scholarly journals Fusion of clathrin and caveolae endocytic vesicles revealed by line-switching dual-color STED microscopy

2021 ◽  
pp. 2150017
Author(s):  
Hefei Ruan ◽  
Jianqiang Yu ◽  
Yayun Wu ◽  
Xiaojun Tang ◽  
Jinghe Yuan ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Protein Interactions ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Membrane Receptors ◽  
Dual Color ◽  
Imaging Results ◽  
Imaging Mode ◽  
Endocytic Vesicles ◽  
Line Switching

Clathrin- and caveolae-mediated endocytosis are the most commonly used pathways for the internalization of cell membrane receptors. However, due to their dimensions are within the diffraction limit, traditional fluorescence microscopy cannot distinguish them and little is known about their interactions underneath cell membrane. In this study, we proposed the line-switching scanning imaging mode for dual-color triplet-state relaxation (T-Rex) stimulated emission depletion (STED) super-resolution microscopy. With this line-switching mode, the cross-talk between the two channels, the side effects from pulse picker and image drift in frame scanning mode can be effectively eliminated. The dual-color super-resolution imaging results in mixed fluorescent beads validated the excellent performance. With this super-resolution microscope, not only the ring-shaped structure of clathrin and caveolae endocytic vesicles, but also their semi-fused structures underneath the cell membrane were distinguished clearly. The resultant information will greatly facilitate the study of clathrin- and caveolae-mediated receptor endocytosis and signaling process and also our home-built dual-color T-Rex STED microscope with this line-switching imaging mode provides a precise and convenient way to study subcellular-scale protein interactions.

Super-resolution optical microscopy of lipid plasma membrane dynamics

2015 ◽  
Vol 57 ◽  
pp. 69-80 ◽  
Author(s):  
Christian Eggeling
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Molecular Diffusion ◽  
Super Resolution ◽  
Optical Microscope ◽  
Membrane Dynamics ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Diffusion Dynamics ◽  
Lipid Protein Interactions

Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. The present chapter depicts the combination of optical super-resolution STED (stimulated emission depletion) microscopy with FCS, and why it is an important tool for investigating molecular membrane dynamics in living cells. Compared with conventional FCS, the STED-FCS approach demonstrates an improved possibility to distinguish free from anomalous molecular diffusion, and thus to give new insights into lipid–protein interactions and the traditional lipid ‘raft’ theory.

scholarly journals Comparing Super-Resolution Microscopy Techniques to Analyze Chromosomes

2021 ◽  
Vol 22 (4) ◽  
pp. 1903
Author(s):  
Ivona Kubalová ◽  
Alžběta Němečková ◽  
Klaus Weisshart ◽  
Eva Hřibová ◽  
Veit Schubert
Keyword(s):  
Single Molecule ◽  
Imaging Techniques ◽  
Chromatin Condensation ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Wide Field ◽  
Structured Illumination Microscopy ◽  
Metaphase Chromosomes ◽  
Localization Microscopy ◽  
Super Resolution Microscopy

The importance of fluorescence light microscopy for understanding cellular and sub-cellular structures and functions is undeniable. However, the resolution is limited by light diffraction (~200–250 nm laterally, ~500–700 nm axially). Meanwhile, super-resolution microscopy, such as structured illumination microscopy (SIM), is being applied more and more to overcome this restriction. Instead, super-resolution by stimulated emission depletion (STED) microscopy achieving a resolution of ~50 nm laterally and ~130 nm axially has not yet frequently been applied in plant cell research due to the required specific sample preparation and stable dye staining. Single-molecule localization microscopy (SMLM) including photoactivated localization microscopy (PALM) has not yet been widely used, although this nanoscopic technique allows even the detection of single molecules. In this study, we compared protein imaging within metaphase chromosomes of barley via conventional wide-field and confocal microscopy, and the sub-diffraction methods SIM, STED, and SMLM. The chromosomes were labeled by DAPI (4′,6-diamidino-2-phenylindol), a DNA-specific dye, and with antibodies against topoisomerase IIα (Topo II), a protein important for correct chromatin condensation. Compared to the diffraction-limited methods, the combination of the three different super-resolution imaging techniques delivered tremendous additional insights into the plant chromosome architecture through the achieved increased resolution.

