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| Open AccessSingle-photon microscopy to study biomolecular condensates
The wide variety of cellular processes involving biomolecular condensation makes their quantification a challenging task. Here, the authors present an integrated platform based on single-photon microscopy to study complex biomolecular processes.
- Eleonora Perego
- , Sabrina Zappone
- & Giuseppe Vicidomini
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Article
| Open AccessA fungal plant pathogen discovered in the Devonian Rhynie Chert
Here, the authors describe a pathogenic fungus from a 400-million-year-old fossil plant from the Devonian Rhynie Chert in Scotland. They use advanced imaging methods to determine that the fungus belongs to the sac fungi, the most diverse group of Fungi today.
- Christine Strullu-Derrien
- , Tomasz Goral
- & David L. Hawksworth
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Article
| Open AccessMultipole engineering by displacement resonance: a new degree of freedom of Mie resonance
Mie resonances are typically manipulated through varying nanostructure shape/size. Here, authors found that Gaussian beam displacement excites higher-order multipolar modes, not accessible by plane wave, featuring maximal linear and nonlinear scattering efficiency when the focus is misaligned.
- Yu-Lung Tang
- , Te-Hsin Yen
- & Shi-Wei Chu
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Article
| Open AccessMolecular landscape and functional characterization of centrosome amplification in ovarian cancer
The prevalence of centrosome amplification (CA) and the genomic landscape of chromosomal instability in high-grade serous ovarian carcinoma (HGSOC) remain to be explored. Here the authors suggest CA as a potential driver of tumour evolution and a biomarker for treatment response in HGSOC.
- Carolin M. Sauer
- , James A. Hall
- & James D. Brenton
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Article
| Open AccessAn entropy-controlled objective chip for reflective confocal microscopy with subdiffraction-limit resolution
The optimized and disordered structures of planar diffractive lenses enable sub-diffraction limit focusing but destroy wide-field imaging. Here, the authors introduce the information entropy to evaluate the disorder of PDL and achieve good balance between super-focusing and imaging.
- Jun He
- , Dong Zhao
- & Kun Huang
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Article
| Open AccessFluorescent 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 AccessInsights 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 AccessSingle-cell quantification and dose-response of cytosolic siRNA delivery
Endosomal escape and subsequent cytosolic delivery of siRNA therapeutics is inefficient, and quantification is difficult. Here the authors report a confocal microscopy-based method to quantify cytosolic delivery of fluorescently labelled siRNA during lipid-mediated delivery.
- Hampus Hedlund
- , Hampus Du Rietz
- & Anders Wittrup
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Article
| Open AccessDetecting and quantifying liquid–liquid phase separation in living cells by model-free calibrated half-bleaching
It is currently debated how to reliably distinguish liquid–liquid phase separation (LLPS) from other mechanisms. Here the authors report model-free calibrated half-FRAP (MOCHA-FRAP) to probe the barrier at the condensate interface that is responsible for preferential internal mixing in LLPS.
- Fernando Muzzopappa
- , Johan Hummert
- & Fabian Erdel
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Article
| Open AccessFocus image scanning microscopy for sharp and gentle super-resolved microscopy
Super-resolution microscopy techniques can be challenging for live cells and thick samples. Here, the authors propose a method to reduce beam intensity and remove out-of-focus fluorescence background in image-scanning microscopy (ISM) and its combination with stimulated emission depletion (STED).
- Giorgio Tortarolo
- , Alessandro Zunino
- & Giuseppe Vicidomini
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Article
| Open AccessExtending resolution within a single imaging frame
The presented Mean-Shift Super Resolution (MSSR) algorithm can extend spatial resolution within a single microscopy image. Its applicability extends across a wide range of experimental and instrumental configurations and it is compatible with other super-resolution microscopy approaches.
- Esley Torres-García
- , Raúl Pinto-Cámara
- & Adán Guerrero
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Article
| Open AccessThe BrightEyes-TTM as an open-source time-tagging module for democratising single-photon microscopy
The authors developed an open-source, low-cost, multi-channel time-tagging module for fluorescence lifetime image scanning microscopy and correlation spectroscopy that can tag in parallel multiple single-photon events with 30 ps precision.
- Alessandro Rossetta
- , Eli Slenders
- & Giuseppe Vicidomini
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Article
| Open AccessLipid-mediated activation of plasma membrane-localized deubiquitylating enzymes modulate endosomal trafficking
The endocytic degradation of plasma membrane proteins can be modulated by deubiquitylating enzymes (DUBs). Here, the authors describe two plasma membrane localized Arabidopsis DUBs that can be activated by binding to anionic lipids and influence the endocytic transport of plasma membrane proteins.
- Karin Vogel
- , Tobias Bläske
- & Erika Isono
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Article
| Open AccessResolving subcellular pH with a quantitative fluorescent lifetime biosensor
Measuring sub-cellular pH with high accuracy and spatiotemporal resolution remains challenging. Here, Johnston and co-workers develop a pH biosensor that combines the pH dependant fluorescent lifetime of mApple with deep learning to accurately determine sub-cellular pH in individual vesicles.
