Live-Cell Probe for In Situ Single-Cell Monitoring of Mitochondrial DNA Methylation

ACS Sensors ◽  
2021 ◽  
Author(s):  
Han Zhao ◽  
Donghan Ma ◽  
Junkai Xie ◽  
Oscar Sanchez ◽  
Fang Huang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mitochondrial Dna ◽  
Single Cell ◽  
Live Cell ◽  
Cell Monitoring

scholarly journals A time-resolved imaging-based CRISPRi screening method

2019 ◽  
Author(s):  
Daniel Camsund ◽  
Michael J. Lawson ◽  
Jimmy Larsson ◽  
Daniel Jones ◽  
Spartak Zikrin ◽  
...  
Keyword(s):  
Single Cell ◽  
Screening Method ◽  
Replication Initiation ◽  
Live Cell ◽  
Time Resolved ◽  
E Coli ◽  
Live Cell Microscopy ◽  
The Impact ◽  
Cell Microscopy

AbstractOur ability to connect genotypic variation to biologically important phenotypes has been seriously limited by the gap between live cell microscopy and library-scale genomic engineering. Specifically, this has restricted studies of intracellular dynamics to one strain at a time and thus, generally, to the impact of genes with known function. Here we show how in situ genotyping of a library of E. coli strains after time-lapse imaging in a microfluidic device overcomes this problem. We determine how 235 different CRISPR interference (CRISPRi) knockdowns impact the coordination of the replication and division cycles of E. coli by monitoring the location of replication forks throughout on average >500 cell cycles per knockdown. The single-cell time-resolved assay allows us to determine the distribution of single-cell growth rates, cell division sizes, and replication initiation volumes. Subsequent in situ genotyping allows us to map each phenotype distribution to a specific genetic perturbation in order to determine which genes are important for cell cycle control. The technology presented in this study enables genome-scale screens of virtually all live-cell microscopy assays and, therefore, constitutes a qualitatively new approach to cellular biophysics.

scholarly journals Single Cell Spatial Chromatin Analysis of Fixed Immunocytochemically Identified Neuronal Cells

2019 ◽  
Author(s):  
Jaehee Lee ◽  
Youtao Lu ◽  
Jinchun Wang ◽  
Jifen Li ◽  
Stephen A. Fisher ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Human Brain ◽  
Single Cell ◽  
Single Cells ◽  
K562 Cells ◽  
Brain Cells ◽  
Open Chromatin ◽  
Single Stranded Dna ◽  
Primary Mouse

AbstractAssays examining the open-chromatin landscape in single cells require isolation of the nucleus, resulting in the loss of spatial/microenvironment information. Here we describe CHEX-seq (CHromatin EXposed) for identifying single-stranded open-chromatin DNA regions in paraformaldehyde-fixed single cells. CHEX-seq uses light-activated DNA probes that binds to single-stranded DNA in open chromatin. In situ laser activation of the annealed probes’ 3’-Lightning Terminator™ in selected cells permits the probe to act as a primer for in situ enzymatic copying of single-stranded DNA that is then sequenced. CHEX-seq is benchmarked with human K562 cells and its utility is demonstrated in dispersed primary mouse and human brain cells, and immunostained cells in mouse brain sections. Further, CHEX-seq queries the openness of mitochondrial DNA in single cells. Evaluation of an individual cell’s chromatin landscape in its tissue context enables “spatial chromatin analysis”.One Sentence SummaryA new method, CHEX-seq (CHromatin eXposed), identifies the open-chromatin landscape in single fixed cells thereby allowing spatial chromatin analysis of selected cells in complex cellular environments.

In-Situ Dual Beam (FIBSEM) Techniques for Probe Pad Deposition and Dielectric Integrity Inspection in 0.2 μm Technology DRAM Single Cells

1999 ◽  
Author(s):  
Gunnar Zimmermann ◽  
Richard Chapman
Keyword(s):  
Electron Beam ◽  
Single Cell ◽  
Single Cells ◽  
Ion Beam ◽  
Gate Oxide ◽  
Ion Milling ◽  
Dual Beam ◽  
Sem Imaging ◽  
Innovative Techniques

Abstract Dual beam FIBSEM systems invite the use of innovative techniques to localize IC fails both electrically and physically. For electrical localization, we present a quick and reliable in-situ FIBSEM technique to deposit probe pads with very low parasitic leakage (Ipara < 4E-11A at 3V). The probe pads were Pt, deposited with ion beam assistance, on top of highly insulating SiOx, deposited with electron beam assistance. The buried plate (n-Band), p-well, wordline and bitline of a failing and a good 0.2 μm technology DRAM single cell were contacted. Both cells shared the same wordline for direct comparison of cell characteristics. Through this technique we electrically isolated the fail to a single cell by detecting leakage between the polysilicon wordline gate and the cell diffusion. For physical localization, we present a completely in-situ FIBSEM technique that combines ion milling, XeF2 staining and SEM imaging. With this technique, the electrically isolated fail was found to be a hole in the gate oxide at the bad cell.

scholarly journals Microfluidic extraction and stretching of chromosomal DNA from single cell nuclei for DNA fluorescence in situ hybridization

2012 ◽  
Vol 14 (3) ◽  
pp. 443-451 ◽  
Author(s):  
Xiaozhu Wang ◽  
Shin-ichiro Takebayashi ◽  
Evans Bernardin ◽  
David M. Gilbert ◽  
Ravindran Chella ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Cell ◽  
Cell Nuclei ◽  
Chromosomal Dna
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