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Microenvironmental reorganization in brain tumors following radiotherapy and recurrence revealed by hyperplexed immunofluorescence imaging
Improved imaging techniques are required to help advance our understanding of the complex role of the tumour microenvironment (TME). Here, the authors develop a high-throughput, highly multiplexed tissue visualisation workflow and demonstrate its utility by characterising the response of the TME to radiotherapy in preclinical models of glioblastoma.
- Spencer S. Watson
- , Benoit Duc
- & Johanna A. Joyce
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| Open Access
Biomimetic NIR-II fluorescent proteins created from chemogenic protein-seeking dyes for multicolor deep-tissue bioimaging
Near-infrared-I/II fluorescent proteins (NIR-I/II FPs) are crucial for in vivo imaging, but their availability is still scarce. Here, the authors make use of protein-seeking NIR-II dyes as chromophores, which covalently bind to tag proteins and thus creating biomimetic NIR-II FPs.
- Jiajun Xu
- , Ningning Zhu
- & Shoujun Zhu
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Article
| Open Access
Projective light-sheet microscopy with flexible parameter selection
Projection imaging for multi-cellular samples can be hindered by several factors, including low contrast. Here, the authors propose projective light-sheet imaging with parameter selection (props) of imaging depth, position and viewing angle.
- Bingying Chen
- , Bo-Jui Chang
- & Reto Fiolka
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Article
| Open Access
Benchtop mesoSPIM: a next-generation open-source light-sheet microscope for cleared samples
The demand to image large biological samples at high resolution requires improvement in current light-sheet microscopy tools. Here, the authors present an improved, benchtop mesoSPIM with a significantly increased field-of-view, improved resolution and improved throughput.
- Nikita Vladimirov
- , Fabian F. Voigt
- & Fritjof Helmchen
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| Open Access
Drug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers
The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Here, the authors show that EGFR lung cancer mutations promote the assembly of kinase-active dimers within ligand-free EGFR oligomers. These dimers bind ligand with high affinity and promote tumor growth.
- R. Sumanth Iyer
- , Sarah R. Needham
- & Marisa L. Martin-Fernandez
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| Open Access
Desymmetric homologating annulation to access chiral pentafulvenes and their application in bioimaging
Polycyclic/multisubstituted pentafulvenes are of interest as they frequently occur in biologically relevant motifs, electrochromic dyes, and solar cells. Here, the authors report an enantioselective homologating annulation of cyclopent-4-ene-dione with 3-cyano-4-methylcoumarins through L-tert-leucine derived thiourea catalysis, affording a wide range of enantioenriched polycyclic multi-substituted embedded aminopentafulvenes.
- Sanjay Singh
- , Ravi Saini
- & Ravi P. Singh
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| Open Access
SEMORE: SEgmentation and MORphological fingErprinting by machine learning automates super-resolution data analysis
There is a lack of universal tools to analyse protein assemblies and quantify underlying structures in single-molecule localization microscopy. Here, the authors present SEMORE, a semi-automatic machine learning framework for system- and input-dependent analysis of super-resolution data.
- Steen W. B. Bender
- , Marcus W. Dreisler
- & Nikos S. Hatzakis
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| Open Access
Open-source microscope add-on for structured illumination microscopy
Researchers developed an open-hardware structured illumination microscopy add-on. This affordable upgrade provides super-resolution capabilities for normal optical microscopes. Detailed instructions enable easy reproduction to help democratize advanced microscopy.
- Mélanie T. M. Hannebelle
- , Esther Raeth
- & Georg E. Fantner
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| Open Access
Mesoscopic calcium imaging in a head-unrestrained male non-human primate using a lensless microscope
Current systems for imaging calcium dynamics in the brains of non-human primates require the animal’s movement to be restricted. Here, the authors demonstrate a mesoscale calcium imaging device in a freely moving non-human primate which features a 20 mm2 field of view.
- Jimin Wu
- , Yuzhi Chen
- & Jacob T. Robinson
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Article
| Open Access
Geometric transformation adaptive optics (GTAO) for volumetric deep brain imaging through gradient-index lenses
The GRIN lenses widely used for deep brain functional imaging suffer from a small measurement field of view due to strong fourth-order astigmatism. Here the authors report Geometric Transformation Adaptive Optics (GTAO) that corrects field-dependent astigmatism and enables large-volume in vivo imaging of deep mouse brain through 0.5 mm GRIN lenses.