- Joshua J. Rennick
- , Cameron J. Nowell
- & Angus P. R. Johnston
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Article
| Open AccessDNA damage-induced transcription stress triggers the genome-wide degradation of promoter-bound Pol II
DNA damage inhibits elongating RNA polymerase II, but also initiates genome-wide transcriptional responses. Here the authors reveal that particularly promoter-bound Pol II is degraded upon DNA damage in a GSK3 signaling-mediated response.
- Barbara Steurer
- , Roel C. Janssens
- & Jurgen Marteijn
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Article
| Open AccessDeep learning enables reference-free isotropic super-resolution for volumetric fluorescence microscopy
Volumetric fluorescence microscopy is often limited by anisotropic spatial resolution. Here, the authors present an unsupervised deep-learning approach that enhances axial resolution by learning from high-resolution lateral images, and demonstrate isotropic resolution and restoration of suppressed visual details.
- Hyoungjun Park
- , Myeongsu Na
- & Jong Chul Ye
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Article
| Open AccessPhase separation and zinc-induced transition modulate synaptic distribution and association of autism-linked CTTNBP2 and SHANK3
Autism impacts synapses. This study reports that autism-linked mutations of CTTNBP2 regulate phase separation to control synaptic enrichment of that protein. A zinc-induced liquid-to-gel transition improves synaptic retention of CTTNBP2 and SHANK3.
- Pu-Yun Shih
- , Yu-Lun Fang
- & Yi-Ping Hsueh
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Article
| Open AccessIn vitro reconstitution of Escherichia coli divisome activation
In E. coli, FtsA and FtsZ control the place and time of cell division. Here, the authors use in vitro experiments to show how FtsA can follow FtsZ treadmilling and that downstream proteins form dynamic copolymers with FtsA to initiate division.
- Philipp Radler
- , Natalia Baranova
- & Martin Loose
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Article
| Open AccessPICASSO allows ultra-multiplexed fluorescence imaging of spatially overlapping proteins without reference spectra measurements
Ultra-multiplexed fluorescence imaging is currently difficult. Here the authors report PICASSO which enables 15-colour imaging of spatially overlapping proteins in a single-round of imaging; they combine it with cyclic immunofluorescence to achieve 45-colour imaging of the mouse brain in 3 cycles.
- Junyoung Seo
- , Yeonbo Sim
- & Jae-Byum Chang
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Article
| Open AccessMre11-Rad50 oligomerization promotes DNA double-strand break repair
The Mre11-Rad50 (MR) complex has key functions in the detection, signaling and repair of DNA breaks. Here the authors use transmission electron microscopy to show MR oligomerization is governed by a small beta-sheet protruding from the head domain of Rad50 at the base of the MR structure, and reveal MR head domain oligomerization is required for efficient DNA end resection.
- Vera M. Kissling
- , Giordano Reginato
- & Matthias Peter
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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 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 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 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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| Open AccessProbing the biogenesis pathway and dynamics of thylakoid membranes
Cyanobacterial thylakoid membranes host the molecular machinery for the light-dependent reactions of photosynthesis and respiratory electron flow. Here, the authors show that newly synthesized thylakoids emerge between the plasma membrane and pre-existing thylakoids and describe the time-dependent assembly process of photosynthetic complexes.
- Tuomas Huokko
- , Tao Ni
- & Lu-Ning Liu
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Article
| Open AccessMicrothermal-induced subcellular-targeted protein damage in cells on plasmonic nanosilver-modified surfaces evokes a two-phase HSP-p97/VCP response
Existing methods for inflicting cellular heat shock are limited by the time delay in achieving the desired temperature and the spatial precision that can be achieved. Here the authors report a method to induce focused thermal protein damage using plasmonic silver nanoparticles.
- Martin Mistrik
- , Zdenek Skrott
- & Jiri Bartek
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Article
| Open AccessLaser scanning reflection-matrix microscopy for aberration-free imaging through intact mouse skull
Microscopic imaging of the brain usually requires thinning of the skull, as it causes complex aberration. Here, the authors introduce a label-free imaging modality termed laser scanning reflection-matrix microscopy, which allows for correcting these aberrations and in vivo imaging through an intact mouse skull.
- Seokchan Yoon
- , Hojun Lee
- & Wonshik Choi
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Article
| Open AccessDeep tissue space-gated microscopy via acousto-optic interaction
In ideal diffraction-limited optical microscopy imaging depth is limited by the multiply scattered wave. Here, the authors present a space gating method, based on an acoustic focus in the object plane and reconstruction using only the acousto-optically modulated ballistic wave, and demonstrate increased imaging depth in scattering samples.
