scholarly journals GFP-Tagged Protein Detection by Electron Microscopy Using a GBP-APEX Tool in Drosophila

2021 ◽  
Vol 9 ◽  
Author(s):  
Fred Bernard ◽  
Julie Jouette ◽  
Catherine Durieu ◽  
Rémi Le Borgne ◽  
Antoine Guichet ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Optical Microscopy ◽  
Cell Biology ◽  
Plasma Membranes ◽  
Protein Detection ◽  
Specific Protein ◽  
Specific Antibodies ◽  
Efficient Detection ◽  
Endocytic Vesicles ◽  
Microscopy Techniques

In cell biology, detection of protein subcellular localizations is often achieved by optical microscopy techniques and more rarely by electron microscopy (EM) despite the greater resolution offered by EM. One of the possible reasons was that protein detection by EM required specific antibodies whereas this need could be circumvented by using fluorescently-tagged proteins in optical microscopy approaches. Recently, the description of a genetically encodable EM tag, the engineered ascorbate peroxidase (APEX), whose activity can be monitored by electron-dense DAB precipitates, has widened the possibilities of specific protein detection in EM. However, this technique still requires the generation of new molecular constructions. Thus, we decided to develop a versatile method that would take advantage of the numerous GFP-tagged proteins already existing and create a tool combining a nanobody anti-GFP (GBP) with APEX. This GBP-APEX tool allows a simple and efficient detection of any GFP fusion proteins without the needs of specific antibodies nor the generation of additional constructions. We have shown the feasibility and efficiency of this method to detect various proteins in Drosophila ovarian follicles such as nuclear proteins, proteins associated with endocytic vesicles, plasma membranes or nuclear envelopes. Lastly, we expressed this tool in Drosophila with the UAS/GAL4 system that enables spatiotemporal control of the protein detection.

scholarly journals Micro-Stratigraphical Investigation on Corrosion Layers in Ancient Bronze Artefacts of Urartian Period by Scanning Electron Microscopy, Energy-Dispersive Spectrometry, and Optical Microscopy

Heritage ◽  
2021 ◽  
Vol 4 (3) ◽  
pp. 2526-2543
Author(s):  
Yeghis Keheyan ◽  
Giancarlo Lanterna
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Optical Microscopy ◽  
Energy Dispersive Spectrometry ◽  
Alloy Composition ◽  
Archaeological Site ◽  
Energy Dispersive ◽  
Microscopy Techniques ◽  
Tin Content ◽  
Scanning Electron

The results of the analysis on some fragments of bronze belts and a bowl discovered from southwestern Armenia at the Yegheghnadzor archaeological site are discussed. The samples are dated to the 7–6th millennium BCE from the Urartian period. The artefacts were corroded, and a multilayer structure was formed. To study the stratigraphy of layers and their composition, the samples have been analyzed using SEM-EDS (Scanning Electron Microscopy, Energy-Dispersive Spectrometry) and OM (Optical Microscopy) techniques. The bronze finds appear with the typical incrustations rich in alloy alteration compounds. Concentrations of copper and tin in the alloys were quantified by SEM-EDS: the pattern and the percentage of the alloy are the same for the belts. Regarding the bowl sample, it is constituted by two foils perfectly in contact but different in color, thickness, and composition. The results evidenced that only two elements participate in forming the alloy composition in the samples: Cu and Sn. The tin content is variable from 7.75% to 13.56%. Other elements such as Ag, As, Fe, Ni, P, Pb, Sb, and Zn make up less than 1% and can be considered as impurities.

scholarly journals Three-dimensional nanostructure of an intact microglia cell

2018 ◽  
Author(s):  
Giulia Bolasco ◽  
Laetitia Weinhard ◽  
Tom Boissonnet ◽  
Ralph Neujahr ◽  
Cornelius T. Gross
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Microglia Cell ◽  
Cell Structures ◽  
Transmission Electron ◽  
Morphological Criteria ◽  
Membrane Protrusions ◽  
Microscopy Techniques