- Yuting Li
- , Zongyue Cheng
- & Meng Cui
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Article
| Open Access
Fluorogenic CRISPR for genomic DNA imaging
Conventional CRISPR-based approaches to monitor genomic loci can be hampered by high background and nonspecific nucleolar signal. Here, the authors propose a fluorogenic CRISPR (fCRISPR) tool that allows for high-contrast and sensitive imaging of genomic DNA.
- Zhongxuan Zhang
- , Xiaoxiao Rong
- & Xing Li
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Article
| Open Access
A one-two punch targeting reactive oxygen species and fibril for rescuing Alzheimer’s disease
Toxic amyloid-beta plaque and harmful inflammation are two leading hallmarks of Alzheimer’s disease (AD), and precise AD therapy is elusive due to the lack of dual-targeting therapy function, limited blood-brain barrier penetration, and low imaging sensitivity. Here, the authors address these issues by designing a near-infrared-II aggregation-induced emission nanotheranostic for precise AD therapy.
- Jiefei Wang
- , Ping Shangguan
- & Ben Zhong Tang
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| Open Access
Muscle abnormalities worsen after post-exertional malaise in long COVID
In this longitudinal, case-controlled, cohort design study, authors show that post-exertional malaise is associated with severe exercise-induced myopathy, local and systemic metabolic disturbances and infiltration of amyloid-containing deposits in skeletal muscles of patients with long COVID.
- Brent Appelman
- , Braeden T. Charlton
- & Rob C. I. Wüst
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| Open Access
Dynamic stability of Sgt2 enables selective and privileged client handover in a chaperone triad
Newly synthesized tail-anchored membrane proteins (TAs) are relayed in a chaperone triad, Hsp70, Sgt2, and Get3, for delivery to the endoplasmic reticulum. Here, the authors show how the conformational dynamics of the cochaperone Sgt2 generates a decision point to enable efficient and selective TA targeting.
- Hyunju Cho
- , Yumeng Liu
- & Shu-ou Shan
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Article
| Open Access
Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms
Photolabeling of intracellular molecules is an invaluable approach to study multiple cellular processes. Here, the authors report on the near-infrared to far-red photoconversion in the miRFP family of fluorescent proteins, which enables photolabeling entirely performed in the near-infrared range.
- Francesca Pennacchietti
- , Jonatan Alvelid
- & Ilaria Testa
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Article
| Open Access
The separation pin distinguishes the pro– and anti–recombinogenic functions of Saccharomyces cerevisiae Srs2
Here the authors report that the main role of the protein Srs2 in homologous recombination is to remove Rad51 from single stranded DNA, rather than to drive synthesis-dependent strand annealing.
- Aviv Meir
- , Vivek B. Raina
- & Eric C. Greene
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| Open Access
Lipofuscin-like autofluorescence within microglia and its impact on studying microglial engulfment
Microglia are brain macrophages that engulf and clear cellular material and protein aggregates. Here, the authors show that lipofuscin-like autofluorescence can confound microglial engulfment analyses, which they can resolve with a photobleaching protocol.
- Jacob M. Stillman
- , Francisco Mendes Lopes
- & Dorothy P. Schafer
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| Open Access
Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging
l-Lactate is increasingly recognized as a key metabolite and signalling molecule in mammals, but the methods to investigate it in vivo have been limited. Here, authors report a pair of improved biosensors—one green and one red—for visualizing l-lactate both inside and outside of cells.
- Yusuke Nasu
- , Abhi Aggarwal
- & Robert E. Campbell
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| Open Access
Visualization of accessible cholesterol using a GRAM domain-based biosensor
Regulated cholesterol transport is essential for the maintenance of cellular cholesterol distribution and homeostasis, but tools to monitor this process are limited. Here, the authors develop a genetically encoded cholesterol biosensor and demonstrate its use for visualising cellular cholesterol distribution in various live cells in real time.
- Dylan Hong Zheng Koh
- , Tomoki Naito
- & Yasunori Saheki
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Article
| Open Access
Bone disease imaging through the near-infrared-II window
Skeletal disorders are commonly diagnosed by X-ray imaging, but the radiation limits its use. Here, the authors show that intravital NIR-II bone imaging is effective in diagnosis of a series of common bone diseases non-invasively in mice.
