Astrocyte nanoscale morphology controls Ca2+ signals at tripartite synapses

AbstractCa2+ signals in astrocytes can trigger the modulation of neuronal activity. Recent developments in Ca2+ imaging and super-resolution microscopy have allowed to characterize the complex morphology of astrocyte branchlets that communicate with neurons and the associated Ca2+ microdomains. Here, we use computational tools to investigate the causal relationship between branchlet morphology and spatio-temporal profile of Ca2+ signals. 3D reticular branchlet geometries were designed, alternating between large (nodes) and thinner cellular compartments (shafts). Simulations confirm experimental observations that a decreased shaft width is associated with a decreased diffusion flux from nodes, enhancing local Ca2+ activity. Upon successive neuronal stimuli, a decreased shaft width facilitates signal propagation in astrocyte branchlets. We further identify parameters that decrease local Ca2+ activity, such as a discontinuous ER geometry and an increased Ca2+ buffering. Overall, this study proposes key parameters that regulate Ca2+ activity locally, potentially favoring neuron-astrocyte communication at tripartite synapses.

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Super-resolution microscopy reveals functional organization of dopamine transporters into cholesterol and neuronal activity-dependent nanodomains

Nature Communications ◽

10.1038/s41467-017-00790-3 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 32

Author(s):

Troels Rahbek-Clemmensen ◽

Matthew D. Lycas ◽

Simon Erlendsson ◽

Jacob Eriksen ◽

Mia Apuschkin ◽

...

Keyword(s):

Neuronal Activity ◽

Functional Organization ◽

Super Resolution ◽

Dopamine Transporters ◽

Super Resolution Microscopy ◽

Activity Dependent

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Encoding and decoding spatio-temporal information for super-resolution microscopy

Nature Communications ◽

10.1038/ncomms7701 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 43

Author(s):

Luca Lanzanò ◽

Iván Coto Hernández ◽

Marco Castello ◽

Enrico Gratton ◽

Alberto Diaspro ◽

...

Keyword(s):

Super Resolution ◽

Temporal Information ◽

Spatio Temporal ◽

Super Resolution Microscopy

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Advances in High-Speed Structured Illumination Microscopy

Frontiers in Physics ◽

10.3389/fphy.2021.672555 ◽

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.

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On the impact of competing intra- and intermolecular triplet-state quenching on photobleaching and photoswitching kinetics of organic fluorophores

10.1101/371443 ◽

2018 ◽

Cited By ~ 1

Author(s):

Jochem H. Smit ◽

Jasper H. M. van der Velde ◽

Jingyi Huang ◽

Vanessa Trauschke ◽

Sarah S. Henrikus ◽

...

Keyword(s):

Triplet State ◽

Aromatic Amino Acids ◽

Super Resolution ◽

Self Healing ◽

Fluorescent Markers ◽

Spatio Temporal ◽

Covalently Linked ◽

Super Resolution Microscopy ◽

The Impact ◽

Kinetics Of

AbstractWhile buffer cocktails remain the gold-standard for photostabilization and photoswitching of fluorescent markers, intramolecular triplet-state quenchers emerge as an alternative strategy to impart fluorophores with ‘self-healing’ or even functional properties such as photoswitching. In this contribution, we evaluated various combinations of both approaches and show that inter- and intramolecular triplet-state quenching processes compete with each other rather than being additive or even synergistic. Often intramolecular processes dominate the photophysical situation for combinations of covalently-linked and solution-based photostabilizers and photoswitching agents. In this context we identified a new function of intramolecular photostabilizers, i.e., protection of fluorophores from reversible off-switching events caused by solution-additives, which were previously misinterpreted as photobleaching. Our studies also provide practical guidance for usage of photostabilizer-dye conjugates for STORM-type super-resolution microscopy permitting the exploitation of their improved photophysics for increased spatio-temporal resolution. Finally, we provide evidence that the biochemical environment, e.g., proximity of aromatic amino-acids such as tryptophan, reduces the photostabilization efficiency of commonly used buffer cocktails. Not only have our results important implications for a deeper mechanistic understanding of self-healing dyes, but they will provide a general framework to select label positions for optimal and reproducible photostability or photoswitching kinetics.

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Continuous rearrangement of the postsynaptic gephyrin scaffolding domain: a super-resolution quantified and energetic approach

10.1101/193698 ◽

2017 ◽

Cited By ~ 2

Author(s):

Pamela C. Rodriguez ◽

Leandro G. Almeida ◽

Antoine Triller

Keyword(s):

