In Vivo Imaging with Microsphere-Based Super-Resolution Microscopy

2018 ◽  
Author(s):  
Gergely Huszka ◽  
Roger Krenger ◽  
Martin A. M. Gijs
Keyword(s):  
In Vivo Imaging ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals Corrigendum: Super-Resolution Microscopy: Shedding New Light on In Vivo Imaging

2021 ◽  
Vol 9 ◽  
Author(s):  
Yingying Jing ◽  
Chenshuang Zhang ◽  
Bin Yu ◽  
Danying Lin ◽  
Junle Qu
Keyword(s):  
In Vivo Imaging ◽  
Super Resolution ◽  
Super Resolution Microscopy

scholarly journals Ligand Geometry Controls Adhesion formation via Integrin Clustering

2018 ◽  
Author(s):  
Rishita Changede ◽  
Haogang Cai ◽  
Shalom Wind ◽  
Michael P. Sheetz
Keyword(s):  
Adhesion Formation ◽  
Focal Adhesions ◽  
Super Resolution ◽  
Total Width ◽  
Cellular Processes ◽  
Cell Matrix ◽  
Integrin Clustering ◽  
Super Resolution Microscopy ◽  
Fiber Mesh

AbstractIntegrin-mediated cell matrix adhesions are key to sensing the geometry and rigidity of the extracellular environment to regulate vital cellular processes. In vivo, the extracellular matrix (ECM) is composed of a fibrous mesh. To understand the geometry that supports adhesion formation on fibrous substrates, we patterned 10 nm gold-palladium single lines or pairs of lines (total width within 100 nm), mimicking thin single ECM fibers or a minimal mesh geometry, respectively and functionalized it with integrin binding ligand Arg-Gly-Asp (RGD). Single lines showed reduced focal adhesion kinase (FAK) recruitment and did not support cell spreading or formation of focal adhesions, despite the presence of a high density of integrin-binding ligands. Using super resolution microscopy, we observed transient integrin clusters on single lines, whereas stable 110 nm integrin clusters formed on pairs of lines similar to those on continuous substrates. This indicated that two-dimensional ligand geometry is required for adhesion formation on rigid substrates. A mechanism to form modular 100nm integrin clusters bridging the minimal fiber mesh would require unliganded integrins. We observed that integrin mutants unable to bind ligand co-clustered with ligand-bound integrins when present in an active extended conformation. Thus, these results indicate that functional integrin clusters are required to form focal adhesions and unliganded integrins can co-cluster to bridge between thin matrix fibers and can form stable integrin adhesions on dense fibrous networks.

scholarly journals High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization

2014 ◽  
Vol 111 (12) ◽  
pp. 4566-4571 ◽  
Author(s):  
S. J. Holden ◽  
T. Pengo ◽  
K. L. Meibom ◽  
C. Fernandez Fernandez ◽  
J. Collier ◽  
...  
Keyword(s):  
High Throughput ◽  
Caulobacter Crescentus ◽  
Super Resolution ◽  
Z Ring ◽  
Super Resolution Microscopy

scholarly journals Mutant EGFR is a preferred SMURF2 substrate of ubiquitination: role in enhanced receptor stability and TKI sensitivity

2020 ◽  
Author(s):  
Paramita Ray ◽  
Krishnan Raghunathan ◽  
Aarif Ahsan ◽  
Uday Sankar Allam ◽  
Shirish Shukla ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Super Resolution ◽  
Receptor Internalization ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Steady State Levels ◽  
Epidermal Growth ◽  
Optical Reconstruction ◽  
Super Resolution Microscopy

