Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Visualization and quantification of cardiac mitochondrial protein clusters with STED microscopy.

ABSTRACT: The visualization and quantification of mitochondria-associated proteins with high power microscopy methods is of particular interest to investigate protein architecture in this organelle. We report the usage of a custom-made STimulated Emission Depletion (STED) fluorescence nanoscope with ~30nm lateral resolution for protein mapping of Percoll-purified viable mitochondria from murine heart. Using this approach, we were able to quantify and resolve distinct protein clusters within mitochondria; specifically, cytochrome c oxidase subunit 2 is distributed in clusters of ~28nm; whereas the voltage dependent anion channel 1 displays three size distributions of ~33, ~55 and ~83nm.

SUBMITTER: Singh H

PROVIDER: S-EPMC3258335 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Visualization and quantification of cardiac mitochondrial protein clusters with STED microscopy.

Singh Harpreet H Lu Rong R Rodríguez Pedro Felipe Gardeazábal PF Wu Yong Y Bopassa Jean Chrisostome JC Stefani Enrico E Toro Ligia L

Mitochondrion 20110925 2

The visualization and quantification of mitochondria-associated proteins with high power microscopy methods is of particular interest to investigate protein architecture in this organelle. We report the usage of a custom-made STimulated Emission Depletion (STED) fluorescence nanoscope with ~30nm lateral resolution for protein mapping of Percoll-purified viable mitochondria from murine heart. Using this approach, we were able to quantify and resolve distinct protein clusters within mitochondria; ...[more]

PMID: 21982778

Similar Datasets

Visualization of Bacterial Protein Complexes Labeled with Fluorescent Proteins and Nanobody Binders for STED Microscopy.

Project description:In situ visualization of molecular assemblies near their macromolecular scale is a powerful tool to investigate fundamental cellular processes. Super-resolution light microscopies (SRM) overcome the diffraction limit and allow researchers to investigate molecular arrangements at the nanoscale. However, in bacterial cells, visualization of these assemblies can be challenging because of their small size and the presence of the cell wall. Thus, although conceptually promising, successful application of SRM techniques requires careful optimization in labeling biochemistry, fluorescent dye choice, bacterial biology and microscopy to gain biological insights. Here, we apply Stimulated Emission Depletion (STED) microscopy to visualize cell division proteins in bacterial cells, specifically E. coli and B. subtilis. We applied nanobodies that specifically recognize fluorescent proteins, such as GFP, mCherry2 and PAmCherry, fused to targets for STED imaging and evaluated the effect of various organic fluorescent dyes on the performance of STED in bacterial cells. We expect this research to guide scientists for in situ macromolecular visualization using STED in bacterial systems.

| S-EPMC6678925 | biostudies-literature

Quantification of Internalized Silica Nanoparticles via STED Microscopy.

Project description: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 × 10(10) particles?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 the in vitro sedimentation, diffusion, and dosimetry (ISDD) model 20-27% of the particles sedimented. In comparison, 10(2)-10(3) NPs 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 × 10(12) particles?mL(-1)) of the smaller particles induced cytotoxicity.

| S-EPMC4466362 | biostudies-other

STED super-resolution microscopy reveals an array of MINOS clusters along human mitochondria.

Project description:The mitochondrial inner membrane organizing system (MINOS) is a conserved large hetero-oligomeric protein complex in the mitochondrial inner membrane, crucial for the maintenance of cristae morphology. MINOS has been suggested to represent the core of an extended protein network that controls mitochondrial function and structure, and has been linked to several human diseases. The spatial arrangement of MINOS within mitochondria is ill-defined, however. Using super-resolution stimulated emission depletion (STED) microscopy and immunogold electron microscopy, we determined the distribution of three known human MINOS subunits (mitofilin, MINOS1, and CHCHD3) in mammalian cells. Super-resolution microscopy revealed that all three subunits form similar clusters within mitochondria, and that MINOS is more abundant in mitochondria around the nucleus than in peripheral mitochondria. At the submitochondrial level, mitofilin, a core MINOS subunit, is preferentially localized at cristae junctions. In primary human fibroblasts, mitofilin labeling uncovered a regularly spaced pattern of clusters arranged in parallel to the cell growth surfaces. We suggest that this array of MINOS complexes might explain the observed phenomenon of largely horizontally arranged cristae junctions that connect the inner boundary membrane to lamellar cristae. The super-resolution images demonstrate an unexpectedly high level of regularity in the nanoscale distribution of the MINOS complex in human mitochondria, supporting an integrating role of MINOS in the structural organization of the organelle.

| S-EPMC3670330 | biostudies-literature

Tomographic STED microscopy.

Project description:Stimulated emission depletion (STED) microscopy is a versatile imaging method with diffraction-unlimited resolution. Here, we present a novel STED microscopy variant that achieves either increased resolution at equal laser power or identical super-resolution conditions at significantly lower laser power when compared to the classical implementation. By applying a one-dimensional depletion pattern instead of the well-known doughnut-shaped STED focus, a more efficient depletion is achieved, thereby necessitating less STED laser power to achieve identical resolution. A two-dimensional resolution increase is obtained by recording a sequence of images with different high-resolution directions. This corresponds to a collection of tomographic projections within diffraction-limited spots, an approach that so far has not been explored in super-resolution microscopy. Via appropriate reconstruction algorithms, our method also provides an opportunity to speed up the acquisition process. Both aspects, the necessity of less STED laser power and the feasibility to decrease the recording time, have the potential to reduce photo-bleaching as well as sample damage drastically.

| S-EPMC7316010 | biostudies-literature

Visualization of early influenza A virus trafficking in human dendritic cells using STED microscopy.

