Browse
Submit Data
Databases
API
Help

Dataset Information

22 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Super-resolution Imaging of the T cell Central Supramolecular Signaling Cluster Using Stimulated Emission Depletion Microscopy.

ABSTRACT: Supramolecular signaling assemblies are of interest for their unique signaling properties. A µm scale signaling assembly, the central supramolecular signaling cluster (cSMAC), forms at the center interface of T cells activated by antigen presenting cells (APC). The adaptor protein linker for activation of T cells (LAT) is a key cSMAC component. The cSMAC has widely been studied using total internal reflection fluorescence microscopy of CD4⁺ T cells activated by planar APC substitutes. Here we provide a protocol to image the cSMAC in its cellular context at the interface between a T cell and an APC. Super resolution stimulated emission depletion microscopy (STED) was utilized to determine the localization of LAT, that of its active, phosphorylated form and its entire pool. Agonist peptide-loaded APCs were incubated with TCR transgenic CD4⁺ T cells for 4.5 min before fixation and antibody staining. Fixed cell couples were imaged using a 100x 1.4 NA objective on a Leica SP8 AOBS confocal laser scanning microscope. LAT clustered in multiple supramolecular complexes and their number and size distributions were determined. Using this protocol, cSMAC properties in its cellular context at the interface between a T cell and an APC could be quantified.

SUBMITTER: Tan SL

PROVIDER: S-EPMC7842649 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Exploiting the tunability of stimulated emission depletion microscopy for super-resolution imaging of nuclear structures.

Project description:Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image. This modulation induces spatially encoded variations of the fluorescence emission that can be visualized in the phasor plot and used to improve and quantify the effective spatial resolution of the STED image. We show that the method can be used to remove direct excitation by the STED beam and perform dual color imaging. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells.

| S-EPMC6109149 | biostudies-literature

Super-Resolution Vibrational Imaging Using Expansion Stimulated Raman Scattering Microscopy.

Project description:Stimulated Raman scattering (SRS) microscopy is an emerging technology that provides high chemical specificity for endogenous biomolecules and can circumvent common constraints of fluorescence microscopy including limited capabilities to probe small biomolecules and difficulty resolving many colors simultaneously. However, the resolution of SRS microscopy remains governed by the diffraction limit. To overcome this, a new technique called molecule anchorable gel-enabled nanoscale Imaging of Fluorescence and stimulated Raman scattering microscopy (MAGNIFIERS) that integrates SRS microscopy with expansion microscopy (ExM) is described. MAGNIFIERS offers chemical-specific nanoscale imaging with sub-50 nm resolution and has scalable multiplexity when combined with multiplex Raman probes and fluorescent labels. MAGNIFIERS is used to visualize nanoscale features in a label-free manner with CH vibration of proteins, lipids, and DNA in a broad range of biological specimens, from mouse brain, liver, and kidney to human lung organoid. In addition, MAGNIFIERS is applied to track nanoscale features of protein synthesis in protein aggregates using metabolic labeling of small metabolites. Finally, MAGNIFIERS is used to demonstrate 8-color nanoscale imaging in an expanded mouse brain section. Overall, MAGNIFIERS is a valuable platform for super-resolution label-free chemical imaging, high-resolution metabolic imaging, and highly multiplexed nanoscale imaging, thus bringing SRS to nanoscopy.

| S-EPMC9284179 | biostudies-literature

STED-SPIM: Stimulated emission depletion improves sheet illumination microscopy resolution.

Project description:We demonstrate the first, to our knowledge, integration of stimulated emission depletion (STED) with selective plane illumination microscopy (SPIM). Using this method, we were able to obtain up to 60% improvements in axial resolution with lateral resolution enhancements in control samples and zebrafish embryos. The integrated STED-SPIM method combines the advantages of SPIM with the resolution enhancement of STED, and thus provides a method for fast, high-resolution imaging with >100 ?m deep penetration into biological tissue.

| S-EPMC3077687 | biostudies-other

Stimulated emission depletion live-cell super-resolution imaging shows proliferative remodeling of T-tubule membrane structures after myocardial infarction.

Project description:Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca(2+) release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF).Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction.Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca(2+) release and action potential prolongation.This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca(2+) release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

| S-EPMC4219578 | biostudies-literature

Low-Power Two-Color Stimulated Emission Depletion Microscopy for Live Cell Imaging.

Project description:Stimulated emission depletion (STED) microscopy is a typical laser-scanning super-resolution imaging technology, the emergence of which has opened a new research window for studying the dynamic processes of live biological samples on a nanometer scale. According to the characteristics of STED, a high depletion power is required to obtain a high resolution. However, a high laser power can induce severe phototoxicity and photobleaching, which limits the applications for live cell imaging, especially in two-color STED super-resolution imaging. Therefore, we developed a low-power two-color STED super-resolution microscope with a single supercontinuum white-light laser. Using this system, we achieved low-power two-color super-resolution imaging based on digital enhancement technology. Lateral resolutions of 109 and 78 nm were obtained for mitochondria and microtubules in live cells, respectively, with 0.8 mW depletion power. These results highlight the great potential of the novel digitally enhanced two-color STED microscopy for long-term dynamic imaging of live cells.

| S-EPMC8468006 | biostudies-literature

Tuning donut profile for spatial resolution in stimulated emission depletion microscopy.

