scholarly journals A robust method for particulate detection of a genetic tag for 3D electron microscopy

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
James Rae ◽  
Charles Ferguson ◽  
Nicholas Ariotti ◽  
Richard I Webb ◽  
Han-Hao Cheng ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Fusion Proteins ◽  
Ion Beam ◽  
Expression System ◽  
Internal Standard ◽  
Membrane Vesicles ◽  
Cytoskeletal Proteins ◽  
Electron Microscopic ◽  
Simple Method ◽  
In Cells

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy. Here we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal to noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins and cytoskeletal proteins. The method can be combined with different electron microscopic techniques including fast freezing and freeze substitution, focussed ion beam scanning electron microscopy, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.

scholarly journals APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy

2020 ◽  
Author(s):  
James Rae ◽  
Charles Ferguson ◽  
Nicholas Ariotti ◽  
Richard I. Webb ◽  
Han-Hao Cheng ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Electron Tomography ◽  
Ion Beam ◽  
Expression System ◽  
Internal Standard ◽  
Freeze Substitution ◽  
Ultrastructural Localization ◽  
Cytoskeletal Proteins ◽  
Electron Microscopic ◽  
Simple Method

AbstractGenetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy. Here we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal to noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins and cytoskeletal proteins. The method can be combined with different electron microscopic techniques including fast freezing and freeze substitution, focussed ion beam scanning electron microscopy, and electron tomography. The method allows detection of endogenously expressed proteins in genome-edited cells. We make use of a cell-free expression system to generate membrane particles with a defined quantum of an APEX-fusion protein. These particles can be added to cells to provide an internal standard for estimating absolute density of expressed APEX-fusion proteins.

Novel Method for Preparing Transmission Electron Microscopy Samples of Micrometer-Sized Powder Particles by Using Focused Ion Beam

2017 ◽  
Vol 23 (5) ◽  
pp. 1055-1060 ◽  
Author(s):  
Tae-Hoon Kim ◽  
Min-Chul Kang ◽  
Ga-Bin Jung ◽  
Dong Soo Kim ◽  
Cheol-Woong Yang
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Focused Ion Beam ◽  
Size Range ◽  
Ion Beam ◽  
Preparation Technique ◽  
Simple Method ◽  
Transmission Electron ◽  
Powder Particles ◽  
Novel Method

AbstractThe preparation of transmission electron microscopy (TEM) samples from powders is quite difficult and challenging. For powders with particles in the 1–5 μm size range, it is especially difficult to select an adequate sample preparation technique. Epoxy is commonly used to bind powder, but drawbacks, such as differential milling originating from unequal milling rates between the epoxy and powder, remain. We propose a new, simple method for preparing TEM samples. This method is especially useful for powders with particles in the 1–5 μm size range that are vulnerable to oxidation. The method uses solder as an embedding agent together with focused ion beam (FIB) milling. The powder was embedded in low-temperature solder using a conventional hot-mounting instrument. Subsequently, FIB was used to fabricate thin TEM samples via the lift-out technique. The solder proved to be more effective than epoxy in producing thin TEM samples with large areas. The problem of differential milling was mitigated, and the solder binder was more stable than epoxy under an electron beam. This methodology can be applied for preparing TEM samples from various powders that are either vulnerable to oxidation or composed of high atomic number elements.

scholarly journals Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

1994 ◽  
Vol 126 (4) ◽  
pp. 901-910 ◽  
Author(s):  
T J Deerinck ◽  
M E Martone ◽  
V Lev-Ram ◽  
D P Green ◽  
R Y Tsien ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Substantial Improvement ◽  
Electron Microscopic ◽  
Simple Method ◽  
High Voltage Electron Microscopy

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

scholarly journals An Electron Microscopic Description of Basophilic Stippling in Red Cells

Blood ◽  
1965 ◽  
Vol 25 (6) ◽  
pp. 933-943 ◽  
Author(s):  
WALLACE N. JENSEN ◽  
GIULIANA D. MORENO ◽  
MARCEL C. BESSIS
Keyword(s):  
Electron Microscopy ◽  
Nonheme Iron ◽  
Red Cells ◽  
Blue Dye ◽  
Electron Microscopic ◽  
Microscopic Description ◽  
Phase Microscopy ◽  
Suspended Cells ◽  
Spontaneous Occurrence ◽  
In Cells

