoli
- , Cesar Augusto Valades-Cruz
- & Sophie Brasselet
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Article
| Open AccessDeterminants of synapse diversity revealed by super-resolution quantal transmission and active zone imaging
Super-resolution quantal imaging relates transmission at excitatory synapses to presynaptic molecular composition. The authors find that evoked transmission varies greatly between synapses and is uncorrelated and physically separate from spontaneous transmission, and identify responsible presynaptic proteins.
- Zachary L. Newman
- , Dariya Bakshinskaya
- & Ehud Y. Isacoff
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Article
| Open AccessImaging moiré deformation and dynamics in twisted bilayer graphene
Local variations of twist angle and strain in twisted bilayer graphene (TBG) can produce relevant changes in the electronic properties of the system. Here, high-resolution low energy electron microscopy is used to characterize the spatial and temporal deformations of moiré patterns in TBG at high temperatures, showing the stability of these structures up to 600 ∘C.
- Tobias A. de Jong
- , Tjerk Benschop
- & Sense Jan van der Molen
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Article
| Open AccessSpatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis
Spatial-omics methods with ease-of-use and high multiplexing are in demand. Here the authors report Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA) which uses Spectral and Fluorescence Lifetime Imaging and Microscopy; they apply this to co-detection of mRNA and protein.
- Tam Vu
- , Alexander Vallmitjana
- & Weian Zhao
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Article
| Open AccessA cryo-electron microscopy support film formed by 2D crystals of hydrophobin HFBI
The amphiphilic low-molecular-weight protein hydrophobin (HFBI) can self-assemble into a crystalline monolayer film. Here, the authors present a cryo-EM support film using such 2D crystals of hydrophobin HFBI and show that it protects specimen from the air-water interface and can help to overcome the preferred orientation problem of particles in cryo-EM.
- Hongcheng Fan
- , Bo Wang
- & Fei Sun
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Article
| Open AccessSingle molecule kinetics of bacteriorhodopsin by HS-AFM
Here, the authors use high-speed atomic force microscopy (HS-AFM) methods to characterize the single molecule kinetics of wild-type bacteriorhodopsin (bR) with millisecond temporal resolution, providing new insights into the bR conformational cycle.
- Alma P. Perrino
- , Atsushi Miyagi
- & Simon Scheuring
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Article
| Open AccessA turquoise fluorescence lifetime-based biosensor for quantitative imaging of intracellular calcium
Currently, genetically encoded calcium indicators are not suitable for direct quantification. Here the authors engineer a fluorescence lifetime imaging calcium biosensor, Turquoise Calcium Fluorescence LIfeTime Sensor (Tq-Ca-FLITS), and measure intracellular calcium concentrations in human-derived organoids.
- Franka H. van der Linden
- , Eike K. Mahlandt
- & Joachim Goedhart
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Article
| Open AccessNanosecond-resolution photothermal dynamic imaging via MHZ digitization and match filtering
Photothermal microscopy is limited for imaging of thermal dynamics. Here, the authors introduce a lock-in free, mid-infrared photothermal dynamic imaging system, which significantly increases SNR and imaging speed, and demonstrate metabolism analysis at single-cell level and background removal.
- Jiaze Yin
- , Lu Lan
- & Ji-Xin Cheng
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Article
| Open AccessTwo-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics
Revealing mechanisms of complex protein machines requires simultaneous exploration of multiple structural coordinates. Here the authors report two-colour fluorescence microscopy combined with photoinduced electron transfer probes to simultaneously detect two structural coordinates in single protein molecules.
- Jonathan Schubert
- , Andrea Schulze
- & Hannes Neuweiler
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Article
| Open AccessTowards non-blind optical tweezing by finding 3D refractive index changes through off-focus interferometric tracking
Optical manipulation of large objects is challenging as optical trap positions are blindly chosen. Here, the authors present off-focus interferometric tracking, which localises optimal grabbing positions with increased refractive index, by analysing the beam deformations of several holographic optical traps.
- Benjamin Landenberger
- , Yatish
- & Alexander Rohrbach
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Article
| Open AccessPlanar photonic chips with tailored angular transmission for high-contrast-imaging devices
The authors design a planar photonic chip with several multilayers of photonic band gaps and a region of dielectric nanoparticles for tailored angular transmission. They use it as sample substrate for high-contrast darkfield and total internal reflection microscopy on a conventional microscope.
- Yan Kuai
- , Junxue Chen
- & Douguo Zhang
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Article
| Open Access6 nm super-resolution optical transmission and scattering spectroscopic imaging of carbon nanotubes using a nanometer-scale white light source
The authors present a super-resolution hyperspectral imaging technique using a nanoscale white light source generated by superfocusing light from a tungsten-halogen lamp. They achieve 6 nm resolution, measuring longitudinal and transverse optical electronic transitions in single-walled carbon nanotubes.