- Chao Mi
- , Xun Zhang
- & Dayong Jin
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Article
| Open Access
Fluorescent sensors for imaging of interstitial calcium
The study of interstitial calcium remains challenging due to scarce methodology. Here, authors present ultra-low affinity genetically encoded fluorescent biosensors for monitoring calcium in intercellular spaces in living tissues.
- Ariel A. Valiente-Gabioud
- , Inés Garteizgogeascoa Suñer
- & Oliver Griesbeck
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| Open Access
Biomolecular condensates modulate membrane lipid packing and hydration
Nano-environmental probes and advance imaging microscopy provide deep insight into protein phase separation and the interaction of condensates with membranes, revealing that wetting by condensates can modulate membrane lipid packing and hydration.
- Agustín Mangiarotti
- , Macarena Siri
- & Rumiana Dimova
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Engineered NIR-II fluorophores with ultralong-distance molecular packing for high-contrast deep lesion identification
To achieve high-contrast in fluorescence imaging of deep tissues is challenging. Here, the authors develop NIR-II fluorescent small molecules with high brightness and emission extending to 1900 nm, enabling in vivo imaging of deep tissues with enhanced signal-to-background ratios.
- Zhe Feng
- , Yuanyuan Li
- & Jun Qian
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Article
| Open Access
Fluorescent protein lifetimes report densities and phases of nuclear condensates during embryonic stem-cell differentiation
Fluorescent proteins can report on many cellular variables. Here, authors develop a method for reporting high local densities, and use it to show that density distribution of heterochromatin in mouse embryonic stem cells are not in a liquid phase.
- Khalil Joron
- , Juliane Oliveira Viegas
- & Eitan Lerner
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Article
| Open Access
A mosaic adeno-associated virus vector as a versatile tool that exhibits high levels of transgene expression and neuron specificity in primate brain
Viral vectors with intense and long-lasting transgene expression are essential for manipulating and imaging of neuronal activity in the primate brain. Here, the authors created a mosaic vector composed of capsid proteins derived from AAV1 and AAV2 that exhibits improved transgene expression and neuronal specificity.
- Kei Kimura
- , Yuji Nagai
- & Masahiko Takada
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Article
| Open Access
Self-assembly of CIP4 drives actin-mediated asymmetric pit-closing in clathrin-mediated endocytosis
CIP4 drives an asymmetric closing process in clathrin-mediated endocytosis by LLPS-driven self assembly and stereospecific interaction with the curved membrane and actin-regulating proteins which generates an actin-rich microenvironment near the pit.
- Yiming Yu
- & Shige H. Yoshimura
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Article
| Open Access
Dual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions
Single-particle tracking (SPT) has revolutionised studies of protein interactions but is often limited by photobleaching. Here, the authors evolve DNA-PAINT-SPT to enable simultaneous dual-colour detection for the quantification of protein dimerization and live cell membrane protein tracking.
- Christian Niederauer
- , Chikim Nguyen
- & Kristina A. Ganzinger
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| Open Access
Ratiometric measurement of MAM Ca2+ dynamics using a modified CalfluxVTN
MAM Ca2+ dynamics play an important role in diverse biological processes, but directly and specifically measuring Ca2+ concentrations in this region is technically challenging. Here the authors report a MAM-specific BRET-based Ca2+ indicator called MAM-Calflux, which works as both a Ca2+ indicator and a structural marker due to its ratiometric nature.
- Eunbyul Cho
- , Youngsik Woo
- & Sang Ki Park
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Article
| Open Access
N-Acryloylindole-alkyne (NAIA) enables imaging and profiling new ligandable cysteines and oxidized thiols by chemoproteomics
Cysteine is a popular target of covalent drugs and can undergo redox modifications. Here, the authors developed cysteine probes, N-acryloylindole-alkynes, for imaging and chemoproteomics to study cysteine oxidation and to identify targetable hotspots by small molecule compounds.
- Tin-Yan Koo
- , Hinyuk Lai
- & Clive Yik-Sham Chung
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Article
| Open Access
Analysis and modeling of cancer drug responses using cell cycle phase-specific rate effects
Understanding the impact of anti-cancer therapies on cell cycle progression could contribute to the discovery of effective therapeutic treatments. Here, the authors use genetically engineered breast cancer cell lines and computational models to analyse drug effects on specific cell cycle phases and identify effective combination treatments.
