In situ mRNA isolation from a microfluidic single-cell array using an external AFM nanoprobe

mRNA probing from single cells within microfluidic arrays, combining the non-destructive and precise-control of a single-cell mRNA probe with sealed microfluidic systems' multifunctional capability.

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

ISTFA 1999: Conference Proceedings from the 25th International Symposium for Testing and Failure Analysis ◽

10.31399/asm.cp.istfa1999p0311 ◽

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.

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Label-Free and Quantitative Dry Mass Monitoring for Single Cells during In Situ Culture

Cells ◽

10.3390/cells10071635 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1635

Author(s):

Ya Su ◽

Rongxin Fu ◽

Wenli Du ◽

Han Yang ◽

Li Ma ◽

...

Keyword(s):

Cell Growth ◽

Single Cell ◽

Hela Cells ◽

Quantitative Measurement ◽

Single Cells ◽

Dry Mass ◽

Quantitative Detection ◽

Label Free ◽

Mass Sensitivity

Quantitative measurement of single cells can provide in-depth information about cell morphology and metabolism. However, current live-cell imaging techniques have a lack of quantitative detection ability. Herein, we proposed a label-free and quantitative multichannel wide-field interferometric imaging (MWII) technique with femtogram dry mass sensitivity to monitor single-cell metabolism long-term in situ culture. We demonstrated that MWII could reveal the intrinsic status of cells despite fluctuating culture conditions with 3.48 nm optical path difference sensitivity, 0.97 fg dry mass sensitivity and 2.4% average maximum relative change (maximum change/average) in dry mass. Utilizing the MWII system, different intrinsic cell growth characteristics of dry mass between HeLa cells and Human Cervical Epithelial Cells (HCerEpiC) were studied. The dry mass of HeLa cells consistently increased before the M phase, whereas that of HCerEpiC increased and then decreased. The maximum growth rate of HeLa cells was 11.7% higher than that of HCerEpiC. Furthermore, HeLa cells were treated with Gemcitabine to reveal the relationship between single-cell heterogeneity and chemotherapeutic efficacy. The results show that cells with higher nuclear dry mass and nuclear density standard deviations were more likely to survive the chemotherapy. In conclusion, MWII was presented as a technique for single-cell dry mass quantitative measurement, which had significant potential applications for cell growth dynamics research, cell subtype analysis, cell health characterization, medication guidance and adjuvant drug development.

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An acoustofluidic trap and transfer approach for organizing a high density single cell array

Lab on a Chip ◽

10.1039/c8lc00196k ◽

2018 ◽

Vol 18 (14) ◽

pp. 2124-2133 ◽

Cited By ~ 6

Author(s):

Korine A. Ohiri ◽

Sean T. Kelly ◽

Jeffrey D. Motschman ◽

Kevin H. Lin ◽

Kris C. Wood ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Microfluidic System ◽

High Density ◽

Cell Array

We demonstrate a hybrid microfluidic system that combines fluidic trapping and acoustic switching to organize an array of single cells at high density.

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AFM and Fluorescence Microscopy of Single Cells with Simultaneous Mechanical Stimulation via Electrically Stretchable Substrates

Materials ◽

10.3390/ma14154131 ◽

2021 ◽

Vol 14 (15) ◽

pp. 4131

Author(s):

Natalia Becerra ◽

Barbara Salis ◽

Mariateresa Tedesco ◽

Susana Moreno Flores ◽

Pasquale Vena ◽

...

Keyword(s):

Cell Culture ◽

Fluorescence Microscopy ◽

Single Cell ◽

Spatial Resolution ◽

Single Cells ◽

Optical Microscope ◽

Average Cell ◽

Atomic Force ◽

Cell Stretcher

We have developed a novel experimental set-up that simultaneously, (i) applies static and dynamic deformations to adherent cells in culture, (ii) allows the visualization of cells under fluorescence microscopy, and (iii) allows atomic force microscopy nanoindentation measurements of the mechanical properties of the cells. The cell stretcher device relies on a dielectric elastomer film that can be electro-actuated and acts as the cell culture substrate. The shape and position of the electrodes actuating the film can be controlled by design in order to obtain specific deformations across the cell culture chamber. By using optical markers we characterized the strain fields under different electrode configurations and applied potentials. The combined setup, which includes the cell stretcher device, an atomic force microscope, and an inverted optical microscope, can assess in situ and with sub-micron spatial resolution single cell topography and elasticity, as well as ion fluxes, during the application of static deformations. Proof of performance on fibroblasts shows a reproducible increase in the average cell elastic modulus as a response to applied uniaxial stretch of just 4%. Additionally, high resolution topography and elasticity maps on a single fibroblast can be acquired while the cell is deformed, providing evidence of long-term instrumental stability. This study provides a proof-of-concept of a novel platform that allows in situ and real time investigation of single cell mechano-transduction phenomena with sub-cellular spatial resolution.