Abstract Red cells which contain ribosomes presumably have the potential to form stippled cells. The ribosomes in cells from animals with phenylhydrazine and postphlebotomy anemia may form stippling when blood films are dried slowly or when stained supravitally with dilute concentrations of New Methylene Blue dye. The ribosomes of cells from animals with lead intoxication have a greater propensity to form aggregates and therefore to result in stippling. Since the formation of stippling in cells is dependent on the dessication of cells, they are not seen in plasma suspended cells nor in cells prepared in routine fashion for electron microscopy. The basophilic stippled material produced in certain blood cells by the action of supravital dye consists exclusively of ribosomes. The association of nonheme iron with basophilic stippling is usually the consequence of the presence of two separate morphologic structures within the cells which may not be resolved, one from the other, in light or phase microscopy, but may be distinguished by the use of electron microscopy. The "spontaneous" occurrence of the basophilic stippling may be interpreted as evidence of ribosomal abnormality, but the multiplicity of cellular injuries induced by lead, other chemicals, drugs or disease processes may indicate that ribosomal aggregation results from alteration of nonribosomal organelles or other cellular constituents.

scholarly journals Unraveling the antiviral activity of plitidepsin by subcellular and morphological analysis

2021 ◽  
Author(s):  
Martin Sachse ◽  
Raquel Tenorio ◽  
Isabel Fernandez de Castro ◽  
Jordana Munoz-Basagoiti ◽  
Daniel Perez-Zsolt ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Morphological Analysis ◽  
Membrane Vesicles ◽  
Antitumor Agent ◽  
Host Protein ◽  
Genome Replication ◽  
Emerging Viruses ◽  
Eukaryotic Translation ◽  
N Protein ◽  
In Cells

The pandemic caused by the new coronavirus SARS-CoV-2 has made evident the need for broad-spectrum, efficient antiviral treatments to combat emerging and re-emerging viruses. Plitidepsin is an antitumor agent of marine origin that has also shown a potent pre-clinical efficacy against SARS-CoV-2. Plitidepsin targets the host protein eEF1A (eukaryotic translation factor 1 alpha 1) and affects viral infection at an early, post-entry step. Because electron microscopy is a valuable tool to study virus-cell interactions and the mechanism of action of antiviral drugs, in this work we have used transmission electron microscopy (TEM) to evaluate the effects of plitidepsin in SARS-CoV-2 infection in cultured Vero E6 cells 24 and 48h post-infection. In the absence of plitidepsin, TEM morphological analysis showed double-membrane vesicles (DMVs), organelles that support coronavirus genome replication, single-membrane vesicles with viral particles, large vacuoles with groups of viruses and numerous extracellular virions attached to the plasma membrane. When treated with plitidepsin, no viral structures were found in SARS-CoV-2-infected Vero E6 cells. Immunogold detection of SARS-CoV-2 nucleocapsid (N) protein and double-stranded RNA (dsRNA) provided clear signals in cells infected in the absence of plitidepsin, but complete absence in cells infected and treated with plitidepsin. The present study shows that plitidepsin completely blocks the biogenesis of viral replication organelles and the morphogenesis of virus progeny. Electron microscopy morphological analysis coupled to immunogold labeling of SARS-CoV-2 products offers a unique approach to understand how antivirals such as plitidepsin work.

Fluorescent and Immuno-Electron Microscopy of Detergent Extracted Cytoskeletal and Associated Antigens

1985 ◽  
Vol 43 ◽  
pp. 582-583
Author(s):  
E.J. Basgall ◽  
M.M. Soong ◽  
W.A.F. Tompkins
Keyword(s):  
Electron Microscopy ◽  
Cultured Cells ◽  
Leukemia Virus ◽  
Protein A ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Simple Method ◽  
Viral Antigens ◽  
Associated Proteins ◽  
Nih 3T3

Triton X-100 detergent extraction has been shown to be a relatively simple method for revealing the internal architecture of cultured cells. This method has proven valuable in studies of cytoskeleton-associated proteins and their functions in regulating cell activities. Exposed cytoskeletal elements can be examined using either transmission or high-resolution scanning electron microscopy. Immunochemical studies at the electron microscopic level have been simplified by the availability of Protein A-colloidal gold as a high affinity marker for immunoglobulins, specifically IgG.Several investigators have reported an association between the cytoskeleton and viral antigens. Evidence has indicated that, in some systems, the cytoskeleton plays a significant role in virus infection. Immunofluorescent studies on canine distemper virus infected, extracted cells have revealed altered cytoskeletal staining patterns, as compared to non-infected controls. In our laboratory, immuno-electronmicroscopy studies of extracted NIH/3T3 cells infected with Maloney-murine leukemia virus have indicated an association between cytoskeletal actin and the viral antigens gp70 and pl5E.

scholarly journals Correlation of organelle dynamics between light microscopic live imaging and electron microscopic 3D architecture using FIB-SEM