- Xuezhi Ma
- , Qiushi Liu
- & Ming Liu
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Article
| Open AccessLightwave-driven scanning tunnelling spectroscopy of atomically precise graphene nanoribbons
Here, the authors perform lightwave-driven terahertz scanning tunnelling microscopy and spectroscopy of graphene nanoribbons with atomic resolution in three dimensions, revealing localized wavefunctions that are inaccessible by conventional scanning tunnelling microscopy.
- S. E. Ammerman
- , V. Jelic
- & T. L. Cocker
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Article
| Open AccessBioorthogonal labeling of transmembrane proteins with non-canonical amino acids unveils masked epitopes in live neurons
Visualisation of TARP localisation is hindered by existing imaging tools. Here the authors report a labelling and imaging platform using genetic code expansion and non-canonical amino acids; they use this to fluorescently label live neurons and localise TARP proteins using super resolution microscopy.
- Diogo Bessa-Neto
- , Gerti Beliu
- & Daniel Choquet
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Article
| Open AccessTwinning-assisted dynamic adjustment of grain boundary mobility
Grain boundary can change its structure upon deformation. Here, the authors show that during this process, grain boundary mobility can be tuned dynamically via a self-stimulated twinning process.
- Qishan Huang
- , Qi Zhu
- & Jiangwei Wang
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Article
| Open AccessFlexible simultaneous mesoscale two-photon imaging of neural activity at high speeds
Functional brain imaging with two-photon microscopy is limited by a tradeoff between imaging area and acquisition speed. Here, the authors present Quadroscope, a flexible microscope which allows for simultaneous video rate acquisition of four independently targetable brain regions across 5 mm.
- Mitchell Clough
- , Ichun Anderson Chen
- & Jerry L. Chen
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Article
| Open AccessDiesel2p mesoscope with dual independent scan engines for flexible capture of dynamics in distributed neural circuitry
Imaging of neuronal activity across distant brain regions is challenging. Here, the authors introduce a two-photon microscope with two independently controlled scan engines, and demonstrate calcium imaging with subcellular resolution in brain regions up to 7 mm apart simultaneously.
- Che-Hang Yu
- , Jeffrey N. Stirman
- & Spencer L. Smith
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Article
| Open AccessProgrammable viscoelasticity in protein-RNA condensates with disordered sticker-spacer polypeptides
Here the authors show that disordered polypeptide-RNA condensates exhibit rheological properties similar to that of a viscoelastic Maxwell fluid, and use simple polypeptide design rules to create microcondensates with tunable viscoelasticity.
- Ibraheem Alshareedah
- , Mahdi Muhammad Moosa
- & Priya R. Banerjee
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Article
| Open AccessIn vivo volumetric imaging of calcium and glutamate activity at synapses with high spatiotemporal resolution
Adaptive optics (AO) corrects sample aberrations and allows high spatial resolution at depth in vivo. Here the authors report an AO method for Bessel focus; they apply AO Bessel focus scanning fluorescence microscopy to volumetric imaging and measure synaptic calcium and glutamate activity in vivo.
- Wei Chen
- , Ryan G. Natan
- & Na Ji
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Article
| Open AccessFull spectrum fluorescence lifetime imaging with 0.5 nm spectral and 50 ps temporal resolution
High data volumes from multidimensional imaging techniques can lead to slow collection and processing times. Here, the authors implement multispectral fluorescence lifetime imaging microscopy (FLIM) that uses time-correlated photon counting technology to reach simultaneously high imaging rates combined with high spectral and temporal resolution.
- Gareth O. S. Williams
- , Elvira Williams
- & Mark Bradley
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Article
| Open AccessThermoresponsive polymer assemblies via variable temperature liquid-phase transmission electron microscopy and small angle X-ray scattering
Thermoresponsive polymers are used in numerous technological applications but well established, direct techniques for elucidating their elevated temperature, solution-phase, nanoscale morphologies and dynamics are lacking. Here, the authors examine thermoresponsive polymeric materials by liquid-cell transmission electron microscopy and gain insight into their thermal-responsive behaviour.
- Joanna Korpanty
- , Lucas R. Parent
- & Nathan C. Gianneschi
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Article
| Open AccessFast wide-field upconversion luminescence lifetime thermometry enabled by single-shot compressed ultrahigh-speed imaging
Photoluminescence lifetime imaging of upconverting nanoparticles is useful for optical thermometry, but is limited for dynamic samples. Here, the authors present a wide-field and single shot approach based on compressive sensing, for video-rate upconversion temperature sensing of moving samples.
