Non-scanning in-vivo three-dimensional hybrid structured illumination microscopy

2018 ◽  
Author(s):  
Ju-Hsuan Chien ◽  
Chen Yen Lin ◽  
Yuan Luo
Keyword(s):  
Three Dimensional ◽  
Structured Illumination ◽  
Structured Illumination Microscopy

scholarly journals Biosensor-Expressing Spheroid Cultures for Imaging of Drug-Induced Effects in Three Dimensions

2013 ◽  
Vol 18 (6) ◽  
pp. 736-743 ◽  
Author(s):  
Rainer Wittig ◽  
Verena Richter ◽  
Stephanie Wittig-Blaich ◽  
Petra Weber ◽  
Wolfgang S. L. Strauss ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Fluorescent Protein ◽  
Temporal Dynamics ◽  
Three Dimensional ◽  
Three Dimensions ◽  
Structured Illumination ◽  
Three Dimensional Model ◽  
Structured Illumination Microscopy ◽  
Drug Induced

In the past, the majority of antitumor compound-screening approaches had been performed in two-dimensional (2D) cell cultures. Although easy to standardize, this method provides results of limited significance because cells are surrounded by an artificial microenvironment, are not exposed to hypoxia gradients, and lack cell-cell contacts. These nonphysiological conditions directly affect relevant parameters such as the resistance to anticancer drugs. Multicellular tumor spheroids more closely resemble the in vivo situation in avascularized tumors. To monitor cellular reactions within this three-dimensional model system, we stably transfected a spheroid-forming glioblastoma cell line with Grx1-roGFP2, a green fluorescent protein (GFP)–based glutathione-specific redox sensor that detects alterations in the glutathione redox potential. Functionality and temporal dynamics of the sensor were verified with redox-active substances in 2D cell culture. Based on structured illumination microscopy using nonphototoxic light doses, ratio imaging was then applied to monitor the response of the glutathione system to exogenous hydrogen peroxide in optical sections of a tumor spheroid. Our approach provides a proof of concept for biosensor-based imaging in 3D cell cultures.

scholarly journals Mitofusin-2 stabilizes adherens junctions and suppresses endothelial inflammation via modulation of β-catenin signaling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Young-Mee Kim ◽  
Sarah Krantz ◽  
Ankit Jambusaria ◽  
Peter T. Toth ◽  
Hyung-Geun Moon ◽  
...  
Keyword(s):  
Adherens Junctions ◽  
Mitochondrial Protein ◽  
Three Dimensional ◽  
Adherens Junction ◽  
Inflammatory Cells ◽  
Endothelial Barrier ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Mitofusin 2

AbstractEndothelial barrier integrity is ensured by the stability of the adherens junction (AJ) complexes comprised of vascular endothelial (VE)-cadherin as well as accessory proteins such as β-catenin and p120-catenin. Disruption of the endothelial barrier due to disassembly of AJs results in tissue edema and the influx of inflammatory cells. Using three-dimensional structured illumination microscopy, we observe that the mitochondrial protein Mitofusin-2 (Mfn2) co-localizes at the plasma membrane with VE-cadherin and β-catenin in endothelial cells during homeostasis. Upon inflammatory stimulation, Mfn2 is sulfenylated, the Mfn2/β-catenin complex disassociates from the AJs and Mfn2 accumulates in the nucleus where Mfn2 negatively regulates the transcriptional activity of β-catenin. Endothelial-specific deletion of Mfn2 results in inflammatory activation, indicating an anti-inflammatory role of Mfn2 in vivo. Our results suggest that Mfn2 acts in a non-canonical manner to suppress the inflammatory response by stabilizing cell–cell adherens junctions and by binding to the transcriptional activator β-catenin.

scholarly journals Super-Resolution Imaging by Dual Iterative Structured Illumination Microscopy

2021 ◽  
Author(s):  
Anna Loeschberger ◽  
Yauheni Novikau ◽  
Ralf Netz ◽  
Marie-Christin Spindler ◽  
Ricardo Benavente ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Brain Slices ◽  
Super Resolution ◽  
Reconstruction Algorithm ◽  
Living Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Spatiotemporal Resolution ◽  
Coated Pits ◽  
Resolution Imaging

Three-dimensional (3D) multicolor super-resolution imaging in the 50-100 nm range in fixed and living cells remains challenging. We extend the resolution of structured illumination microscopy (SIM) by an improved nonlinear iterative reconstruction algorithm that enables 3D multicolor imaging with improved spatiotemporal resolution at low illumination intensities. We demonstrate the performance of dual iterative SIM (diSIM) imaging cellular structures in fixed cells including synaptonemal complexes, clathrin coated pits and the actin cytoskeleton with lateral resolutions of 60-100 nm with standard fluorophores. Furthermore, we visualize dendritic spines in 70 micrometer thick brain slices with an axial resolution < 200 nm. Finally, we image dynamics of the endoplasmatic reticulum and microtubules in living cells with up to 255 frames/s.

scholarly journals Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes

2020 ◽  
Author(s):  
Jiji Chen ◽  
Hideki Sasaki ◽  
Hoyin Lai ◽  
Yijun Su ◽  
Jiamin Liu ◽  
...  
Keyword(s):  
Fluorescence Microscopy ◽  
Network Performance ◽  
Resolution Enhancement ◽  
Three Dimensional ◽  
Ground Truth ◽  
Time Lapse ◽  
Stimulated Emission ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Attention Networks