Project description:Influenza A viruses (IAV) primarily target respiratory epithelial cells, but can also replicate in immune cells, including human dendritic cells (DCs). Super-resolution microscopy provides a novel method of visualizing viral trafficking by overcoming the resolution limit imposed by conventional light microscopy, without the laborious sample preparation of electron microscopy. Using three-color Stimulated Emission Depletion (STED) microscopy, we visualized input IAV nucleoprotein (NP), early and late endosomal compartments (EEA1 and LAMP1 respectively), and HLA-DR (DC membrane/cytosol) by immunofluorescence in human DCs. Surface bound IAV were internalized within 5 min of infection. The association of virus particles with early endosomes peaked at 5 min when 50% of NP+ signals were also EEA1+. Peak association with late endosomes occurred at 15 min when 60% of NP+ signals were LAMP1+. At 30 min of infection, the majority of NP signals were in the nucleus. Our findings illustrate that early IAV trafficking in human DCs proceeds via the classical endocytic pathway.

| S-EPMC5462357 | biostudies-literature

Deep learning enables fast, gentle STED microscopy.

Project description:STED microscopy is widely used to image subcellular structures with super-resolution. Here, we report that restoring STED images with deep learning can mitigate photobleaching and photodamage by reducing the pixel dwell time by one or two orders of magnitude. Our method allows for efficient and robust restoration of noisy 2D and 3D STED images with multiple targets and facilitates long-term imaging of mitochondrial dynamics.

| S-EPMC10300082 | biostudies-literature

Synergizing Exchangeable Fluorophore Labels for Multitarget STED Microscopy.

Project description:Investigating the interplay of cellular proteins with optical microscopy requires multitarget labeling. Spectral multiplexing using high-affinity or covalent labels is limited in the number of fluorophores that can be discriminated in a single imaging experiment. Advanced microscopy methods such as STED microscopy additionally demand balanced excitation, depletion, and emission wavelengths for all fluorophores, further reducing multiplexing capabilities. Noncovalent, weak-affinity labels bypass this "spectral barrier" through label exchange and sequential imaging of different targets. Here, we combine exchangeable HaloTag ligands, weak-affinity DNA hybridization, and hydrophophic and protein-peptide interactions to increase labeling flexibility and demonstrate six-target STED microscopy in single cells. We further show that exchangeable labels reduce photobleaching as well as facilitate long acquisition times and multicolor live-cell and high-fidelity 3D STED microscopy. The synergy of different types of exchangeable labels increases the multiplexing capabilities in fluorescence microscopy, and by that, the information content of microscopy images.

| S-EPMC9706782 | biostudies-literature

QUANTIFICATION OF MITOCHONDRIAL MEMBRANE CURVATURE BY THREE-DIMENSIONAL LOCALIZATION MICROSCOPY.

Project description:Mitochondrial membrane organization is important for many biological functions, and is implicated in a number of diseases, but conventional microscopy has insufficient resolution to image biologically relevant structures. We present methods to quantify nanoscale membrane curvature using three-dimensional localization-based super-resolution microscopy. Localizations are analyzed using a cluster algorithm followed by principal component analysis to determine local membrane curvature. Results are shown for mitochondria in C2C12 mouse myotubes labeled with Tom20-Dendra2.

| S-EPMC8992009 | biostudies-literature

Live-cell imaging of dendritic spines by STED microscopy.

Project description:Time lapse fluorescence imaging has become one of the most important approaches in neurobiological research. In particular, both confocal and two-photon microscopy have been used to study activity-dependent changes in synaptic morphology. However, the diffraction-limited resolution of light microscopy is often inadequate, forcing researchers to complement the live cell imaging strategy by EM. Here, we report on the first use of a far-field optical technique with subdiffraction resolution to noninvasively image activity-dependent morphological plasticity of dendritic spines. Specifically we show that time lapse stimulated emission depletion imaging of dendritic spines of YFP-positive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. The technique substantially improves the quantification of morphological parameters, such as the neck width and the curvature of the heads of spines, which are thought to play critical roles for the function and plasticity of synaptic connections.

| S-EPMC2585941 | biostudies-literature

Two-Photon Excitation STED Microscopy with Time-Gated Detection.

Project description:We report on a novel two-photon excitation stimulated emission depletion (2PE-STED) microscope based on time-gated detection. The time-gated detection allows for the effective silencing of the fluorophores using moderate stimulated emission beam intensity. This opens the possibility of implementing an efficient 2PE-STED microscope with a stimulated emission beam running in a continuous-wave. The continuous-wave stimulated emission beam tempers the laser architecture's complexity and cost, but the time-gated detection degrades the signal-to-noise ratio (SNR) and signal-to-background ratio (SBR) of the image. We recover the SNR and the SBR through a multi-image deconvolution algorithm. Indeed, the algorithm simultaneously reassigns early-photons (normally discarded by the time-gated detection) to their original positions and removes the background induced by the stimulated emission beam. We exemplify the benefits of this implementation by imaging sub-cellular structures. Finally, we discuss of the extension of this algorithm to future all-pulsed 2PE-STED implementationd based on time-gated detection and a nanosecond laser source.

| S-EPMC4725939 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

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