- Mooseok Jang
- , Hakseok Ko
- & Wonshik Choi
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Article
| Open AccessPre-processing visualization of hyperspectral fluorescent data with Spectrally Encoded Enhanced Representations
Spectral phasor analysis allows unmixing fluorescence microscopy images, but it requires user involvement and has a limited number of labels that can be analyzed and displayed. Here the authors present a semi-automated solution to visualise multiple spectral components of hyperspectral fluorescence images, simultaneously.
- Wen Shi
- , Daniel E. S. Koo
- & Francesco Cutrale
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Article
| Open Access3D sub-diffraction imaging in a conventional confocal configuration by exploiting super-linear emitters
Super-resolution microscopy is a valuable tool in bioimaging, but often requires complex systems or post-processing. Here, the authors present super-linear excitation-emission (SEE) microscopy, which overcomes these limitations by taking advantage of markers with super-linear dependence between emission and excitation power.
- Denitza Denkova
- , Martin Ploschner
- & James A. Piper
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Article
| Open AccessStudying nucleic envelope and plasma membrane mechanics of eukaryotic cells using confocal reflectance interferometric microscopy
Biomechanical studies of eukaryotic cells have been limited due to low sensitivity and axial resolution in interferometric imaging. Here, the authors present depth-resolved confocal reflectance interferometric microscopy with high sensitivity and temporal resolution, which enables quantification of nucleic envelope and plasma membrane fluctuations.
- Vijay Raj Singh
- , Yi An Yang
- & Peter T. C. So
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Article
| Open AccessMonitoring drug nanocarriers in human blood by near-infrared fluorescence correlation spectroscopy
While nanocarrier-based drug delivery is a promising therapeutic approach, in situ characterization of drug nanocarriers in blood remains difficult. Here, the authors demonstrate how the fluorescence correlation spectroscopy can be used to directly characterize drug nanocarriers in flowing blood.
- Inka Negwer
- , Andreas Best
- & Kaloian Koynov
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Article
| Open AccessHeterogeneity in tumor chromatin-doxorubicin binding revealed by in vivo fluorescence lifetime imaging confocal endomicroscopy
The engagement of DNA-binding drugs to their target is difficult to study, particularly in vivo. Here the authors develop an in vivo fluorescence lifetime imaging confocal laparo/endomicroscope to show intra- and inter-tumor heterogeneity in doxorubicin binding to peritoneal metastases, which depends on the route of administration.
- Hugh Sparks
- , Hiroshi Kondo
- & Erik Sahai
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Article
| Open AccessQuantitative spatial analysis of haematopoiesis-regulating stromal cells in the bone marrow microenvironment by 3D microscopy
The bone marrow microenvironment modulates haematopoiesis, stem cell maintenance and differentiation. Here, the authors use 3D microscopy to map the topography of haematopoietic stem cell niche stromal components.
- Alvaro Gomariz
- , Patrick M. Helbling
- & César Nombela-Arrieta
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Article
| Open AccessMultiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT
Existing methods for nanoscale visualization of biological targets in thick samples require complex hardware. Here, the authors combine the standard spinning disk confocal (SDC) microscopy with DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) to image proteins, DNA and RNA deep in cells.
- Florian Schueder
- , Juanita Lara-Gutiérrez
- & Ralf Jungmann
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| Open AccessNegri bodies are viral factories with properties of liquid organelles
Negative strand RNA viruses, such as rabies virus, induce formation of cytoplasmic inclusions for genome replication. Here, Nikolic et al. show that these so-called Negri bodies (NBs) have characteristics of liquid organelles and they identify the minimal protein domains required for NB formation.
- Jovan Nikolic
- , Romain Le Bars
- & Danielle Blondel
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| Open AccessSizing nanomaterials in bio-fluids by cFRAP enables protein aggregation measurements and diagnosis of bio-barrier permeability
Measuring the size distribution of nanomaterials in biological fluids is crucial to understand their properties in vivo. Here, the authors apply fluorescence recovery after photobleaching to measure protein aggregation in serum and to study permeability of biological membranes in mouse models.
- Ranhua Xiong
- , Roosmarijn E. Vandenbroucke
- & Kevin Braeckmans
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Article
| Open AccessDrosophila wing imaginal discs respond to mechanical injury via slow InsP3R-mediated intercellular calcium waves
It is unclear what role calcium signalling plays in the Drosophila wing disc. Here, the authors show that on mechanical stress, slow, long-range intercellular calcium waves are initiated in vivo and ex vivo, mediated by the inositol-3-phosphate receptor, the calcium pump SERCA and gap junction component Inx2.
- Simon Restrepo
- & Konrad Basler
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Measuring the size of individual particles from three-dimensional imaging experiments
The degree of polydispersity of colloidal suspensions is known to have consequences for their physical properties. Kuritaet al. present a general method for determining the sizes of individual particles, and thus the polydispersity, using only the coordinates of the centre positions of spherical particles.
- Rei Kurita
- , David B. Ruffner
- & Eric R. Weeks