ABSTRACTWe previously reported that differential protein degradation of TKI-sensitive [L858R, del(E746-A750)] and resistant (T790M) epidermal growth factor receptor (EGFR) mutants upon erlotinib treatment correlates with drug sensitivity. However, the molecular mechanism remains unclear. We also reported SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. Here, using in vitro and in vivo ubiquitination assays, mass spectrometry, and super-resolution microscopy, we show SMURF2-EGFR functional interaction is critical in receptor stability and TKI sensitivity. We found that L858R/T790M EGFR is a preferred substrate of SMURF2-UBCH5 (an E3-E2) complex-mediated K63-linked polyubiquitination, which preferentially stabilizes mutant receptor. We identified four lysine (K) residues (K721, 846, 1037 and 1164) as the sites of ubiquitination and replacement of K to acetylation-mimicking asparagine (Q) at K1037 position in L858R/T790M background converts the stable protein sensitive to erlotinib-induced degradation. Using STochastic Optical Reconstruction Microscopy (STORM) imaging, we show that SMURF2 presence allows longer membrane retention of activated EGFR upon EGF treatment, whereas, siRNA-mediated SMURF2 knockdown fastens receptor endocytosis and lysosome enrichment. In an erlotinib-sensitive PC9 cells, SMURF2 overexpression increased EGFR levels with improved erlotinib tolerance, whereas, SMURF2 knockdown decreased EGFR steady state levels in NCI-H1975 and PC9-AR cells to overcome erlotinib and AZD-9291 resistance respectively. Additionally, by genetically altering the SMURF2-UBCH5 complex formation destabilized EGFR. Together, we propose that SMURF2-mediated preferential polyubiquitination of L858R/T790M EGFR may be competing with acetylation-mediated receptor internalization to provide enhanced receptor stability and that disruption of the E3-E2 complex may be an attractive alternate to overcome TKI resistance.

scholarly journals Binding of the RNA Chaperone Hfq on Target mRNAs Promotes the Small RNA RyhB-Induced Degradation in Escherichia coli

2021 ◽  
Vol 7 (4) ◽  
pp. 64
Author(s):  
David Lalaouna ◽  
Karine Prévost ◽  
Seongjin Park ◽  
Thierry Chénard ◽  
Marie-Pier Bouchard ◽  
...  
Keyword(s):  
Complex Formation ◽  
Super Resolution ◽  
Rnase E ◽  
Rna Chaperone ◽  
Target Mrnas ◽  
Mrna Targets ◽  
Super Resolution Microscopy

Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation.

scholarly journals Super-Resolution Microscopy: Shedding New Light on In Vivo Imaging

2021 ◽  
Vol 9 ◽  
Author(s):  
Yingying Jing ◽  
Chenshuang Zhang ◽  
Bin Yu ◽  
Danying Lin ◽  
Junle Qu
Keyword(s):  
Biological Activities ◽  
Super Resolution ◽  
Spatiotemporal Resolution ◽  
The Past ◽  
Relevant Field ◽  
Living Species ◽  
Resolution Imaging ◽  
Super Resolution Microscopy ◽  
Conventional Light Microscopy

Over the past two decades, super-resolution microscopy (SRM), which offered a significant improvement in resolution over conventional light microscopy, has become a powerful tool to visualize biological activities in both fixed and living cells. However, completely understanding biological processes requires studying cells in a physiological context at high spatiotemporal resolution. Recently, SRM has showcased its ability to observe the detailed structures and dynamics in living species. Here we summarized recent technical advancements in SRM that have been successfully applied to in vivo imaging. Then, improvements in the labeling strategies are discussed together with the spectroscopic and chemical demands of the fluorophores. Finally, we broadly reviewed the current applications for super-resolution techniques in living species and highlighted some inherent challenges faced in this emerging field. We hope that this review could serve as an ideal reference for researchers as well as beginners in the relevant field of in vivo super resolution imaging.

scholarly journals Analysis of Preplatelets and Their Barbell Platelet Derivatives by Imaging Flow Cytometry

2021 ◽  
Author(s):  
Samuel Kemble ◽  
Amanda L Dalby ◽  
Gillian C Lowe ◽  
Phillip LR Nicolson ◽  
Steve P Watson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ex Vivo ◽  
Super Resolution ◽  
Thiazole Orange ◽  
Healthy Human ◽  
Platelet Counts ◽  
Human Volunteers ◽  
Immature Platelets ◽  
Super Resolution Microscopy

