scholarly journals Single cell epigenetic visualization assay

2021 ◽  
Author(s):  
Sam Kint ◽  
Wim Van Criekinge ◽  
Linos Vandekerckhove ◽  
Winnok H De Vos ◽  
Karol Bomsztyk ◽  
...  
Keyword(s):  
Target Gene ◽  
Single Cells ◽  
Epigenetic Mark ◽  
Epigenetic Marks ◽  
Individual Gene ◽  
Rna Fish ◽  
Limited Coverage ◽  
Hiv 1

Abstract Characterization of the epigenetic status of individual cells remains a challenge. Current sequencing approaches have limited coverage, and it is difficult to assign an epigenetic status to the transcription state of individual gene alleles in the same cell. To address these limitations, a targeted microscopy-based epigenetic visualization assay (EVA) was developed for detection and quantification of epigenetic marks at genes of interest in single cells. The assay is based on an in situ biochemical reaction between an antibody-conjugated alkaline phosphatase bound to the epigenetic mark of interest, and a 5′-phosphorylated fluorophore-labeled DNA oligo tethered to a target gene by gene-specific oligonucleotides. When the epigenetic mark is present at the gene, phosphate group removal by the phosphatase protects the oligo from λ-exonuclease activity providing a quantitative fluorescent readout. We applied EVA to measure 5-methylcytosine (5mC) and H3K9Ac levels at different genes and the HIV-1 provirus in human cell lines. To link epigenetic marks to gene transcription, EVA was combined with RNA-FISH. Higher 5mC levels at the silenced compared to transcribed XIST gene alleles in female somatic cells validated this approach and demonstrated that EVA can be used to relate epigenetic marks to the transcription status of individual gene alleles.

scholarly journals Visualizing adenosine to inosine RNA editing in single mammalian cells

2016 ◽  
Author(s):  
Ian A. Mellis ◽  
Rohit K. Gupte ◽  
Arjun Raj ◽  
Sara H. Rouhanifard
Keyword(s):  
Subcellular Localization ◽  
Rna Editing ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Single Cells ◽  
Frequent Type ◽  
Rna Fish

AbstractConversion of adenosine bases to inosine in RNA is a frequent type of RNA editing, but important details about its biology, including subcellular localization, remain unknown due to a lack of imaging tools. We developed an RNA FISH strategy we called inoFISH that enables us to directly visualize and quantify adenosine-to-inosine edited transcripts in situ. Applying this tool to three edited transcripts (GRIA2, EIF2AK2 and NUP43), we found that editing of these transcripts is not correlated with nuclear localization nor paraspeckle association, and that NUP43 exhibits constant editing rates between single cells while the rates for GRIA2 vary.

scholarly journals Flow Virometry Quantification of Host Proteins on the Surface of HIV-1 Pseudovirus Particles

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1296
Author(s):  
Jonathan Burnie ◽  
Vera A. Tang ◽  
Joshua A. Welsh ◽  
Arvin T. Persaud ◽  
Laxshaginee Thaya ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Single Cells ◽  
Surface Current ◽  
High Sensitivity ◽  
Viral Envelope ◽  
High Throughput Analysis ◽  
Associated Proteins ◽  
Hiv 1

The HIV-1 glycoprotein spike (gp120) is typically the first viral antigen that cells encounter before initiating immune responses, and is often the sole target in vaccine designs. Thus, characterizing the presence of cellular antigens on the surfaces of HIV particles may help identify new antiviral targets or impact targeting of gp120. Despite the importance of characterizing proteins on the virion surface, current techniques available for this purpose do not support high-throughput analysis of viruses, and typically only offer a semi-quantitative assessment of virus-associated proteins. Traditional bulk techniques often assess averages of viral preparations, which may mask subtle but important differences in viral subsets. On the other hand, microscopy techniques, which provide detail on individual virions, are difficult to use in a high-throughput manner and have low levels of sensitivity for antigen detection. Flow cytometry is a technique that traditionally has been used for rapid, high-sensitivity characterization of single cells, with limited use in detecting viruses, since the small size of viral particles hinders their detection. Herein, we report the detection and surface antigen characterization of HIV-1 pseudovirus particles by light scattering and fluorescence with flow cytometry, termed flow virometry for its specific application to viruses. We quantified three cellular proteins (integrin α4β7, CD14, and CD162/PSGL-1) in the viral envelope by directly staining virion-containing cell supernatants without the requirement of additional processing steps to distinguish virus particles or specific virus purification techniques. We also show that two antigens can be simultaneously detected on the surface of individual HIV virions, probing for the tetraspanin marker, CD81, in addition to α4β7, CD14, and CD162/PSGL-1. This study demonstrates new advances in calibrated flow virometry as a tool to provide sensitive, high-throughput characterization of the viral envelope in a more efficient, quantitative manner than previously reported techniques.