Microscopy ◽  
2020 ◽  
Author(s):  
Keisuke Ohta ◽  
Shingo Hirashima ◽  
Yoshihiro Miyazono ◽  
Akinobu Togo ◽  
Kei-ichiro Nakamura
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Optical Microscope ◽  
Live Imaging ◽  
Reconstruction Technique ◽  
Electron Microscopic

Abstract Correlative light and electron microscopy (CLEM) methods combined with live imaging can be applied to understand the dynamics of organelles. Although recent advances in cell biology and light microscopy have helped in visualizing the details of organelle activities, observing their ultrastructure or organization of surrounding microenvironments is a challenging task. Therefore, CLEM, which allows us to observe the same area as an optical microscope with an electron microscope, has become a key technique in cell biology. Unfortunately, most CLEM methods have technical drawbacks, and many researchers face difficulties in applying CLEM methods. Here, we propose a live three-dimensional CLEM method, combined with a three-dimensional reconstruction technique using focused ion beam scanning electron microscopy tomography, as a solution to such technical barriers. We review our method, the associated technical limitations and the options considered to perform live CLEM.

scholarly journals Volume electron microscopy: analyzing the lung

2020 ◽  
Author(s):  
Jan Philipp Schneider ◽  
Jan Hegermann ◽  
Christoph Wrede
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Three Dimensional ◽  
Alveolar Epithelium ◽  
Practical Implementation ◽  
Electron Microscopic ◽  
Array Tomography ◽  
Three Dimensional Models

AbstractSince its entry into biomedical research in the first half of the twentieth century, electron microscopy has been a valuable tool for lung researchers to explore the lung’s delicate ultrastructure. Among others, it proved the existence of a continuous alveolar epithelium and demonstrated the surfactant lining layer. With the establishment of serial sectioning transmission electron microscopy, as the first “volume electron microscopic” technique, electron microscopy entered the third dimension and investigations of the lung’s three-dimensional ultrastructure became possible. Over the years, further techniques, ranging from electron tomography over serial block-face and focused ion beam scanning electron microscopy to array tomography became available. All techniques cover different volumes and resolutions, and, thus, different scientific questions. This review gives an overview of these techniques and their application in lung research, focusing on their fields of application and practical implementation. Furthermore, an introduction is given how the output raw data are processed and the final three-dimensional models can be generated.

Electron Microscopic Demonstration of Adenovirus in Appendix Vermiformis in a Case of Ileocecal Intussusception

PEDIATRICS ◽  
1973 ◽  
Vol 51 (3) ◽  
pp. 566-570
Author(s):  
Eduardo J. Yunis ◽  
Yoshie Hashida
Keyword(s):  
Electron Microscopy ◽  
Electron Microscopic ◽  
Simple Method ◽  
Appendix Vermiformis ◽  
Viral Particles ◽  
History Of ◽  
Ileocecal Intussusception ◽  
Electron Microscopic Demonstration ◽  
Hematoxylin Eosin ◽  
Selection Of

Viral particles identical to those of adenovirus were demonstrated in the intranuclear inclusion bodies of the epithelial cells of an appendix vermiformis removed from a boy with a recent history of intussusception. Viral particles were demonstrated by electron microscopy following reprocessing of a formaldehyde-fixed, paraffin embedded and hematoxylin-eosin stained section. This simple method permits precise selection of the tissue for electron microscopy and in instances such as this gives sufficient tissue preservation to demonstrate viral particles. Our findings support the idea that some cases of intussusception may be the result of the adenovirus infection.

scholarly journals Direct Synthesis of Gold Nanoparticles on Cysteine-rich Tags in Mammalian Cells

2020 ◽  
Author(s):  
Zhaodi Jiang ◽  
Xiumei Jin ◽  
Yuhua Li ◽  
Sitong Liu ◽  
Xiao-Man Liu ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Gold Nanoparticles ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Mammalian Cells ◽  
Direct Synthesis ◽  
Single Molecule Detection ◽  
Chemical Fixation ◽  
High Pressure Freezing ◽  
In Cells

Abstract We developed a novel auto-nucleation suppressed mechanism (ANSM) for direct synthesis of EM-visible gold nanoparticles (AuNPs) on cysteine-rich tags (e.g., metallothionein) in cells for single-molecule detection with electron microscopy (it accompanies our Nature Method manuscript, Jiang et al. 2020 [1]). Both tagged-fusion proteins expressed in cells (e.g.bacteria, yeast and mammalian cells) and antigens stained with antibody-tag fusion proteins can be visualized by this protocol. Here we describe the typical protocols (both the chemical fixation and the high pressure freezing cases) developed for ANSM-based AuNP synthesis in HeLa cells expressing metallothionein (MTn) tags (Figure 1). This approach should be widely applicable to many systems for EM visualization of single-molecule in mammalian cells.

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