scholarly journals Revealing t-tubules in striated muscle with new optical super-resolution microscopy techniques

2014 ◽  
Vol 25 (1) ◽  
Author(s):  
Isuru D. Jayasinghe ◽  
Alexander H. Clowsley ◽  
Michelle Munro ◽  
Yufeng Hou ◽  
David J. Crossman ◽  
...  
Keyword(s):  
Striated Muscle ◽  
Super Resolution ◽  
T Tubules ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

scholarly journals Revealing t-tubules in striated muscle with new optical super-resolution microscopy techniques

2014 ◽  
Vol 25 (1) ◽  
pp. 15 ◽  
Author(s):  
Isuru D. Jayasinghe ◽  
Alexander H. Clowsley ◽  
Michelle Munro ◽  
Yufeng Hou ◽  
David J. Crossman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Single Molecule ◽  
Striated Muscle ◽  
Calcium Signalling ◽  
Super Resolution ◽  
Muscle Cells ◽  
Muscle Fibres ◽  
T Tubules ◽  
Microscopy Techniques

The t-tubular system plays a central role in the synchronisation of calcium signalling and excitation-contraction coupling in most striated muscle cells. Light microscopy has been used for imaging t-tubules for well over 100 years and together with electron microscopy (EM), has revealed the three-dimensional complexities of the t-system topology within cardiomyocytes and skeletal muscle fibres from a range of species. The emerging super-resolution single molecule localisation microscopy (SMLM) techniques are offering a near 10-fold improvement over the resolution of conventional fluorescence light microscopy methods, with the ability to spectrally resolve nanometre scale distributions of multiple molecular targets. In conjunction with the next generation of electron microscopy, SMLM has allowed the visualisation and quantification of intricate t-tubule morphologies within large areas of muscle cells at an unprecedented level of detail. In this paper, we review recent advancements in the t-tubule structural biology with the utility of various microscopy techniques. We outline the technical considerations in adapting SMLM to study t-tubules and its potential to further our understanding of the molecular processes that underlie the sub-micron scale structural alterations observed in a range of muscle pathologies.

scholarly journals Storm-Lite: An Inexpensive Tool to Demonstrate Stochastic Super Resolution Microscopy Techniques in the Classroom

2017 ◽  
Vol 112 (3) ◽  
pp. 464a
Author(s):  
Anthony Wu ◽  
Lark Moreno ◽  
Maxim Prigozhin ◽  
Sharlene Denos
Keyword(s):  
Super Resolution ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

scholarly journals How did correlative atomic force microscopy and super-resolution microscopy evolve in the quest for unravelling enigmas in biology?

Nanoscale ◽  
2021 ◽  
Author(s):  
Adelaide Miranda ◽  
Ana I. Gómez-Varela ◽  
Andreas Stylianou ◽  
Liisa M. Hirvonen ◽  
Humberto Sánchez ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Super Resolution ◽  
Force Microscopy ◽  
Atomic Force ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Detailed Picture

This review provides a detailed picture of the innovative efforts to combine atomic force microscopy and different super-resolution microscopy techniques to elucidate biological questions.

scholarly journals Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)

2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
Author(s):  
Donna R. Whelan ◽  
Thorge Holm ◽  
Markus Sauer ◽  
Toby D. M. Bell
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Set Up ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Microscopic Techniques

The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.

scholarly journals Nanoscopic dopamine transporter distribution and conformation are inversely regulated by excitatory drive and D2-autoreceptor activity

2021 ◽  
Author(s):  
Matthew D. Lycas ◽  
Aske L. Ejdrup ◽  
Andreas T. Sørensen ◽  
Nicolai O. Haahr ◽  
Søren H. Jørgensen ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
Single Molecule ◽  
Dynamic Equilibrium ◽  
Super Resolution ◽  
Striatal Slices ◽  
D2 Autoreceptor ◽  
Molecular Components ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Excitatory Drive

SUMMARYThe nanoscopic organization and regulation of individual molecular components in presynaptic varicosities of neurons releasing modulatory volume neurotransmitters like dopamine (DA) remain largely elusive. Here we show by application of several single-molecule sensitive super-resolution microscopy techniques to cultured neurons and mouse striatal slices, that the dopamine transporter (DAT), a key protein in varicosities of dopaminergic neurons, exists in the membrane in dynamic equilibrium between an inward-facing nanodomain-localized and outward-facing unclustered configuration. The balance between these configurations is inversely regulated by excitatory drive and by DA D2-autoreceptor activation in manner dependent on Ca2+-influx via N-type voltage-gated Ca2+-channels. The DAT nanodomains contain tens of transporters molecules and overlap with nanodomains of PIP2 (phosphatidylinositol-4,5-bisphosphate) but show little overlap with D2-autoreceptor, syntaxin-1 and clathrin nanodomains. By demonstrating that nanoscopic reorganizations with putative major impact on transmitter homeostasis can take place in dopaminergic varicosities, the data have important implications for understanding modulatory neurotransmitter physiology.

scholarly journals Dynamics and nanoscale organization of the postsynaptic endocytic zone at excitatory synapses

2021 ◽  
Author(s):  
Lisa A.E. Catsburg ◽  
Manon Westra ◽  
Annemarie M. L. van Schaik ◽  
Harold D. MacGillavry
Keyword(s):  
Synaptic Plasticity ◽  
Actin Cytoskeleton ◽  
Glutamate Receptors ◽  
Super Resolution ◽  
Live Cell ◽  
Excitatory Synapses ◽  
Membrane Interactions ◽  
Synaptic Receptors ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

