scholarly journals A specific hybridisation internalisation probe (SHIP) enables precise live-cell and super-resolution imaging of internalized cargo

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Sara Hernández-Pérez ◽  
Pieta K. Mattila
Keyword(s):  
Super Resolution ◽  
Cell Receptor ◽  
Live Cell ◽  
Receptor Ligand ◽  
Vesicle Traffic ◽  
Specific Hybridization ◽  
Multiple Imaging ◽  
Resolution Imaging ◽  
Membrane Bound ◽  
Quenching Probe

AbstractFacilitated by the advancements in microscopy, our understanding of the complexity of intracellular vesicle traffic has dramatically increased in recent years. However, distinguishing between plasma membrane-bound or internalised ligands remains a major challenge for the studies of cargo sorting to endosomal compartments, especially in small and round cells such as lymphocytes. The specific hybridization internalisation probe (SHIP) assay, developed for flow cytometry studies, employs a ssDNA fluorescence internalisation probe and a complementary ssDNA quenching probe to unambiguously detect the internalized receptors/cargo. Here, we adopted the SHIP assay to study the trafficking of receptor/ligand complexes using B lymphocytes and B cell receptor-mediated antigen internalization as a model system. Our study demonstrates the potential of the SHIP assay for improving the imaging of internalized receptor/ligand complexes and establishes the compatibility of this assay with multiple imaging modalities, including live-cell imaging and super-resolution microscopy.

scholarly journals A B cell actomyosin arc network couples integrin co-stimulation to mechanical force-dependent immune synapse formation

2021 ◽  
Author(s):  
Jia C Wang ◽  
Yang-In Yim ◽  
Xufeng Wu ◽  
Valentin Jaumouillé ◽  
Clare M Waterman ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Activation ◽  
Synapse Formation ◽  
Super Resolution ◽  
Cell Receptor ◽  
B Cell Activation ◽  
Protein Distribution ◽  
Immune Synapse ◽  
Resolution Imaging ◽  
Membrane Bound

B-cell activation and immune synapse (IS) formation with membrane-bound antigens are actin-dependent processes that scale positively with the strength of antigen-induced signals. Importantly, ligating the B-cell integrin, LFA-1, with ICAM-1 promotes IS formation when antigen is limiting. Whether the actin cytoskeleton plays a specific role in integrin-dependent IS formation is unknown. Here we show using super-resolution imaging of primary B cells that LFA-1: ICAM-1 interactions promote the formation of an actomyosin network that dominates the B-cell IS. This network is created by the formin mDia1, organized into concentric, contractile arcs by myosin 2A, and flows inward at the same rate as B-cell receptor (BCR): antigen clusters. Consistently, individual BCR microclusters are swept inward by individual actomyosin arcs. Under conditions where integrin is required for synapse formation, inhibiting myosin impairs synapse formation, as evidenced by reduced antigen centralization, diminished BCR signaling, and defective signaling protein distribution at the synapse. Together, these results argue that a contractile actomyosin arc network plays a key role in the mechanism by which LFA-1 co-stimulation promotes B-cell activation and IS formation.

Instant Live-Cell Super-Resolution Imaging of Cellular Structures by Nanoinjection of Fluorescent Probes

Nano Letters ◽  
2015 ◽  
Vol 15 (2) ◽  
pp. 1374-1381 ◽  
Author(s):  
Simon Hennig ◽  
Sebastian van de Linde ◽  
Martina Lummer ◽  
Matthias Simonis ◽  
Thomas Huser ◽  
...  
Keyword(s):  
Fluorescent Probes ◽  
Super Resolution ◽  
Live Cell ◽  
Cellular Structures ◽  
Resolution Imaging

Live-Cell Super-Resolution Imaging with Synthetic Fluorophores

2012 ◽  
Vol 63 (1) ◽  
pp. 519-540 ◽  
Author(s):  
Sebastian van de Linde ◽  
Mike Heilemann ◽  
Markus Sauer
Keyword(s):  
Super Resolution ◽  
Live Cell ◽  
Resolution Imaging

scholarly journals Customizable live-cell imaging chambers for multimodal and multiplex fluorescence microscopy

2020 ◽  
Vol 98 (5) ◽  
pp. 612-623
Author(s):  
Adam Tepperman ◽  
David Jiao Zheng ◽  
Maria Abou Taka ◽  
Angela Vrieze ◽  
Austin Le Lam ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Imaging Modalities ◽  
Experimental Conditions ◽  
Microscopy Imaging ◽  
Glass Coverslip ◽  
Multiple Imaging ◽  
Microscopy Techniques

Using multiple imaging modalities while performing independent experiments in parallel can greatly enhance the throughput of microscopy-based research, but requires the provision of appropriate experimental conditions in a format that meets the optical requirements of the microscope. Although customized imaging chambers can meet these challenges, the difficulty of manufacturing custom chambers and the relatively high cost and design inflexibility of commercial chambers has limited the adoption of this approach. Herein, we demonstrate the use of 3D printing to produce inexpensive, customized, live-cell imaging chambers that are compatible with a range of imaging modalities, including super-resolution microscopy. In this approach, biocompatible plastics are used to print imaging chambers designed to meet the specific needs of an experiment, followed by adhesion of the printed chamber to a glass coverslip, producing a chamber that is impermeant to liquids and that supports the growth and imaging of cells over multiple days. This approach can also be used to produce moulds for casting microfluidic devices made of polydimethylsiloxane. The utility of these chambers is demonstrated using designs for multiplex microscopy, imaging under shear, chemotaxis, and general cellular imaging. Together, this approach represents an inexpensive yet highly customizable approach for producing imaging chambers that are compatible with modern microscopy techniques.

Live cell super resolution imaging by radial fluctuations using fluorogen binding tags

Nanoscale ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 3626-3632 ◽  
Author(s):  
Muthukumaran Venkatachalapathy ◽  
Vivek Belapurkar ◽  
Mini Jose ◽  
Arnaud Gautier ◽  
Deepak Nair
Keyword(s):  
Super Resolution ◽  
Time Lapse ◽  
Live Cell ◽  
Resolution Imaging

Combination of SRRF and stochastic labeling based on FAST:Fluorogen complexes to achieve super-resolution in 2D, 3D and in time-lapse.

scholarly journals FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

2020 ◽  
Author(s):  
Matthieu Lagardère ◽  
Ingrid Chamma ◽  
Emmanuel Bouilhol ◽  
Macha Nikolski ◽  
Olivier Thoumine
Keyword(s):  
Real Time ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Imaging Techniques ◽  
Simulated Data ◽  
Super Resolution ◽  
Molecular Complexes ◽  
Live Cell ◽  
Single Molecule Level ◽  
Resolution Imaging

AbstractFluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these limitations, we present here a unified computer program that allows one to model and predict membrane protein dynamics at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments. FluoSim is an interactive real-time simulator of protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. The software, thoroughly validated against experimental data on the canonical neurexin-neuroligin adhesion complex, integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the distribution of thousands of independent molecules in 2D cellular geometries, providing simulated data of protein dynamics and localization directly comparable to actual experiments.

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