Neuronal Activity ◽

Single Molecule ◽

Morphological Changes ◽

Super Resolution ◽

Scaffold Protein ◽

Synaptic Function ◽

Inhibitory Synapses ◽

Spatial Reorganization ◽

Nanoscopic Structure ◽

Super Resolution Microscopy

AbstractSynaptic function and plasticity requires a delicate balance between overall structural stability and the continuous rearrangement of the components that make up the presynaptic active zone and the postsynaptic density (PSD). Photoactivated localization microscopy (PALM) has provided a detailed view of the nanoscopic structure and organization of some of these synaptic elements. Still lacking, are tools to address the morphing and stability of such complexes at super-resolution. We describe an approach to quantify morphological changes and energetic states of multimolecular assemblies over time. With this method, we studied the scaffold protein gephyrin, which forms postsynaptic clusters that play a key role in the stabilization of receptors at inhibitory synapses. Postsynaptic gephyrin clusters exhibit an internal microstructure composed of nanodomains. We found, that within the PSD, gephyrin molecules continuously undergo spatial reorganization. This dynamic behavior depends on neuronal activity and cytoskeleton integrity. The proposed approach also allowed access to the effective energy responsible for the tenacity of the PSD despite molecular instability.Significant statementSuper-resolution microscopy has become an important tool for the study of biological systems, allowing detailed, nano-scale structural reconstruction, single molecule tracking, particle counting, and interaction studies. However, quantification tools that take full advantage of the information provided by this technology are still lacking. We describe a novel quantification method to obtain information related to the size, directionality, dynamics, and stability of clustered structures from super-resolution microscopy. With this method, we studied the stability of gephyrin clusters, the main inhibitory scaffold protein. We found that gephyrin molecules continuously undergo reorganization based on neuronal activity and changes in the cytoskeleton.

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Structured illumination microscopy and its new developments

Journal of Innovative Optical Health Sciences ◽

10.1142/s179354581630010x ◽

2016 ◽

Vol 09 (03) ◽

pp. 1630010 ◽

Cited By ~ 1

Author(s):

Jianling Chen ◽

Caimin Qiu ◽

Minghai You ◽

Xiaogang Chen ◽

Hongqin Yang ◽

...

Keyword(s):

Optical Microscopy ◽

Spatial Resolution ◽

Imaging Techniques ◽

Super Resolution ◽

Living Cells ◽

The Other ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Recent Developments ◽

Super Resolution Microscopy

Optical microscopy allows us to observe the biological structures and processes within living cells. However, the spatial resolution of the optical microscopy is limited to about half of the wavelength by the light diffraction. Structured illumination microscopy (SIM), a type of new emerging super-resolution microscopy, doubles the spatial resolution by illuminating the specimen with a patterned light, and the sample and light source requirements of SIM are not as strict as the other super-resolution microscopy. In addition, SIM is easier to combine with the other imaging techniques to improve their imaging resolution, leading to the developments of diverse types of SIM. SIM has great potential to meet the various requirements of living cells imaging. Here, we review the recent developments of SIM and its combination with other imaging techniques.

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Super-resolution structured illumination microscopy: past, present and future

Philosophical Transactions of The Royal Society A Mathematical Physical and Engineering Sciences ◽

10.1098/rsta.2020.0143 ◽

2021 ◽

Vol 379 (2199) ◽

Author(s):

Kirti Prakash ◽

Benedict Diederich ◽

Stefanie Reichelt ◽

Rainer Heintzmann ◽

Lothar Schermelleh

Keyword(s):

Three Dimensional ◽

Super Resolution ◽

Computational Imaging ◽

Biological Applications ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Recent Developments ◽

Resolution Imaging ◽

Microscopy Techniques ◽

Super Resolution Microscopy

Structured illumination microscopy (SIM) has emerged as an essential technique for three-dimensional (3D) and live-cell super-resolution imaging. However, to date, there has not been a dedicated workshop or journal issue covering the various aspects of SIM, from bespoke hardware and software development and the use of commercial instruments to biological applications. This special issue aims to recap recent developments as well as outline future trends. In addition to SIM, we cover related topics such as complementary super-resolution microscopy techniques, computational imaging, visualization and image processing methods.This article is part of the Theo Murphy meeting issue ‘Super-resolution structured illumination microscopy (part 1)’.

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Glyoxal Does Not Preserve Cellular Proteins as Accurately as PFA: A Microscopical Survey of Epitopes

10.1101/625863 ◽

2019 ◽

Author(s):

Ferda Topal Celikkan ◽

Ceren Mungan ◽

Merve Sucu ◽

Fatma Uysal ◽

Selda Kahveci ◽

...

Keyword(s):

Cellular Localization ◽

Super Resolution ◽

Cell Types ◽

Cellular Proteins ◽

Human Stem Cells ◽

Cellular Targets ◽

Molecular Alterations ◽

Recent Developments ◽

Spatio Temporal ◽

Fixed Cell

AbstractChemical fixation is one of the most critical steps to retaining cellular targets as naturally as possible. Recent developments in microscopy allow sophisticated detection and measuring techniques with which spatio-temporal molecular alterations is conceivable. Here, we document the fixation competence of glyoxal (Gly), a less-toxic dialdehyde molecule, and paraformaldehyde (PFA) side-by-side (with or without Triton-X 100 permealization) in live- and fixed-cell preparations including human stem cells, spermatozoa, mouse oocytes/embryos using super-resolution microscopy. Although Gly seemed to act faster than PFA, catastrophic consequences were found not acceptable, especially in oocytes and embryos. Due to cell lysate and immunocytochemistry surveys, it was obvious that PFA is superior to Gly in retaining cellular proteins in situ with little/no background staining. In many samples, PFA revealed more reliable and consistent results regarding the protein quantity and cellular localization corresponding to previously defined patterns in the literature. Although the use of Gly is beneficial as indicated by previous reports, we concluded that it does not meet the requirement for proper fixation, at least for the tested cell types and proteins.

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