ABSTRACTAt postsynaptic sites of neurons, a prominent clathrin-coated structure, the endocytic zone (EZ), controls the trafficking of glutamate receptors and is essential for synaptic plasticity. Despite its importance, little is known about how this clathrin structure is organized to mediate endocytosis. We used live-cell and super-resolution microscopy techniques to reveal the dynamic organization of this poorly understood clathrin structure. We found that a subset of endocytic proteins only transiently appeared at postsynaptic sites. In contrast, other proteins, including Eps15, intersectin1L, and β2-adaptin, were persistently enriched and partitioned at the edge of the EZ. We found that uncoupling the EZ from the synapse led to the loss of most of these components, while disrupting the actin cytoskeleton or AP2-membrane interactions did not alter EZ positioning. We conclude that the EZ is a stable, highly organized molecular platform where components are differentially recruited and positioned to orchestrate the endocytosis of synaptic receptors.

scholarly journals Microscope-AOtools: A generalised adaptive optics implementation

2020 ◽  
Author(s):  
Nicholas Hall ◽  
Josh Titlow ◽  
Martin J. Booth ◽  
Ian M. Dobbie
Keyword(s):  
Image Quality ◽  
Adaptive Optics ◽  
Super Resolution ◽  
Biological Imaging ◽  
New Techniques ◽  
Reduce Image Quality ◽  
Set Up ◽  
And Function ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

AbstractMicroscope-AOtools is a software package which allows for a simple, robust and generalised implementation of adaptive optics (AO) elements. It contains all the necessary methods for set-up, calibration, and aberration correction which are simple to use and function in a robust manner. Aberrations arising from sources such as sample hetero-geneity and refractive index mismatches are constant problems in biological imaging. These aberrations reduce image quality and the achievable depth of imaging, particularly in super-resolution microscopy techniques. AO technology has been proven to be effective in correcting for these aberrations and thereby improving the image quality. However, it has not been widely adopted by the biological imaging community due, in part, to difficulty in set-up and operation of AO, particularly by non-specialist users. Microscope-AOtools offers a robust, easy-to-use implementation of the essential methods for set-up and use of AO techniques. These methods are constructed in a generalised manner that can utilise a range of adaptive optics elements, wavefront sensing techniques and sensorless AO correction methods. Furthermore, the methods are designed to be easily extensible as new techniques arise, leading to a streamlined pipeline for new AO technology and techniques to be adopted by the wider microscopy community.

scholarly journals Embracing the uncertainty: the evolution of SOFI into a diverse family of fluctuation-based super-resolution microscopy methods

2021 ◽  
Author(s):  
Monika Pawlowska ◽  
Ron Tenne ◽  
Bohnishikha Ghosh ◽  
Adrian Makowski ◽  
Radek Lapkiewicz
Keyword(s):  
3D Imaging ◽  
Deep Neural Networks ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Significant Progress ◽  
Spatial Correlations ◽  
Order Of Magnitude ◽  
Temporal And Spatial ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy

Abstract Super-resolution microscopy techniques have pushed the limits of resolution in optical imaging by more than an order of magnitude. However, these methods often require long acquisition times as well as complex setups and sample preparation protocols. Super-resolution Optical Fluctuation Imaging (SOFI) emerged over ten years ago as an approach that exploits temporal and spatial correlations within the acquired images to obtain increased resolution with less strict requirements. This review follows the progress of SOFI from its first demonstration to the development of a branch of methods that treat fluctuations as a source of contrast, rather than noise. Among others, we highlight the implementation of SOFI with standard fluorescent proteins as well as the microscope modification that facilitate 3D imaging and the application of modern cameras. Going beyond the classical framework of SOFI, we explore different innovative concepts from deep neural networks all the way to a quantum analogue of SOFI, antibunching microscopy. While SOFI has not reached the same level of ubiquity as other super-resolution methods, our overview finds significant progress and substantial potential for the concept of leveraging fluorescence fluctuations to obtain super-resolved images.

Tools and limitations to study the molecular composition of synapses by fluorescence microscopy

2016 ◽  
Vol 473 (20) ◽  
pp. 3385-3399 ◽  
Author(s):  
Manuel Maidorn ◽  
Silvio O. Rizzoli ◽  
Felipe Opazo
Keyword(s):  
Super Resolution ◽  
Molecular Composition ◽  
Synaptic Organization ◽  
Synaptic Physiology ◽  
Copy Numbers ◽  
Resolution Imaging ◽  
And Function ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Affinity Probes

The synapse is densely packed with proteins involved in various highly regulated processes. Synaptic protein copy numbers and their stoichiometric distribution have a drastic influence on neuronal integrity and function. Therefore, the molecular analysis of synapses is a key element to understand their architecture and function. The overall structure of the synapse has been revealed with an exquisite amount of details by electron microscopy. However, the molecular composition and the localization of proteins are more easily addressed with fluorescence imaging, especially with the improved resolution achieved by super-resolution microscopy techniques. Notably, the fast improvement of imaging instruments has not been reflected in the optimization of biological sample preparation. During recent years, large efforts have been made to generate affinity probes smaller than conventional antibodies adapted for fluorescent super-resolution imaging. In this review, we briefly discuss the current views on synaptic organization and necessary key technologies to progress in the understanding of synaptic physiology. We also highlight the challenges faced by current fluorescent super-resolution methods, and we describe the prerequisites for an ideal study of synaptic organization.

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

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)’.

Sign in / Sign up

Export Citation Format

Share Document