scholarly journals Autophagy-Related Proteins GABARAP and LC3B Label Structures of Similar Size but Different Shape in Super-Resolution Imaging

Molecules ◽  
2019 ◽  
Vol 24 (9) ◽  
pp. 1833 ◽  
Author(s):  
Iman Abdollahzadeh ◽  
Johnny Hendriks ◽  
Julia L. Sanwald ◽  
Indra M. Simons ◽  
Silke Hoffmann ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Vesicular Trafficking ◽  
Hek293 Cells ◽  
Wide Field ◽  
Size Distributions ◽  
Similar Shape ◽  
Resolution Imaging ◽  
Localisation Microscopy ◽  
Related Proteins

Subcellular structures containing autophagy-related proteins of the Atg8 protein family have been investigated with conventional wide-field fluorescence and single molecule localisation microscopy. Fusion proteins of GABARAP and LC3B, respectively, with EYFP were overexpressed in HEK293 cells. While size distributions of structures labelled by the two proteins were found to be similar, shape distributions appeared quite disparate, with EYFP-GABARAP favouring circular structures and elliptical structures being dominant for EYFP-LC3B. The latter also featured a nearly doubled fraction of U-shape structures. The experimental results point towards highly differential localisation of the two proteins, which appear to label structures representing distinct stages or even specific channels of vesicular trafficking pathways. Our data also demonstrate that the application of super-resolution techniques expands the possibilities of fluorescence-based methods in autophagy studies and in some cases can rectify conclusions obtained from conventional fluorescence microscopy with diffraction-limited resolution.

scholarly journals Virtual-'Light-Sheet' Single-Molecule Localisation Microscopy Enables Quantitative Optical Sectioning for Super-Resolution Imaging

PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0125438 ◽  
Author(s):  
Matthieu Palayret ◽  
Helen Armes ◽  
Srinjan Basu ◽  
Adam T. Watson ◽  
Alex Herbert ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Light Sheet ◽  
Optical Sectioning ◽  
Resolution Imaging ◽  
Localisation Microscopy

scholarly journals ThX – A next-generation probe for the early detection of amyloid aggregates

2019 ◽  
Author(s):  
Lisa-Maria Needham ◽  
Judith Weber ◽  
Juan A. Varela ◽  
James W. B. Fyfe ◽  
Dung T. Do ◽  
...  
Keyword(s):  
Fluorescent Probe ◽  
Single Molecule ◽  
Protein Misfolding ◽  
Super Resolution ◽  
Fold Increase ◽  
Next Generation ◽  
Aggregation Processes ◽  
Resolution Imaging ◽  
Localisation Microscopy ◽  
Protein Misfolding And Aggregation

AbstractNeurodegenerative diseases such as Alzheimer’s and Parkinson’s are associated with protein misfolding and aggregation. Recent studies suggest that the small, rare and heterogeneous oligomeric species, formed early on in the aggregation process, may be a source of cytotoxicity. Thioflavin T (ThT) is currently the gold-standard fluorescent probe for the study of amyloid proteins and aggregation processes. However, the poor photophysical and binding properties of ThT impairs the study of oligomers. To overcome this challenge, we have designed Thioflavin X, (ThX), a next-generation fluorescent probe which displays superior properties; including a 5-fold increase in brightness and 7-fold increase in binding affinity to amyloidogenic proteins. As an extrinsic dye, this can be used to study unique structural amyloid features both in bulk and on a single-aggregate level. Furthermore, ThX can be used as a super-resolution imaging probe in single-molecule localisation microscopy. Finally, we demonstrate that ThX can be used to detect a distinct oligomeric species, not observed via traditional ThT imaging.

scholarly journals Ground-State Depletion Nanoscopy of Nitrogen-Vacancy Centres in Nanodiamonds

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Jelle Storterboom ◽  
Martina Barbiero ◽  
Stefania Castelletto ◽  
Min Gu
Keyword(s):  
Single Molecule ◽  
Ground State ◽  
Nitrogen Concentration ◽  
Super Resolution ◽  
Optical Power ◽  
Stimulated Emission ◽  
Nitrogen Vacancy ◽  
Resolution Imaging ◽  
Localisation Microscopy ◽  
Bulk Diamond