- Sean M. Gross
- , Farnaz Mohammadi
- & Laura M. Heiser
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Article
| Open Access
Transparent tissue in solid state for solvent-free and antifade 3D imaging
Current liquid-based optical clearing protocols can suffer from solvent evaporation and photobleaching. Here, the authors develop a solid high-refractive-index polymer to embed mouse and human tissues for clearing and antifade high-resolution 3D imaging.
- Fu-Ting Hsiao
- , Hung-Jen Chien
- & Shiue-Cheng Tang
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| Open Access
Evaluation of therapeutic PD-1 antibodies by an advanced single-molecule imaging system detecting human PD-1 microclusters
Immune checkpoint inhibitors are now routinely used in cancer therapy, however, the dosage and integration into conventional cancer therapy is determined via empirical experience rather than mechanistic rationale. Here authors establish an advanced single-molecule imaging method, by with which they are directly monitoring and evaluating the effect of immune checkpoint inhibitors on T cell signaling.
- Wataru Nishi
- , Ei Wakamatsu
- & Tadashi Yokosuka
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Article
| Open Access
Quantitative structured illumination microscopy via a physical model-based background filtering algorithm reveals actin dynamics
Quantitative live-cell superresolution imaging that maintains the linearity of fluorescence signals remains difficult. Here, the authors propose a physical model-based background filtering method for 2D-SIM, which allows for quantitative imaging and high signal completeness.
- Yanquan Mo
- , Kunhao Wang
- & Liangyi Chen
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Article
| Open Access
Single-frame deep-learning super-resolution microscopy for intracellular dynamics imaging
Multi-frame super-resolution microscopy is hampered by long acquisition times and phototoxicity, which hinder its use for live-cell imaging. Here, authors propose a deep-learning-based single-frame super-resolution approach to image cellular dynamics with high spatiotemporal resolution.
- Rong Chen
- , Xiao Tang
- & Shuhuai Yao
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Article
| Open Access
A local water molecular-heating strategy for near-infrared long-lifetime imaging-guided photothermal therapy of glioblastoma
Neodymium (Nd)-doped nanoparticles have been described for imaging-guided photothermal therapy. Here the authors design a Nd-Yb co-doped nanomaterial as nearinfrared long-lifetime imaging-guided waterheating probe, showing photothermal ablation in a glioblastoma pre-clinical mode
- Dongkyu Kang
- , Hyung Shik Kim
- & Joonseok Lee
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| Open Access
Insights into receptor structure and dynamics at the surface of living cells
It is challenging to approach protein structures in living cells. Here the authors investigate Interleukin-4 receptor alpha, which has a noncanonical amino acid incorporated at different locations, and see that evaluating click efficiency with calibrated imaging gives information on structure-related properties.
- Frederik Steiert
- , Peter Schultz
- & Thomas Weidemann
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Article
| Open Access
Multiplex translaminar imaging in the spinal cord of behaving mice
Fluorescence imaging of the spinal cord poses challenges, including depth of imaging. Here the authors describe a custom microscope and chronically implanted microprism that enables multicolor translaminar imaging of sensory and motor evoked activity in behaving mice, and show that spinal astrocytes show sensorimotor program-dependent calcium excitation.
- Pavel Shekhtmeyster
- , Erin M. Carey
- & Axel Nimmerjahn
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Article
| Open Access
The structural flexibility of MAD1 facilitates the assembly of the Mitotic Checkpoint Complex
The formation of the mitotic checkpoint complex requires an intricate spatial coordination amongst the proteins Bub1, Mad1, Cdc20, and Mad2. Chen et al show that a structural flexibility in Mad1 plays an essential role in achieving this coordination.
- Chu Chen
- , Valentina Piano
- & Ajit P. Joglekar
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Article
| Open Access
Spatial profiling of microbial communities by sequential FISH with error-robust encoding
Spatial analysis of microbiomes at single cell resolution is challenging. Here the authors report a highly multiplexed method for spatial profiling, sequential error-robust fluorescence in situ hybridisation (SEER-FISH), and show that this allows mapping of microbial communities at micron-scale.
- Zhaohui Cao
- , Wenlong Zuo
- & Lei Dai
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Article
| Open Access
1,2,4,5-Tetrazine-tethered probes for fluorogenically imaging superoxide in live cells with ultrahigh specificity
Specific detection of cellular superoxide is challenging. Here, the authors designed 1,2,4,5-tetrazine based fluorogenic probes for specific and sensitive imaging of superoxide, and applied them in high throughput screening of modulators of oxidative stress.