Microglia are non-neuronal cells of the myeloid lineage that invade and take up long-term residence in the brain during development (Ginhoux et al. 2010) and are increasingly implicated in neuronal maturation, homeostasis, and pathology (Bessis et al. 2007; Paolicelli et al. 2011; Li et al. 2012; Aguzzi et al. 2013, Cunningham 2013, Cunningham et al. 2013). Since the early twentieth century several methods for staining and visualizing microglia have been developed. Scientists in Ramón y Cajal’s group (Achúcarro 1913, Río-Hortega 1919) pioneered these methods and their work led to the christening of microglia as the third element of the nervous system, distinct from astrocytes and neurons. More recently, a combination of imaging, genetic, and immunological tools has been used to visualize microglia in living brain (Davalos et al. 2005; Nimmerjahn et al. 2005). It was found that microglia are highly motile under resting conditions and rapidly respond to injuries (Kettenmann et al. 2011) suggesting a role for microglia in both brain homeostasis and pathology. Transmission Electron microscopy (TEM) has provided crucial complementary information on microglia morphology and physiology but until recently EM analyses have been limited to single or limited serial section studies (Tremblay et al. 2010; Paolicelli et al. 2011; Schafer et al. 2012; Tremblay et al. 2012; Sipe et al. 2016). TEM studies were successful in defining a set of morphological criteria for microglia: a polygonal nucleus with peripheral condensed chromatin, a relatively small cytoplasm with abundant presence of rough endoplasmic reticulum (RER), and a large volume of lysosomes and inclusions in the perikaryon. Recent advances in volumetric electron microscopy techniques allow for 3D reconstruction of large samples at nanometer-resolution, thus opening up new avenues for the understanding of cell biology and architecture in intact tissues. At the same time, correlative light and electron microscopy (CLEM) techniques have been extended to 3D brain samples to help navigate and identify critical molecular landmarks within large EM volumes (Briggman and Denk 2006; Maco et al. 2013; Blazquez-Llorca et al. 2015, Bosch et al. 2015). Here we present the first volumetric ultrastructural reconstruction of an entire mouse hippocampal microglia using serial block face scanning electron microscopy (SBEM). Using CLEM we have ensured the inclusion of both large, small, and filopodial microglia processes. Segmentation of the dataset allowed us to carry out a comprehensive inventory of microglia cell structures, including vesicles, organelles, membrane protrusions, and processes. This study provides a reference that can serve as a data mining resource for investigating microglia cell biology.

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 THE ULTRASTRUCTURE OF THE NEXUS

1970 ◽  
Vol 47 (3) ◽  
pp. 666-688 ◽  
Author(s):  
N. Scott McNutt ◽  
Ronald S. Weinstein
Keyword(s):  
Electron Microscopy ◽  
Plasma Membranes ◽  
Close Apposition ◽  
Lanthanum Hydroxide ◽  
Thin Sections ◽  
Macromolecular Complexes ◽  
Subunit Assembly ◽  
Microscopy Techniques ◽  
Electron Lucent ◽  
Colloidal Lanthanum

A correlation is made between the appearances of the nexus ("gap junction") as revealed by thin-section and by freeze-cleave electron microscopy techniques. These methods reveal different aspects of a complex subunit assembly forming the nexus membranes. In thin sections, the nexus is formed by the very close apposition of two "unit" membranes. The electron-opaque tracer, colloidal lanthanum hydroxide, outlines an aspect of electron-lucent subunits that project into the central region of the nexus. The freeze-cleave technique demonstrates novel membrane faces that are generated from within the interior of plasma membranes by splitting them into two lamellae (Lm): Lm 1 adjacent to the cytoplasm, and Lm 2 adjacent to the extracellular space. Each of the two membranes forming the nexus can be split into these two lamellae. On the new face of Lm 1, particles approximately 50 A in diameter are closely packed in an array which is often hexagonal with a 90–100 A center-to-center spacing. The two apposed lamellae (Lm 2-Lm 2) of the nexus are constructed of sheets of subunits in a similar array. The Lm 1 particles appear to extend into the Lm 2 subunits to form macromolecular complexes. The Lm 2 subunits extend to the center of the nexus to form the contacts outlined by lanthanum in sections. It is postulated that central hydrophilic channels may extend through the subunit assembly to provide a direct route for intercellular communication.

scholarly journals Conventional transmission electron microscopy

2014 ◽  
Vol 25 (3) ◽  
pp. 319-323 ◽  
Author(s):  
Mark Winey ◽  
Janet B. Meehl ◽  
Eileen T. O'Toole ◽  
Thomas H. Giddings
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Experimental Design ◽  
Sample Preparation ◽  
Cell Biology ◽  
Conventional Transmission Electron Microscopy ◽  
Point Potential ◽  
Transmission Electron ◽  
Microscopy Techniques

Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project.

scholarly journals A comparative study on the use of microscopy in pharmacology and cell biology research

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245795
Author(s):  
Agatha M. Reigoto ◽  
Sarah A. Andrade ◽  
Marianna C. R. R. Seixas ◽  
Manoel L. Costa ◽  
Claudia Mermelstein
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Live Cell ◽  
Biomedical Sciences ◽  
Bright Field ◽  
Optical And Electron Microscopy ◽  
Microscopy Techniques ◽  
The Impact