AbstractThe negatively charged nitrogen-vacancy ($${\text{NV}}^{ - }$$ NV - ) centre in nanodiamonds (NDs) has been recently studied for applications in cellular imaging due to its better photo-stability and biocompatibility if compared to other fluorophores. Super-resolution imaging achieving 20-nm resolution of $${\text{NV}}^{ - }$$ NV - in NDs has been proved over the years using sub-diffraction limited imaging approaches such as single molecule stochastic localisation microscopy and stimulated emission depletion microscopy. Here we show the first demonstration of ground-state depletion (GSD) nanoscopy of these centres in NDs using three beams, a probe beam, a depletion beam and a reset beam. The depletion beam at 638 nm forces the $${\text{NV}}^{ - }$$ NV - centres to the metastable dark state everywhere but in the local minimum, while a Gaussian beam at 594 nm probes the $${\text{NV}}^{ - }$$ NV - centres and a 488-nm reset beam is used to repopulate the excited state. Super-resolution imaging of a single $${\text{NV}}^{ - }$$ NV - centre with a full width at half maximum of 36 nm is demonstrated, and two adjacent $${\text{NV}}^{ - }$$ NV - centres separated by 72 nm are resolved. GSD microscopy is here applied to $${\text{NV}}^{ - }$$ NV - in NDs with a much lower optical power compared to bulk diamond. This work demonstrates the need to control the NDs nitrogen concentration to tailor their application in super-resolution imaging methods and paves the way for studies of $${\text{NV}}^{ - }$$ NV - in NDs’ nanoscale interactions.

scholarly journals Super-resolving light fields in microscopy: depth from disparity

2019 ◽  
Author(s):  
Ruth R. Sims ◽  
Sohaib Abdul Rehman ◽  
Martin O. Lenz ◽  
Leila Mureşan ◽  
Kevin O'Holleran
Keyword(s):  
Single Molecule ◽  
Light Field ◽  
Super Resolution ◽  
Depth Of Field ◽  
Source Position ◽  
Multiple Perspective ◽  
Point Emitter ◽  
Simulation And Experiment ◽  
Resolution Imaging ◽  
Localisation Microscopy

Single molecule localisation microscopy (SMLM) has opened a new window for imaging fluorescently labelled biological specimens. Common 3D SMLM techniques enable data collection across an axial range of 1 - 5μm with high precision. Despite the success of 3D single molecule imaging there is a real need to image larger volumes. Here we demonstrate, through simulation and experiment, the potential of Single Molecule Light Field Microscopy (SMLFM) for extended depth-of-field super-resolution imaging, extracting 3D point source position by measuring the disparity between localizations of a point emitter in multiple perspective views.

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 Comparing lifeact and phalloidin for super-resolution imaging of actin in fixed cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0246138
Author(s):  
Hanieh Mazloom-Farsibaf ◽  
Farzin Farzam ◽  
Mohamadreza Fazel ◽  
Michael J. Wester ◽  
Marjolein B. M. Meddens ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cell Biology ◽  
Cell Movement ◽  
Super Resolution ◽  
Actin Binding ◽  
Thin Filaments ◽  
Reversible Binding ◽  
Multiple Regions ◽  
Resolution Imaging ◽  
Division Cell

Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a dSTORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

scholarly journals Differential FSH Glycosylation Modulates FSHR Oligomerization and Subsequent cAMP Signaling

2021 ◽  
Vol 12 ◽  
Author(s):  
Uchechukwu T. Agwuegbo ◽  
Emily Colley ◽  
Anthony P. Albert ◽  
Viktor Y. Butnev ◽  
George R. Bousfield ◽  
...  
Keyword(s):  
Super Resolution ◽  
Hek293 Cells ◽  
Camp Signaling ◽  
Camp Production ◽  
Functional Roles ◽  
Menopausal Women ◽  
Resolution Imaging ◽  
Post Menopausal

Follicle-stimulating hormone (FSH) and its target G protein-coupled receptor (FSHR) are essential for reproduction. Recent studies have established that the hypo-glycosylated pituitary FSH glycoform (FSH21/18), is more bioactive in vitro and in vivo than the fully-glycosylated variant (FSH24). FSH21/18 predominates in women of reproductive prime and FSH24 in peri-post-menopausal women, suggesting distinct functional roles of these FSH glycoforms. The aim of this study was to determine if differential FSH glycosylation modulated FSHR oligomerization and resulting impact on cAMP signaling. Using a modified super-resolution imaging technique (PD-PALM) to assess FSHR complexes in HEK293 cells expressing FSHR, we observed time and concentration-dependent modulation of FSHR oligomerization by FSH glycoforms. High eFSH and FSH21/18 concentrations rapidly dissociated FSHR oligomers into monomers, whereas FSH24 displayed slower kinetics. The FSHR β-arrestin biased agonist, truncated eLHβ (Δ121-149) combined with asparagine56-deglycosylated eLHα (dg-eLHt), increased FSHR homomerization. In contrast, low FSH21/18 and FSH24 concentrations promoted FSHR association into oligomers. Dissociation of FSHR oligomers correlated with time points where higher cAMP production was observed. Taken together, these data suggest that FSH glycosylation may modulate the kinetics and amplitude of cAMP production, in part, by forming distinct FSHR complexes, highlighting potential avenues for novel therapeutic targeting of the FSHR to improve IVF outcomes.

Single molecule Coordinate and Height super-resolution Imaging with Dithering and Orientation (CHIDO)

2020 ◽  
Author(s):  
Luis A. Alemán-Castañeda ◽  
Valentina Curcio ◽  
Thomas G. Brown ◽  
Sophie Brasselet ◽  
Miguel A. Alonso
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Resolution Imaging
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