scholarly journals Bacteria Single-Cell and Photosensitizer Interaction Revealed by Quantitative Phase Imaging

2021 ◽  
Vol 22 (10) ◽  
pp. 5068
Author(s):  
Igor Buzalewicz ◽  
Agnieszka Ulatowska-Jarża ◽  
Aleksandra Kaczorowska ◽  
Marlena Gąsior-Głogowska ◽  
Halina Podbielska ◽  
...  
Keyword(s):  
Imaging Techniques ◽  
Single Cells ◽  
Three Dimensional ◽  
Bacterial Biofilm ◽  
Phase Imaging ◽  
Label Free ◽  
Biophysical Processes ◽  
Contemporary Medicine

Quantifying changes in bacteria cells in the presence of antibacterial treatment is one of the main challenges facing contemporary medicine; it is a challenge that is relevant for tackling issues pertaining to bacterial biofilm formation that substantially decreases susceptibility to biocidal agents. Three-dimensional label-free imaging and quantitative analysis of bacteria–photosensitizer interactions, crucial for antimicrobial photodynamic therapy, is still limited due to the use of conventional imaging techniques. We present a new method for investigating the alterations in living cells and quantitatively analyzing the process of bacteria photodynamic inactivation. Digital holographic tomography (DHT) was used for in situ examination of the response of Escherichia coli and Staphylococcus aureus to the accumulation of the photosensitizers immobilized in the copolymer revealed by the changes in the 3D refractive index distributions of single cells. Obtained results were confirmed by confocal microscopy and statistical analysis. We demonstrated that DHT enables real-time characterization of the subcellular structures, the biophysical processes, and the induced local changes of the intracellular density in a label-free manner and at sub-micrometer spatial resolution.

scholarly journals Enzyme-based synthesis of single molecule RNA FISH probes

2018 ◽  
Author(s):  
Christian Lanctôt
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Low Cost ◽  
Single Cells ◽  
Rna Sequences ◽  
Rna Fish ◽  
Terminal Deoxynucleotide Transferase ◽  
Target Rna ◽  
Subcellular Level

ABSTRACTSingle molecule RNA fluorescence in situ hybridization (smRNA FISH) allows the quantitative analysis of gene expression in single cells. The technique relies on the use of pools of end-labeled fluorescent oligonucleotides to detect specific cellular RNA sequences. These fluorescent probes are currently chemically synthesized. Here I describe a novel technique based on the use of routine molecular biology enzymes to generate smRNA FISH probes without the need for chemical synthesis of pools of oligonucleotides. The protocol comprises 3 main steps: purification of phagemid-derived single stranded DNA molecules comprising a segment complementary to a target RNA sequence; fragmentation of these molecules by limited DNase I digestion; and end-labeling of the resulting oligonucleotides with terminal deoxynucleotide transferase and fluorescent dideoxynucleotides. smRNA FISH probes that are obtained using the technique presented here are shown to perform as well as conventional probes. The main advantages of the method are the low cost of probes and the flexibility it affords in the choice of labels. Enzyme-based synthesis of probes should further increase the popularity of smRNA FISH as a tool to investigate gene expression at the cellular or subcellular level.

scholarly journals In Situ Characterization of Genetically Targeted (Green Fluorescent) Single Cells and Their Microenvironment in an Adoptive Host

2001 ◽  
Vol 158 (6) ◽  
pp. 1975-1983 ◽  
Author(s):  
Frank Leithäuser ◽  
Zlatko Trobonjaca ◽  
Jörg Reimann ◽  
Peter Möller
Keyword(s):  
Single Cells ◽  
In Situ Characterization ◽  
Green Fluorescent

scholarly journals Dual cationic–anionic profiling of metabolites in a single identified cell in a live Xenopus laevis embryo by microprobe CE-ESI-MS

The Analyst ◽  
2019 ◽  
Vol 144 (3) ◽  
pp. 892-900 ◽  
Author(s):  
Erika P. Portero ◽  
Peter Nemes
Keyword(s):  
Mass Spectrometry ◽  
Capillary Electrophoresis ◽  
Xenopus Laevis ◽  
Electrospray Ionization ◽  
Single Cells ◽  
Laevis Embryo ◽  
Xenopus Laevis Embryo ◽  
Esi Mass Spectrometry

In situ capillary microsampling with capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry (MS) enabled the characterization of cationic and anionic metabolites in single cells in complex tissues and organisms, such as the live frog (X. laevis) embryo.

