scholarly journals New advances in the research of stimulated emission depletion super-resolution microscopy

2020 ◽  
Vol 69 (10) ◽  
pp. 108702
Author(s):  
Jia-Lin Wang ◽  
Wei Yan ◽  
Jia Zhang ◽  
Lu-Wei Wang ◽  
Zhi-Gang Yang ◽  
...  
Keyword(s):  
Super Resolution ◽  
Stimulated Emission ◽  
Stimulated Emission Depletion ◽  
Super Resolution Microscopy

scholarly journals Expansion stimulated emission depletion microscopy (ExSTED)

2018 ◽  
Author(s):  
Mengfei Gao ◽  
Riccardo Maraspini ◽  
Oliver Beutel ◽  
Amin Zehtabian ◽  
Britta Eickholt ◽  
...  
Keyword(s):  
Excited State ◽  
Optical Resolution ◽  
Super Resolution ◽  
Stimulated Emission ◽  
High Fidelity ◽  
Sted Microscopy ◽  
Structural Details ◽  
Stimulated Emission Depletion ◽  
Conventional Microscopy ◽  
Super Resolution Microscopy

AbstractStimulated emission depletion (STED) microscopy is routinely used to resolve the ultra-structure of cells with a ∼10-fold higher resolution compared to diffraction limited imaging. While STED microscopy is based on preparing the excited state of fluorescent probes with light, the recently developed expansion microscopy (ExM) provides sub-diffraction resolution by physically enlarging the sample before microscopy. Expansion of fixed cells by crosslinking and swelling of hydrogels easily enlarges the sample ∼4-fold and hence increases the effective optical resolution by this factor. To overcome the current limits of these complimentary approaches, we here combined ExM with STED (ExSTED) and demonstrate an increase in resolution of up to 30-fold compared to conventional microscopy (<10 nm lateral and ∼50 nm isotropic). While the increase in resolution is straight forward, we found that high fidelity labelling via multi-epitopes is required to obtain emitter densities that allow to resolve ultra-structural details with ExSTED. Our work provides a robust template for super resolution microscopy of entire cells in the ten nanometer range.

scholarly journals Reticulon and CLIMP-63 control nanodomain organization of peripheral ER tubules

2019 ◽  
Author(s):  
Guang Gao ◽  
Chengjia Zhu ◽  
Emma Liu ◽  
Ivan R. Nabi
Keyword(s):  
Endoplasmic Reticulum ◽  
Confocal Microscopy ◽  
High Speed ◽  
Super Resolution ◽  
Live Cell ◽  
Stimulated Emission ◽  
Preferential Segregation ◽  
Stimulated Emission Depletion ◽  
Super Resolution Microscopy ◽  
Local Accumulation

AbstractThe endoplasmic reticulum (ER) is an expansive, membrane-enclosed organelle composed of smooth peripheral tubules and rough, ribosome-studded central ER sheets whose morphology is determined, in part, by the ER-shaping proteins, reticulon and CLIMP-63, respectively. Here, STimulated Emission Depletion (STED) super-resolution microscopy shows that reticulon and CLIMP-63 also control the organization and dynamics of peripheral ER tubule nanodomains. STED imaging shows that lumenal ERmoxGFP, membrane Sec61βGFP, knock-in calreticulin-GFP and antibody-labeled ER resident proteins calnexin and derlin-1 are all localized to periodic puncta along the length of peripheral ER tubules that are not readily observable by diffraction limited confocal microscopy. Reticulon segregates away from and restricts lumenal blob length while CLIMP-63 associates with and increases lumenal blob length. Reticulon and CLIMP-63 also regulate the nanodomain distribution of ER resident proteins, being required for the preferential segregation of calnexin and derlin-1 puncta away from lumenal ERmoxGFP blobs. High-speed (40 ms/frame) live cell STED imaging shows that reticulon and CLIMP-63 control nanoscale compartmentalization of lumenal flow in peripheral ER tubules. Reticulon enhances and CLIMP-63 disrupts the local accumulation of lumenal ERmoxGFP at spatially defined sites along ER tubules. The ER shaping proteins reticulon and CLIMP-63 therefore control lumenal ER nanodomain dynamics, heterogeneity and interaction with ER resident proteins in peripheral ER tubules.

Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach

Nanoscale ◽  
2018 ◽  
Vol 10 (34) ◽  
pp. 16252-16260 ◽  
Author(s):  
Luwei Wang ◽  
Bingling Chen ◽  
Wei Yan ◽  
Zhigang Yang ◽  
Xiao Peng ◽  
...  
Keyword(s):  
Low Power ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Resolution Limit ◽  
Sted Microscopy ◽  
Microscopy Technique ◽  
Resolution Improvement ◽  
Phasor Plot ◽  
Stimulated Emission Depletion ◽  
Super Resolution Microscopy

Stimulated emission depletion (STED) microscopy is a powerful super-resolution microscopy technique that has achieved significant results in breaking the resolution limit and relevant applications.

scholarly journals Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins

2016 ◽  
Vol 291 (33) ◽  
pp. 16948-16962 ◽  
Author(s):  
Silvia Galiani ◽  
Dominic Waithe ◽  
Katharina Reglinski ◽  
Luis Daniel Cruz-Zaragoza ◽  
Esther Garcia ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Protein Import ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Peroxisomal Membrane ◽  
Heterogeneous Distribution ◽  
Biological Studies ◽  
Stimulated Emission Depletion ◽  
Sterol Carrier Protein 2 ◽  
Super Resolution Microscopy

Membrane-associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super-resolution optical stimulated emission depletion microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14, and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11 but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.

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.

Recent advances in luminescent materials for super-resolution imaging via stimulated emission depletion nanoscopy

2021 ◽  
Author(s):  
Yanzi Xu ◽  
Ruohan Xu ◽  
Zhi Wang ◽  
Yu Zhou ◽  
Qifei Shen ◽  
...  
Keyword(s):  
Recent Progress ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Luminescent Materials ◽  
New Materials ◽  
Recent Advances ◽  
Resolution Imaging ◽  
Stimulated Emission Depletion

Recent progress on STED fluorophores for super-resolution imaging and also their characteristics are outlined here, thus providing some guidelines to select proper probes and even develop new materials for super-resolution imaging via STED nanoscopy.

Long-term STED imaging of amyloid fibers with exchangeable Thioflavin T

Nanoscale ◽  
2020 ◽  
Vol 12 (28) ◽  
pp. 15050-15053
Author(s):  
Joaquim Torra ◽  
Patricia Bondia ◽  
Sylvia Gutierrez-Erlandsson ◽  
Begoña Sot ◽  
Cristina Flors
Keyword(s):  
Super Resolution ◽  
Stimulated Emission ◽  
Thioflavin T ◽  
Amyloid Fibers ◽  
Resolution Imaging ◽  
Stimulated Emission Depletion

We report the use of the amyloid probe Thioflavin T (ThT) as a specific and exchangeable fluorophore for stimulated emission depletion (STED) super-resolution imaging of amyloid fibers.

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