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 Precision of fiducial marker alignment for correlative super‐resolution fluorescence and transmission electron microscopy

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Adeeba Fathima ◽  
César Augusto Quintana-Cataño ◽  
Christoph Heintze ◽  
Michael Schlierf
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Specific Protein ◽  
Fiducial Markers ◽  
Transmission Electron ◽  
Microscopy Techniques

AbstractRecent advances in microscopy techniques enabled nanoscale discoveries in biology. In particular, electron microscopy reveals important cellular structures with nanometer resolution, yet it is hard, and sometimes impossible to resolve specific protein localizations. Super-resolution fluorescence microscopy techniques developed over the recent years allow for protein-specific localization with ~ 20 nm precision are overcoming this limitation, yet it remains challenging to place those in cells without a reference frame. Correlative light and electron microscopy (CLEM) approaches have been developed to place the fluorescence image in the context of a cellular structure. However, combining imaging methods such as super resolution microscopy and transmission electron microscopy necessitates a correlation using fiducial markers to locate the fluorescence on the structures visible in electron microscopy, with a measurable precision. Here, we investigated different fiducial markers for super-resolution CLEM (sCLEM) by evaluating their shape, intensity, stability and compatibility with photoactivatable fluorescent proteins as well as the electron density. We further carefully determined limitations of correlation accuracy. We found that spectrally-shifted FluoSpheres are well suited as fiducial markers for correlating single-molecule localization microscopy with transmission electron microscopy.

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

2013 Microscopy Today Innovation Awards

2013 ◽  
Vol 21 (5) ◽  
pp. 40-45
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Scanning Probe Microscopy ◽  
Scanning Probe ◽  
Probe Microscopy ◽  
Analysis Methods ◽  
Microscopy Analysis ◽  
Ion Microscopy ◽  
Microscopy Techniques

Microscopy Today congratulates the fourth annual group of Innovation Award winners. The ten innovations described below move several microscopy techniques forward: light microscopy, scanning probe microscopy, electron microscopy, ion microscopy, and hybrid microscopy-analysis methods. These innovations will make imaging and analysis more powerful, more flexible, more productive, and easier to accomplish.

Electron Microscopy Applied to Fine-Needle Aspiration. A Report of Six Cases from Various Sites

1983 ◽  
Vol 69 (5) ◽  
pp. 423-435 ◽  
Author(s):  
Saverio Cinti ◽  
Maurizio Ferretti ◽  
Silvana Amati ◽  
Giancarlo Balercia ◽  
Adalberto Vecchi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Fine Needle Aspiration ◽  
Needle Aspiration ◽  
Ultrastructural Analysis ◽  
Tissue Structure ◽  
Fine Needle ◽  
Neoplastic Lesions ◽  
Microscopy Techniques

The authors report the results obtained from the application of electron microscopy techniques to the cytology of fine-needle-aspirated samples of neoplastic lesions from various body sites. These results show that the tissue structure, which is usually lost during the squashing necessary for light microscopy cytology, is preserved when the samples are processed for ultrastructural analysis. Electron microscopy also allows a highly detailed study of the cell's inner structures. Thus, when this technique is applied, fine needle-aspirated samples can be regarded as actual microbiopsies. However, because of the high cost of ultrastructural techniques, we suggest that actual analysis be performed only in selected cases, whereas fixation and inclusion for electron microscopy could be done routinely.

scholarly journals An Easy Path for Correlative Electron and Super-Resolution Light Microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dorothea Pinotsi ◽  
Simona Rodighiero ◽  
Silvia Campioni ◽  
Gabor Csucs
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Light Microscopy ◽  
Amyloid Fibrils ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Wide Field ◽  
Set Up ◽  
Bio Compatibility ◽  
Scanning Electron

Abstract A number of new Correlative Light and Electron Microscopy approaches have been developed over the past years, offering the opportunity to combine the specificity and bio-compatibility of light microscopy with the high resolution achieved in electron microscopy. More recently, these approaches have taken one step further and also super-resolution light microscopy was combined with transmission or scanning electron microscopy. This combination usually requires moving the specimen between different imaging systems, an expensive set-up and relatively complicated imaging workflows. Here we present a way to overcome these difficulties by exploiting a commercially available wide-field fluorescence microscope integrated in the specimen chamber of a Scanning Electron Microscope (SEM) to perform correlative LM/EM studies. Super-resolution light microscopy was achieved by using a recently developed algorithm - the Super-Resolution Radial Fluctuations (SRRF) - to improve the resolution of diffraction limited fluorescent images. With this combination of hardware/software it is possible to obtain correlative super-resolution light and scanning electron microscopy images in an easy and fast way. The imaging workflow is described and demonstrated on fluorescently labelled amyloid fibrils, fibrillar protein aggregates linked to the onset of multiple neurodegenerative diseases, revealing information about their polymorphism.