Circulating large ″preplatelets″ undergo fission via barbell platelet intermediates into two smaller, mature platelets. In this study, we determine whether preplatelets and/or barbells are equivalent to reticulated/immature platelets by using ImageStream flow cytometry (ISFC) and super-resolution microscopy. Immature platelets, preplatelets and barbells were quantified in healthy and thrombocytopenic mice, healthy human volunteers, and patients with immune thrombocytopenia (ITP) or undergoing chemotherapy. Preplatelets and barbells were 1.9%±0.18/1.7%±0.48 (n=6) and 3.3%-+1.6/0.5%±0.27 (n=12) of total platelet counts in murine and human whole blood, respectively. Both preplatelets and barbells exhibited high expression of HLA-I with high thiazole orange and mitotracker fluorescence. Tracking dye experiments confirmed that preplatelets transform into barbells and undergo fission ex vivo to increase platelet counts, with dependence upon the cytoskeleton and normal mitochondrial respiration. Samples from antibody-induced thrombocytopenia in mice and patients with ITP had increased levels of both preplatelets and barbells correlating with immature platelet levels. Furthermore, barbells were absent post-chemotherapy in patients. In mice, in vivo biotinylation confirmed that barbells, but not all large platelets, were immature. This study demonstrates that a subpopulation of large platelets are immature preplatelets that can transform into barbells and undergo fission during maturation.

scholarly journals New mechanism of fibronectin fibril assembly revealed by live imaging and super-resolution microscopy

2020 ◽  
Author(s):  
Darshika Tomer ◽  
Sudipto Munshi ◽  
Brianna E. Alexander ◽  
Brenda French ◽  
Pavan Vedula ◽  
...  
Keyword(s):  
Cell Fate ◽  
Three Dimensional ◽  
Super Resolution ◽  
Live Imaging ◽  
Modular Architecture ◽  
Ecm Remodeling ◽  
Linear Arrays ◽  
Cell Fate Decisions ◽  
Super Resolution Microscopy

AbstractThe regulation of cell fate decisions, morphogenesis, and responses to injury are intimately linked to the process of Fn1 fibrillogenesis. Live imaging and super-resolution microscopy revealed that Fn1 fibrils are not continuous. Instead, Fn1 fibrils arise from nanodomains containing multiple Fn1 dimers. As they move toward cell center, Fn1 nanodomains become organized into linear arrays with a spacing of 130 nm between the nanodomains, with little Fn1 in between; Fn1 nanodomain arrays are resistant to deoxycholate treatment demonstrating that these beaded assemblies are indeed mature Fn1 fibrils. FUD, a bacterial peptide that disrupts Fn1 fibrillogenesis, does not disrupt nanodomain formation; instead, it interferes with the organization of nanodomains into arrays. The nanodomain composition of Fn1 fibrils is observed in multiple contexts: in three-dimensional ECM in vivo, on substrata of different composition and stiffness, and is retained in the absence of cells. The modular architecture of Fn1 fibrils bears important implications for mechanisms of ECM remodeling and signal transduction.

scholarly journals Dynamic super-resolution structured illumination imaging in the living brain

2019 ◽  
Vol 116 (19) ◽  
pp. 9586-9591 ◽  
Author(s):  
Raphaël Turcotte ◽  
Yajie Liang ◽  
Masashi Tanimoto ◽  
Qinrong Zhang ◽  
Ziwei Li ◽  
...  
Keyword(s):  
Adaptive Optics ◽  
Super Resolution ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Optical Aberrations ◽  
Sample Motion ◽  
Heterogeneous Tissue ◽  
Conventional Microscopy ◽  
Super Resolution Microscopy

Cells in the brain act as components of extended networks. Therefore, to understand neurobiological processes in a physiological context, it is essential to study them in vivo. Super-resolution microscopy has spatial resolution beyond the diffraction limit, thus promising to provide structural and functional insights that are not accessible with conventional microscopy. However, to apply it to in vivo brain imaging, we must address the challenges of 3D imaging in an optically heterogeneous tissue that is constantly in motion. We optimized image acquisition and reconstruction to combat sample motion and applied adaptive optics to correcting sample-induced optical aberrations in super-resolution structured illumination microscopy (SIM) in vivo. We imaged the brains of live zebrafish larvae and mice and observed the dynamics of dendrites and dendritic spines at nanoscale resolution.

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