- Xuefeng Jiang
- , Min Li
- & Xin Li
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Article
| Open Access
Bright Tm3+-based downshifting luminescence nanoprobe operating around 1800 nm for NIR-IIb and c bioimaging
Fluorescence imaging in the near-infrared region yields high-quality images that overcome the current depth limitations. Here, the authors report a Tm3 + -based nanoprobe for NIR-IIb/c imaging, providing references to future bioimaging beyond 1700 nm.
- Yulei Chang
- , Haoren Chen
- & Xianggui Kong
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Article
| Open Access
Visualizing orthogonal RNAs simultaneously in live mammalian cells by fluorescence lifetime imaging microscopy (FLIM)
No multi-color RNA fluorescent tags are currently available for use in live cells. Here, the authors show that fluorescence lifetime imaging microscopy is advantageous for multiplexed RNA visualization while achieving robust cellular contrast.
- Nadia Sarfraz
- , Emilia Moscoso
- & Esther Braselmann
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Article
| Open Access
A FRET-based respirasome assembly screen identifies spleen tyrosine kinase as a target to improve muscle mitochondrial respiration and exercise performance in mice
Mitochondrial supercomplex assembly may efficiently supply energy, yet its role remains controversial. Here, the authors show that SYK inhibitors increase supercomplex assembly and mitochondrial respiration in cells and can enhance exercise performance in mice.
- Ami Kobayashi
- , Kotaro Azuma
- & Satoshi Inoue
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Article
| Open Access
Revealing the tissue-level complexity of endogenous glucagon-like peptide-1 receptor expression and signaling
Visualizing endogenous GPCRs is challenging. Here the authors generate mice with an enzyme self-label genome-edited into the endogenous glucagon-like peptide-1 receptor locus, design fluorescent dyes for specific labelling in complex tissue, and reveal tissue-level organisation and dynamics of an endogenous class B GPCR.
- Julia Ast
- , Daniela Nasteska
- & David J. Hodson
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Article
| Open Access
An optofluidic platform for interrogating chemosensory behavior and brainwide neural representation in larval zebrafish
Studying chemosensory processing desires precise chemical cue presentation, behavioral response monitoring, and large-scale neuronal activity recording. Here, the authors report a fluidics-based toolkit for studying chemosensation in larval zebrafish, and used it to reveal the brainwide neural representations of cadaverine sensing and its binasal input-dependent behavioral avoidance.
- Samuel K. H. Sy
- , Danny C. W. Chan
- & Ho Ko
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Article
| Open Access
Filamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane
GABAB receptors mediate the effects of the main inhibitory neurotransmitter in the brain. Here, authors identify the cytoskeletal protein filamin A as a key player that controls the exact location and function of GABAB receptors at the cell surface.
- Marie-Lise Jobin
- , Sana Siddig
- & Davide Calebiro
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Article
| Open Access
Engineering inducible biomolecular assemblies for genome imaging and manipulation in living cells
Imaging non-repetitive loci in living cells remains challenging. Here, the authors engineered an inducible system whereby biomolecular assemblies can be guided to specific genomic loci by a nuclease-defective Cas9, allowing the simultaneous imaging and manipulation of the loci.
- Qin Peng
- , Ziliang Huang
- & Yingxiao Wang
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Article
| Open Access
Tiltable objective microscope visualizes selectivity for head motion direction and dynamics in zebrafish vestibular system
Signals about head orientation and movement in the vestibular periphery are fundamental to the sense of balance and motion, but difficult to measure systematically during head motion. Here, the authors build a microscope that visualizes neural activity in hair cells and vestibular ganglion cells during 360° head tilt and vibration in zebrafish larvae, and reveal a topographic organization of direction- and static/dynamic stimulus-selective responses.
- Masashi Tanimoto
- , Ikuko Watakabe
- & Shin-ichi Higashijima
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Article
| Open Access
Achieving single nucleotide sensitivity in direct hybridization genome imaging
Visualisation of point mutations in situ is informative for studying genetic diseases. Here the authors report single guide genome oligopaint via local denaturation fluorescence in situ hybridisation, sgGOLDFISH, a direct hybridisation genome imaging method with single-nucleotide sensitivity.
- Yanbo Wang
- , W. Taylor Cottle
- & Taekjip Ha
Cortex-wide transcranial localization microscopy with fluorescently labeled red blood cells