The Use of Advanced Analytical Techniques for Characterization of the Chemical, Physical, and Crystallographic Nature of a Surface

1973 ◽  
Vol 31 ◽  
pp. 132-133 ◽  
Author(s):  
R. E. Herfert
Keyword(s):  
Surface Characterization ◽  
Analytical Techniques ◽  
X Ray Diffraction ◽  
Depth Of Penetration ◽  
X Ray ◽  
The Past ◽  
Transmission Electron ◽  
Scanning Electron

Studies of the nature of a surface, either metallic or nonmetallic, in the past, have been limited to the instrumentation available for these measurements. In the past, optical microscopy, replica transmission electron microscopy, electron or X-ray diffraction and optical or X-ray spectroscopy have provided the means of surface characterization. Actually, some of these techniques are not purely surface; the depth of penetration may be a few thousands of an inch. Within the last five years, instrumentation has been made available which now makes it practical for use to study the outer few 100A of layers and characterize it completely from a chemical, physical, and crystallographic standpoint. The scanning electron microscope (SEM) provides a means of viewing the surface of a material in situ to magnifications as high as 250,000X.

A very rapid in situ Kikuchi technique to determine relative crystal misorientation

1988 ◽  
Vol 46 ◽  
pp. 830-831
Author(s):  
J. I. Bennetch
Keyword(s):  
Electron Beam ◽  
Analytical Techniques ◽  
Superplastic Forming ◽  
The Other ◽  
Kikuchi Pattern ◽  
Kikuchi Line ◽  
Line Analysis

In a recent study of the superplastic forming (SPF) behavior of certain Al-Li-X alloys, the relative misorientation between adjacent (sub)grains proved to be an important parameter. It is well established that the most accurate way to determine misorientation across boundaries is by Kikuchi line analysis. However, the SPF study required the characterization of a large number of (sub)grains in each sample to be statistically meaningful, a very time-consuming task even for comparatively rapid Kikuchi analytical techniques.In order to circumvent this problem, an alternate, even more rapid in-situ Kikuchi technique was devised, eliminating the need for the developing of negatives and any subsequent measurements on photographic plates. All that is required is a double tilt low backlash goniometer capable of tilting ± 45° in one axis and ± 30° in the other axis. The procedure is as follows. While viewing the microscope screen, one merely tilts the specimen until a standard recognizable reference Kikuchi pattern is centered, making sure, at the same time, that the focused electron beam remains on the (sub)grain in question.

Coherent electron nanodiffraction from clean silver nano particles in a UHV STEM

1993 ◽  
Vol 51 ◽  
pp. 1058-1059
Author(s):  
J. Liu ◽  
M. Pan ◽  
G. E. Spinnler
Keyword(s):  
Supported Catalysts ◽  
Imaging Techniques ◽  
Nano Particles ◽  
Metal Particles ◽  
Shape Structure ◽  
Dynamical Simulations ◽  
Small Metal Particles ◽  
Extract Information

Small metal particles have peculiar chemical and physical properties as compared to bulk materials. They are especially important in catalysis since metal particles are common constituents of supported catalysts. The structural characterization of small particles is of primary importance for the understanding of structure-catalytic activity relationships. The shape and size of metal particles larger than approximately 5 nm in diameter can be determined by several imaging techniques. It is difficult, however, to deduce the shape of smaller metal particles. Coherent electron nanodiffraction (CEND) patterns from nano particles contain information about the particle size, shape, structure and defects etc. As part of an on-going program of STEM characterization of supported catalysts we report some preliminary results of CEND study of Ag nano particles, deposited in situ in a UHV STEM instrument, and compare the experimental results with full dynamical simulations in order to extract information about the shape of Ag nano particles.

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