Abstract We demonstrate residual channel attention networks (RCAN) for restoring and enhancing volumetric time-lapse (4D) fluorescence microscopy data. First, we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy 4D super-resolution data, enabling image capture over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables class-leading resolution enhancement, superior to other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling ~2.5-fold lateral resolution enhancement using stimulated emission depletion (STED) microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy ground truth, achieving improvements of ~1.4-fold laterally and ~3.4-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluating and further enhancing network performance.

scholarly journals A novel workflow for three-dimensional analysis of tumour cell migration

2020 ◽  
Vol 10 (2) ◽  
pp. 20190070 ◽  
Author(s):  
Sophie Ketchen ◽  
Arndt Rohwedder ◽  
Sabine Knipp ◽  
Filomena Esteves ◽  
Nina Struve ◽  
...  
Keyword(s):  
Dimensional Analysis ◽  
Three Dimensional ◽  
Two Dimensional ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Tumour Spheroid ◽  
3D Data ◽  
Biological Studies ◽  
Research Findings ◽  
Imagej Plugin

The limitations of two-dimensional analysis in three-dimensional (3D) cellular imaging impair the accuracy of research findings in biological studies. Here, we report a novel 3D approach to acquisition, analysis and interpretation of tumour spheroid images. Our research interest in mesenchymal–amoeboid transition led to the development of a workflow incorporating the generation and analysis of 3D data with instant structured illumination microscopy and a new ImageJ plugin.

scholarly journals High-speed multiplane structured illumination microscopy of living cells using an image-splitting prism

Nanophotonics ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 143-148
Author(s):  
Adrien Descloux ◽  
Marcel Müller ◽  
Vytautas Navikas ◽  
Andreas Markwirth ◽  
Robin van den Eynde ◽  
...  
Keyword(s):  
High Speed ◽  
Three Dimensional ◽  
Super Resolution ◽  
Optical Element ◽  
Spatial Light Modulators ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Image Stack ◽  
Light Modulators ◽  
3D Information

AbstractSuper-resolution structured illumination microscopy (SR-SIM) can be conducted at video-rate acquisition speeds when combined with high-speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live-cell imaging. If, however, three-dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work, we present a multiplane approach to SR-SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multiplane image splitting prism combined with high-speed SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally highly resolved 3D image stack. We demonstrate the performance of multiplane SIM by applying this instrument to imaging the dynamics of mitochondria in living COS-7 cells.

scholarly journals Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse

2016 ◽  
Vol 215 (3) ◽  
pp. 383-399 ◽  
Author(s):  
Sricharan Murugesan ◽  
Jinsung Hong ◽  
Jason Yi ◽  
Dong Li ◽  
Jordan R. Beach ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Immunological Synapse ◽  
Three Dimensional ◽  
Cell Receptor ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Tcr Signaling ◽  
Immune Synapse ◽  
Distal Edge

Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1). Using structured-illumination microscopy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments generated by one or more formins present at the IS distal edge. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs. Three-dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs. Consistently, disrupting actin arc formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T–B cell adhesion, and diminished TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport.

scholarly journals Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

2015 ◽  
Vol 112 (32) ◽  
pp. E4390-E4399 ◽  
Author(s):  
Mathew Stracy ◽  
Christian Lesterlin ◽  
Federico Garza de Leon ◽  
Stephan Uphoff ◽  
Pawel Zawadzki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Molecule ◽  
Spatial Organization ◽  
Search Time ◽  
Population Level ◽  
Bacterial Cells ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Superresolution Microscopy

Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells.

scholarly journals CEP19 cooperates with FOP and CEP350 to drive early steps in the ciliogenesis programme

Open Biology ◽  
2017 ◽  
Vol 7 (6) ◽  
pp. 170114 ◽  
Author(s):  
Bahareh A. Mojarad ◽  
Gagan D. Gupta ◽  
Monica Hasegan ◽  
Oumou Goudiam ◽  
Renata Basto ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Three Dimensional ◽  
Structured Illumination ◽  
Loss Of Function ◽  
Structured Illumination Microscopy ◽  
C Terminus ◽  
Interaction Partners ◽  
Cilia Formation ◽  
Mother Centriole ◽  
Section Electron

Primary cilia are microtubule-based sensory organelles necessary for efficient transduction of extracellular cues. To initiate cilia formation, ciliary vesicles (CVs) are transported to the vicinity of the centrosome where they dock to the distal end of the mother centriole and fuse to initiate cilium assembly. However, to this date, the early steps in cilia formation remain incompletely understood. Here, we demonstrate functional interplay between CEP19, FOP and CEP350 in ciliogenesis. Using three-dimensional structured-illumination microscopy (3D-SIM) imaging, we mapped the relative spatial distribution of these proteins at the distal end of the mother centriole and show that CEP350/FOP act upstream of CEP19 in their recruitment hierarchy. We demonstrate that CEP19 CRISPR KO cells are severely impaired in their ability to form cilia, analogous to the loss of function of CEP19 binding partners FOP and CEP350. Notably, in the absence of CEP19 microtubule anchoring at centromes is similar in manner to its interaction partners FOP and CEP350. Using GFP-tagged deletion constructs of CEP19, we show that the C-terminus of CEP19 is required for both its localization to centrioles and for its function in ciliogenesis. Critically, this region also mediates the interaction between CEP19 and FOP/CEP350. Interestingly, a morbid-obesity-associated R82* truncated mutant of CEP19 cannot ciliate nor interact with FOP and CEP350, indicative of a putative role for CEP19 in ciliopathies. Finally, analysis of CEP19 KO cells using thin-section electron microscopy revealed marked defects in the docking of CVs to the distal end of the mother centrioles. Together, these data demonstrate a role for the CEP19, FOP and CEP350 module in ciliogenesis and the possible effect of disrupting their functions in ciliopathies.

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