Super-resolution fluorescent materials: an insight into design and bioimaging applications

2016 ◽  
Vol 45 (17) ◽  
pp. 4651-4667 ◽  
Author(s):  
Zhigang Yang ◽  
Amit Sharma ◽  
Jing Qi ◽  
Xiao Peng ◽  
Dong Yeop Lee ◽  
...  
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Fluorescent Imaging ◽  
Organic Fluorophores ◽  
Biological Targets ◽  
Fluorescent Materials ◽  
Microscopy Techniques ◽  
Insight Into

With the emerging of super-resolution fluorescent imaging microscopy techniques, biological targets below 200 nm in size are successful to be localized clearly and precisely with unprecedented details. In this tutorial review, the fluorescent materials, including organic fluorophores and nanomaterials, utilized in STED, single molecule localized microscopy (PALM/STORM) and SOFI microscopies, together with their working principles are mainly discussed.

scholarly journals Characterization of the sarcoplasmic reticulum proteins in the thermogenic muscles of fish.

1994 ◽  
Vol 127 (5) ◽  
pp. 1275-1287 ◽  
Author(s):  
B A Block ◽  
J O'Brien ◽  
G Meissner
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Direct Evidence ◽  
Striated Muscle ◽  
High Capacity ◽  
Muscle Cells ◽  
Immunoblot Analysis ◽  
Fluorescent Labeling ◽  
T Tubules ◽  
The Brain

Marlins, sailfish, spearfishes, and swordfish have extraocular muscles that are modified into thermogenic organs beneath the brain. The modified muscle cells, called heater cells, lack organized myofibrils and are densely packed with sarcoplasmic reticulum (SR), transverse (T) tubules, and mitochondria. Thermogenesis in the modified extraocular muscle fibers is hypothesized to be associated with increased energy turnover due to Ca2+ cycling at the SR. In this study, the proteins associated with sequestering and releasing Ca2+ from the SR (ryanodine receptor, Ca2+ ATPase, calsequestrin) of striated muscle cells were characterized in the heater SR using immunoblot and immunofluorescent techniques. Immunoblot analysis with a monoclonal antibody that recognizes both isoforms of nonmammalian RYRs indicates that the fish heater cells express only the alpha RYR isoform. The calcium dependency of [3H]ryanodine binding to the RYR isoform expressed in heater indicates functional identity with the non-mammalian alpha RYR isoform. Fluorescent labeling demonstrates that the RYR is localized in an anastomosing network throughout the heater cell cytoplasm. Measurements of oxalate supported 45Ca2+ uptake, Ca2+ ATPase activity, and [32P]phosphoenzyme formation demonstrate that the SR contains a high capacity for Ca2+ uptake via an ATP dependent enzyme. Immunoblot analysis of calsequestrin revealed a significant amount of the Ca2+ binding protein in the heater cell SR. The present study provides the first direct evidence that the heater SR system contains the proteins necessary for Ca2+ release, re-uptake and sequestration, thus supporting the hypothesis that thermogenesis in the modified muscle cells is achieved via an ATP-dependent cycling of Ca2+ between the SR and cytosolic compartments.

scholarly journals Molecular resolution imaging by post-labeling expansion single-molecule localization microscopy (Ex-SMLM)

2020 ◽  
Author(s):  
Fabian U. Zwettler ◽  
Sebastian Reinhard ◽  
Davide Gambarotto ◽  
Toby D. M. Bell ◽  
Virginie Hamel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Single Molecule ◽  
Super Resolution ◽  
Reference Structure ◽  
Positional Error ◽  
Localization Microscopy ◽  
Resolution Imaging ◽  
Endogenous Proteins ◽  
Super Resolution Microscopy ◽  
Single Molecule Localization Microscopy

AbstractExpansion microscopy (ExM) enables super-resolution fluorescence imaging of physically expanded biological samples with conventional microscopes. By combining expansion microscopy (ExM) with single-molecule localization microscopy (SMLM) it is potentially possible to approach the resolution of electron microscopy. However, current attempts to combine both methods remained challenging because of protein and fluorophore loss during digestion or denaturation, gelation, and the incompatibility of expanded polyelectrolyte hydrogels with photoswitching buffers. Here we show that re-embedding of expanded hydrogels enables dSTORM imaging of expanded samples and demonstrate that post-labeling ExM resolves the current limitations of super-resolution microscopy. Using microtubules as a reference structure and centrioles, we demonstrate that post-labeling Ex-SMLM preserves ultrastructural details, improves the labeling efficiency and reduces the positional error arising from linking fluorophores into the gel thus paving the way for super-resolution imaging of immunolabeled endogenous proteins with true molecular resolution.

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 Ultrastructure of developing human muscle: the problem of multinucleation of striated muscle cells

1986 ◽  
Vol 44 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Guilberto Minguetti ◽  
W. G. P. Mair
Keyword(s):  
Electron Microscopy ◽  
Basement Membrane ◽  
Plasma Membranes ◽  
Striated Muscle ◽  
Muscle Cells ◽  
Human Muscle ◽  
Membrane Tube ◽  
Embryonic Myogenesis ◽  
Human Foetuses

The authors studied by electron microscopy the muscle of 27 human foetuses ranging from 9 weeks to 9 months development. It was possible to observe that disintegration of the plasma membranes of adjacent myoblasts and myotubes which share a common basement membrane tube appears to occur in longitudinally disposed cells of those categories. This may help to explain how further nuclei may be incorporated into well developed myotubes and how the striated muscle cells become multinucleated during embryonic myogenesis and regeneration in vivo.

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