Microscopy is the main technique to visualize and study the structure and function of cells. The impact of optical and electron microscopy techniques is enormous in all fields of biomedical research. It is possible that different research areas rely on microscopy in diverse ways. Here, we analyzed comparatively the use of microscopy in pharmacology and cell biology, among other biomedical sciences fields. We collected data from articles published in several major journals in these fields. We analyzed the frequency of use of different optical and electron microscopy techniques: bright field, phase contrast, differential interference contrast, polarization, conventional fluorescence, confocal, live cell imaging, super resolution, transmission and scanning electron microscopy, and cryoelectron microscopy. Our analysis showed that the use of microscopy has a distinctive pattern in each research area, and that nearly half of the articles from pharmacology journals did not use any microscopy method, compared to the use of microscopy in almost all the articles from cell biology journals. The most frequent microscopy methods in all the journals in all areas were bright field and fluorescence (conventional and confocal). Again, the pattern of use was different: while the most used microscopy methods in pharmacology were bright field and conventional fluorescence, in cell biology the most used methods were conventional and confocal fluorescence, and live cell imaging. We observed that the combination of different microscopy techniques was more frequent in cell biology, with up to 6 methods in the same article. To correlate the use of microscopy with the research theme of each article, we analyzed the proportion of microscopy figures with the use of cell culture. We analyzed comparatively the vocabulary of each biomedical sciences field, by the identification of the most frequent words in the articles. The collection of data described here shows a vast difference in the use of microscopy among different fields of biomedical sciences. The data presented here could be valuable in other scientific and educational contexts.

Biological Labeling and Correlative Microscopy

1999 ◽  
Vol 5 (S2) ◽  
pp. 474-475
Author(s):  
J.M. Robinson ◽  
T. Takizawa
Keyword(s):  
Electron Microscopy ◽  
Optical Microscopy ◽  
Cell Biology ◽  
Spatial Information ◽  
Dynamic Properties ◽  
Imaging Modality ◽  
Cell Structure ◽  
Fluorescence Labeling ◽  
Specific Chemical ◽  
Biological Labeling

A variety of biological labeling techniques has been developed in order to obtain specific chemical and spatial information from cells and tissues. Traditionally theses labeling techniques have been categorized as cytochemistry, immunocytochemistry, and in situ hybridization. Another special category relates to fluorescence analog cytochemistry in which specific fluorescently-labeled molecules become incorporated into the pool of endogenous molecules of the cell. They can thus serve as reporters for analysis of the dynamic properties of the population of molecules of interest. Such molecules are usually introduced into cells by microinjection or expressed within the cell (e.g., green fluorescent protein derivatives).The past few years have witnessed a renaissance in biological optical microscopy. Many of the advances in the elucidation of cell structure-function relationships made through the use of optical microscopy have relied upon fluorescence labeling technology. These advances notwithstanding there remain experimental situations in cell biology that require the higher spatial resolution afforded by electron microscopy. Combining fluorescence and electron microscopy to study the same structures would be very useful in many experimental situations in cell biology. Such an examination of the same structures with more than one imaging modality can be referred to as correlative or integrated microscopy. The number of such studies is relatively small; this is probably due to technical difficulties encountered by various investigators.

A SEM/TEM examination of a ceramic membrane

1986 ◽  
Vol 44 ◽  
pp. 682-683
Author(s):  
C.E. Voegele-Kliewer ◽  
A.D. McMaster ◽  
G.W. Dirks
Keyword(s):  
Electron Microscopy ◽  
Gas Separation ◽  
Ceramic Membrane ◽  
Hydrogen Iodide ◽  
Separation Processes ◽  
Corning Glass ◽  
Gas Transport Properties ◽  
Porous Microstructure ◽  
Microscopy Techniques ◽  
The Relationship

Materials other than polymers, e.g. ceramic silicates, are currently being investigated for gas separation processes. The permeation characteristics of one such material, Vycor (Corning Glass #1370), have been reported for the separation of hydrogen from hydrogen iodide. This paper will describe the electron microscopy techniques applied to reveal the porous microstructure of a Vycor membrane. The application of these techniques has led to an increased understanding in the relationship between the substructure and the gas transport properties of this material.

Defects of Platelet Function Recognized by X-Ray Imaging and Scanning Electron Microscopy

1985 ◽  
Vol 43 ◽  
pp. 610-613
Author(s):  
M.G. Baldini ◽  
S. Morinaga ◽  
D. Minasian ◽  
R. Feder ◽  
D. Sayre ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Cell Biology ◽  
Imaging Technique ◽  
Human Platelets ◽  
X Rays ◽  
X Ray ◽  
X Ray Imaging ◽  
Complex Phenomena ◽  
Scanning Electron

Contact X-ray imaging is presently developing as an important imaging technique in cell biology. Our recent studies on human platelets have demonstrated that the cytoskeleton of these cells contains photondense structures which can preferentially be imaged by soft X-ray imaging. Our present research has dealt with platelet activation, i.e., the complex phenomena which precede platelet appregation and are associated with profound changes in platelet cytoskeleton. Human platelets suspended in plasma were used. Whole cell mounts were fixed and dehydrated, then exposed to a stationary source of soft X-rays as previously described. Developed replicas and respective grids were studied by scanning electron microscopy (SEM).

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