- Xianglei Liu
- , Artiom Skripka
- & Jinyang Liang
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Article
| Open AccessComputational optical sectioning with an incoherent multiscale scattering model for light-field microscopy
Light-field microscopy provides volumetric imaging at high speeds, but suffers from degradation in scattering tissue. Here, the authors present an incoherent multiscale scattering model which allows for quantitative 3D reconstruction in complex environments, and demonstrate dynamic imaging in vivo.
- Yi Zhang
- , Zhi Lu
- & Qionghai Dai
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Article
| Open AccessSimultaneous orientation and 3D localization microscopy with a Vortex point spread function
Molecular orientation is often ignored during single-molecule localisation microscopy. Here, the authors use a Vortex point spread function in order to simultaneously estimate the 3D position, dipole orientation and degree of rotational constraint, within 30% of the Cramér-Rao bound limit.
- Christiaan N. Hulleman
- , Rasmus Ø. Thorsen
- & Bernd Rieger
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Article
| Open AccessSubstrate colonization by an emulsion drop prior to spreading
In classical wetting, the spreading of a drop on a surface is preceded by a bridge directly connecting the drop and the surface, yet it ignores the solubility of the drop phase in the medium. Here, the authors show that dissolved drop fluid from the parent drop can nucleate on the surface as islands, one of which coalesces with the parent drop to effect wetting.
- Suraj Borkar
- & Arun Ramachandran
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Article
| Open AccessA blueprint of the topology and mechanics of the human ovary for next-generation bioengineering and diagnosis
Although the first dissection of the human ovary dates back to the 17th century, its characterization is still limited. Here, the authors have unraveled a unique biophysical and topological phenotype of reproductive-age tissue, bridging biophysics and female fertility and providing a blueprint for the artificial ovary.
- Emna Ouni
- , Alexis Peaucelle
- & Christiani A. Amorim
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Article
| Open AccessSub-diffraction error mapping for localisation microscopy images
Determining the quality of localisation microscopy images is currently challenging. Here the authors report use of the Haar wavelet kernel analysis (HAWK) Method for the Assessment of Nanoscopy, termed HAWKMAN, to assess the reliability of localisation information.
- Richard J. Marsh
- , Ishan Costello
- & Susan Cox
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Article
| Open AccessTerahertz response of monolayer and few-layer WTe2 at the nanoscale
The behaviour of Tungsten ditelluride (WTe2) in few-layer form is not yet fully characterized. Here the authors use a near-field terahertz microscopy technique to observe the electromagnetic responses of WTe2 flakes from one to several layers and to study their semimetallic/ semiconducting behavior.
- Ran Jing
- , Yinming Shao
- & D. N. Basov
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Article
| Open AccessAn active tethering mechanism controls the fate of vesicles
Molecular tethers physically bridge transport vesicles to their target membranes as a prerequisite step for fusion. Here the authors control vesicle tethering using optogenetic approaches to study the interplay between vesicle tethering and fusion.
- Seong J. An
- , Felix Rivera-Molina
- & Derek Toomre
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Article
| Open AccessBowls, vases and goblets—the microcrockery of polymer and nanocomposite morphology revealed by two-photon optical tomography
The morphology of semicrystalline plastics on the 1-100 μm scale, such as spherulites, strongly affect mechanical and other properties of the material but currently only 2D imaging techniques are available. Here, the authors use fluorescence labels and confocal microscopy to visualize the internal structure of neat polymers and composites in 3D and reveal unsuspected morphologies.
- Shu-Gui Yang
- , Zhen-Zhen Wei
- & Goran Ungar
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Article
| Open AccessProbing atom dynamics of excited Co-Mo-S nanocrystals in 3D
The authors introduce an analytical approach for quantitative analysis of 3D atom dynamics during electron microscopy. They image a Co-Mo-S nanocrystal with 1.5 Å resolution, and observe chemical transformations caused by beam-stimulated vibrations.
- Fu-Rong Chen
- , Dirk Van Dyck
- & Stig Helveg
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Article
| Open AccessImaging biological tissue with high-throughput single-pixel compressive holography
Single-pixel holography generates holographic images with a single-pixel detector making this relatively inexpensive. Here the authors report a high-throughput single-pixel compressive holography method for imaging biological tissue which can either provide a large field of view or high resolution.
- Daixuan Wu
- , Jiawei Luo
- & Zhaohui Li
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Article
| Open AccessTurn-key mapping of cell receptor force orientation and magnitude using a commercial structured illumination microscope
The authors have recently developed molecular force microscopy (MFM) which uses fluorescence polarisation to measure cell-surface receptor force orientation. Here they show that structured illumination microscopes, which inherently use fluorescence polarisation, can be used for MFM in a turn-key manner.
- Aaron Blanchard
- , J. Dale Combs
- & Khalid Salaita
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