Project description:In stimulated emission depletion (STED)-based or up-conversion depletion-based super-resolution optical microscopy, the donut-shaped depletion beam profile is of critical importance to its resolution. In this study, we investigate the transformation of the donut-shaped depletion beam focused by a high numerical aperture (NA) microscope objective, and model STED point spread function (PSF) as a function of donut beam profile. We show experimentally that the intensity profile of the dark kernel of the donut can be approximated as a parabolic function, whose slope is determined by the donut beam size before the objective back aperture, or the effective NA. Based on this, we derive the mathematical expression for continuous wave (CW) STED PSF as a function of focal plane donut and excitation beam profiles, as well as dye properties. We find that the effective NA and the residual intensity at the center are critical factors for STED imaging quality and the resolution. The effective NA is critical for STED resolution in that it not only determines the donut shape but also the area the depletion laser power is dispersed. An improperly expanded depletion beam will have negligible improvement in resolution. The polarization of the depletion beam also plays an important role as it affects the residual intensity in the center of the donut. Finally, we construct a CW STED microscope operating at 488 nm excitation and 592 nm depletion with a resolution of 70 nm. Our study provides detailed insight to the property of donut beam, and parameters that are important for the optimal performance of STED microscopes. This paper will provide a useful guide for the construction and future development of STED microscopes.

| S-EPMC3637329 | biostudies-other

Polarity-Sensitive Probes for Superresolution Stimulated Emission Depletion Microscopy.

Project description:The lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed. Polarity-sensitive dyes are powerful tools to characterize such lipid membrane order, employing, for example, confocal and two-photon microscopy. The investigation of potential nanodomains, however, requires the use of superresolution microscopy. Here, we test the performance of the polarity-sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA, and NR12S in superresolution stimulated emission depletion microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles, show the high potential of these dyes for probing nanoscale membrane heterogeneity.

| S-EPMC5607142 | biostudies-literature

Super-resolution vibrational microscopy by stimulated Raman excited fluorescence.

Project description:Inspired by the revolutionary impact of super-resolution fluorescence microscopy, super-resolution Raman imaging has been long pursued because of its much higher chemical specificity than the fluorescence counterpart. However, vibrational contrasts are intrinsically less sensitive compared with fluorescence, resulting in only mild resolution enhancement beyond the diffraction limit even with strong laser excitation power. As such, it is still a great challenge to achieve biocompatible super-resolution vibrational imaging in the optical far-field. In 2019 Stimulated Raman Excited Fluorescence (SREF) was discovered as an ultrasensitive vibrational spectroscopy that combines the high chemical specificity of Raman scattering and the superb sensitivity of fluorescence detection. Herein we developed a novel super-resolution vibrational imaging method by harnessing SREF as the contrast mechanism. We first identified the undesired role of anti-Stokes fluorescence background in preventing direct adoption of super-resolution fluorescence technique. We then devised a frequency-modulation (FM) strategy to remove the broadband backgrounds and achieved high-contrast SREF imaging. Assisted by newly synthesized SREF dyes, we realized multicolor FM-SREF imaging with nanometer spectral resolution. Finally, by integrating stimulated emission depletion (STED) with background-free FM-SREF, we accomplished high-contrast super-resolution vibrational imaging with STED-FM-SREF whose spatial resolution is only determined by the signal-to-noise ratio. In our proof-of-principle demonstration, more than two times of resolution improvement is achieved in biological systems with moderate laser excitation power, which shall be further refined with optimized instrumentation and imaging probes. With its super resolution, high sensitivity, vibrational contrast, and mild laser excitation power, STED-FM-SREF microscopy is envisioned to aid a wide variety of applications.

| S-EPMC8058038 | biostudies-literature

Painting Supramolecular Polymers in Organic Solvents by Super-resolution Microscopy.

Project description:Despite the rapid development of complex functional supramolecular systems, visualization of these architectures under native conditions at high resolution has remained a challenging endeavor. Super-resolution microscopy was recently proposed as an effective tool to unveil one-dimensional nanoscale structures in aqueous media upon chemical functionalization with suitable fluorescent probes. Building upon our previous work, which enabled photoactivation localization microscopy in organic solvents, herein, we present the imaging of one-dimensional supramolecular polymers in their native environment by interface point accumulation for imaging in nanoscale topography (iPAINT). The noncovalent staining, typical of iPAINT, allows the investigation of supramolecular polymers' structure in situ without any chemical modification. The quasi-permanent adsorption of the dye to the polymer is exploited to identify block-like arrangements within supramolecular fibers, which were obtained upon mixing homopolymers that were prestained with different colors. The staining of the blocks, maintained by the lack of exchange of the dyes, permits the imaging of complex structures for multiple days. This study showcases the potential of PAINT-like strategies such as iPAINT to visualize multicomponent dynamic systems in their native environment with an easy, synthesis-free approach and high spatial resolution.

| S-EPMC5968428 | biostudies-literature

Imaging tripartite synapses using super-resolution microscopy.

Project description:Astroglia are vital facilitators of brain development, homeostasis, and metabolic support. In addition, they are also essential to the formation and regulation of synaptic circuits. Due to the extraordinary complex, nanoscopic morphology of astrocytes, the underlying cellular mechanisms have been poorly understood. In particular, fine astrocytic processes that can be found in the vicinity of synapses have been difficult to study using traditional imaging techniques. Here, we describe a 3D three-colour super-resolution microscopy approach to unravel the nanostructure of tripartite synapses. The method is based on the SMLM technique direct stochastic optical reconstruction microscopy (dSTORM) which uses conventional fluorophore-labelled antibodies. This approach enables reconstructing the nanoscale localisation of individual astrocytic glutamate transporter (GLT-1) molecules surrounding presynaptic (bassoon) and postsynaptic (Homer1) protein localisations in fixed mouse brain sections. However, the technique is readily adaptable to other types of targets and tissues.

| S-EPMC7144327 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data