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.

High-speed Live-cell Super-resolution Microscopy with Stochastically Switching Fluorophores

CLEO: 2013 ◽  
2013 ◽  
Author(s):  
Fang Huang ◽  
Tobias M. P. Hartwich ◽  
Felix E. Rivera-Molina ◽  
Yu Lin ◽  
Jordan R. Myers ◽  
...  
Keyword(s):  
High Speed ◽  
Super Resolution ◽  
Live Cell ◽  
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 Advances in High-Speed Structured Illumination Microscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Tianyu Zhao ◽  
Zhaojun Wang ◽  
Tongsheng Chen ◽  
Ming Lei ◽  
Baoli Yao ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
High Speed ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Light Power ◽  
Recent Developments ◽  
Super Resolution Microscopy

Super-resolution microscopy surpasses the diffraction limit to enable the observation of the fine details in sub-cellular structures and their dynamics in diverse biological processes within living cells. Structured illumination microscopy (SIM) uses a relatively low illumination light power compared with other super-resolution microscopies and has great potential to meet the demands of live-cell imaging. However, the imaging acquisition and reconstruction speeds limit its further applications. In this article, recent developments all targeted at improving the overall speed of SIM are reviewed. These comprise both hardware and software improvements, which include a reduction in the number of raw images, GPU acceleration, deep learning and the spatial domain reconstruction. We also discuss the application of these developments in live-cell imaging.

scholarly journals Systematic Tuning of Rhodamine Spirocyclization for Super-Resolution Microscopy

2021 ◽  
Author(s):  
Nicolas Lardon ◽  
Lu Wang ◽  
Aline Tschanz ◽  
Philipp Hoess ◽  
Mai Tran ◽  
...  
Keyword(s):  
Single Molecule ◽  
Large Range ◽  
Dynamic Equilibrium ◽  
Super Resolution ◽  
Live Cell ◽  
Stimulated Emission ◽  
Important Class ◽  
Localization Microscopy ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

Rhodamines are the most important class of fluorophores for applications in live-cell fluorescence microscopy. This is mainly because rhodamines exist in a dynamic equilibrium between a fluorescent zwitterion and a non-fluorescent but cell-permeable spirocyclic form. Different imaging applications require different positions of this dynamic equilibrium, which poses a challenge for the design of suitable probes. We describe here how the conversion of the ortho-carboxy moiety of a given rhodamine into substituted acyl benzenesulfonamides and alkylamides permits the systematic tuning of the equilibrium of spirocyclization with unprecedented accuracy and over a large range. This allows to transform the same rhodamine into either a highly fluorogenic and cell-permeable probe for live-cell stimulated emission depletion (STED) microscopy, or into a spontaneously blinking dye for single molecule localization microscopy (SMLM). We used this approach to generate differently colored probes optimized for different labeling systems and imaging applications.

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

Biosensors ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 330
Author(s):  
Jia Zhang ◽  
Xinwei Gao ◽  
Luwei Wang ◽  
Yong Guo ◽  
Yinru Zhu ◽  
...  
Keyword(s):  
Low Power ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Super Resolution ◽  
Live Cell ◽  
Stimulated Emission ◽  
Live Cells ◽  
Sted Microscopy ◽  
Resolution Imaging ◽  
Stimulated Emission Depletion

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.

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

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.

Sign in / Sign up

Export Citation Format

Share Document