scholarly journals MINSTED fluorescence localization and nanoscopy

2021 ◽  
Author(s):  
Michael Weber ◽  
Marcel Leutenegger ◽  
Stefan Stoldt ◽  
Stefan Jakobs ◽  
Tiberiu S. Mihaila ◽  
...  
Keyword(s):  
Standard Deviation ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stimulated Emission ◽  
Far Field ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Molecular Scale ◽  
Localization Precision ◽  
Super Resolution Microscopy

AbstractWe introduce MINSTED, a fluorophore localization and super-resolution microscopy concept based on stimulated emission depletion (STED) that provides spatial precision and resolution down to the molecular scale. In MINSTED, the intensity minimum of the STED doughnut, and hence the point of minimal STED, serves as a movable reference coordinate for fluorophore localization. As the STED rate, the background and the required number of fluorescence detections are low compared with most other STED microscopy and localization methods, MINSTED entails substantially less fluorophore bleaching. In our implementation, 200–1,000 detections per fluorophore provide a localization precision of 1–3 nm in standard deviation, which in conjunction with independent single fluorophore switching translates to a ~100-fold improvement in far-field microscopy resolution over the diffraction limit. The performance of MINSTED nanoscopy is demonstrated by imaging the distribution of Mic60 proteins in the mitochondrial inner membrane of human cells.

scholarly journals Quantification of Internalized Silica Nanoparticles via STED Microscopy

2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Henrike Peuschel ◽  
Thomas Ruckelshausen ◽  
Christian Cavelius ◽  
Annette Kraegeloh
Keyword(s):  
Silica Nanoparticles ◽  
Super Resolution ◽  
Engineered Nanoparticles ◽  
Stimulated Emission ◽  
Alveolar Type ◽  
Serum Free Medium ◽  
Free Medium ◽  
Essential Information ◽  
Serum Free

The development of safe engineered nanoparticles (NPs) requires a detailed understanding of their interaction mechanisms on a cellular level. Therefore, quantification of NP internalization is crucial to predict the potential impact of intracellular NP doses, providing essential information for risk assessment as well as for drug delivery applications. In this study, the internalization of 25 nm and 85 nm silica nanoparticles (SNPs) in alveolar type II cells (A549) was quantified by application of super-resolution STED (stimulated emission depletion) microscopy. Cells were exposed to equal particle number concentrations (9.2×1010particles mL−1) of each particle size and the sedimentation of particles during exposure was taken into account. Microscopy images revealed that particles of both sizes entered the cells after 5 h incubation in serum supplemented and serum-free medium. According to thein vitrosedimentation, diffusion, and dosimetry (ISDD) model 20–27% of the particles sedimented. In comparison, 102-103NPs per cell were detected intracellularly serum-containing medium. Furthermore, in the presence of serum, no cytotoxicity was induced by the SNPs. In serum-free medium, large agglomerates of both particle sizes covered the cells whereas only high concentrations (≥ 3.8 × 1012particles mL−1) of the smaller particles induced cytotoxicity.

scholarly journals Combined Use of Unnatural Amino Acids Enables Dual-Color Super-Resolution Imaging of Proteins via Click Chemistry

ACS Nano ◽  
2018 ◽  
Vol 12 (12) ◽  
pp. 12247-12254 ◽  
Author(s):  
Kim-A. Saal ◽  
Frank Richter ◽  
Peter Rehling ◽  
Silvio O. Rizzoli
Keyword(s):  
Amino Acids ◽  
Click Chemistry ◽  
Unnatural Amino Acids ◽  
Super Resolution ◽  
Dual Color ◽  
Combined Use ◽  
Resolution Imaging

scholarly journals Supramolecular Structures of the Dictyostelium Lamin NE81

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 162
Author(s):  
Marianne Grafe ◽  
Petros Batsios ◽  
Irene Meyer ◽  
Daria Lisin ◽  
Otto Baumann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Model Organism ◽  
Super Resolution ◽  
Nuclear Lamina ◽  
Stimulated Emission ◽  
Structural Level ◽  
Animal Cells ◽  
Nuclear Lamins

Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.

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