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Integration of a microfluidic chip with a size-based cell bandpass filter for reliable isolation of single cells

Lab on a Chip ◽

10.1039/c5lc00904a ◽

2015 ◽

Vol 15 (21) ◽

pp. 4128-4132 ◽

Cited By ~ 22

Author(s):

Hojin Kim ◽

Sanghyun Lee ◽

Jae-hyung Lee ◽

Joonwon Kim

Keyword(s):

Single Cell ◽

Microfluidic Chip ◽

Bandpass Filter ◽

Single Cells ◽

Cell Array ◽

Novel Approach ◽

Reliable Isolation

A novel approach for reliable arraying of single cells is presented using a size-based cell bandpass filter integrated with a microfluidic single-cell array chip.

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Self-reporting transposons enable simultaneous readout of gene expression and transcription factor binding in single cells

10.1101/538553 ◽

2019 ◽

Cited By ~ 3

Author(s):

Arnav Moudgil ◽

Michael N. Wilkinson ◽

Xuhua Chen ◽

June He ◽

Alex J. Cammack ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Single Cell ◽

Binding Sites ◽

Expression Profiles ◽

Single Cells ◽

Gene Expression Profiles ◽

Cell Types ◽

Specific Cell

AbstractIn situ measurements of transcription factor (TF) binding are confounded by cellular heterogeneity and represent averaged profiles in complex tissues. Single cell RNA-seq (scRNA-seq) is capable of resolving different cell types based on gene expression profiles, but no technology exists to directly link specific cell types to the binding pattern of TFs in those cell types. Here, we present self-reporting transposons (SRTs) and their use in single cell calling cards (scCC), a novel assay for simultaneously capturing gene expression profiles and mapping TF binding sites in single cells. First, we show how the genomic locations of SRTs can be recovered from mRNA. Next, we demonstrate that SRTs deposited by the piggyBac transposase can be used to map the genome-wide localization of the TFs SP1, through a direct fusion of the two proteins, and BRD4, through its native affinity for piggyBac. We then present the scCC method, which maps SRTs from scRNA-seq libraries, thus enabling concomitant identification of cell types and TF binding sites in those same cells. As a proof-of-concept, we show recovery of cell type-specific BRD4 and SP1 binding sites from cultured cells. Finally, we map Brd4 binding sites in the mouse cortex at single cell resolution, thus establishing a new technique for studying TF biology in situ.

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Direct correlation between motile behavior and protein abundance in single cells

10.1101/067918 ◽

2016 ◽

Author(s):

Yann S Dufour ◽

Sébastien Gillet ◽

Nicholas W Frankel ◽

Douglas B Weibel ◽

Thierry Emonet

Keyword(s):

Protein Expression ◽

Single Cell ◽

Phenotypic Diversity ◽

Single Cells ◽

Selective Advantage ◽

Single Cell Protein ◽

Cell Protein ◽

Chemotaxis Proteins

AbstractUnderstanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage.

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Spatial charting of single cell transcriptomes in tissues

10.1101/2021.11.24.469915 ◽

2021 ◽

Author(s):

Nicholas Navin ◽

Runmin Wei ◽

Siyuan He ◽

Shanshan Bai ◽

Emi Sei ◽

...

Keyword(s):

Single Cell ◽

Spatial Organization ◽

Spatial Information ◽

Ductal Carcinoma ◽

Single Cells ◽

Cell Types ◽

Spatial Structures ◽

Tissue Sections ◽

Normal Mouse Brain

Single cell RNA sequencing (scRNA-seq) methods can profile the transcriptomes of single cells but cannot preserve spatial information. Conversely, spatial transcriptomics (ST) assays can profile spatial regions in tissue sections, but do not have single cell genomic resolution. Here, we developed a computational approach called SChart, that combines these two datasets to achieve single cell spatial mapping of cell types, cell states and continuous phenotypes. We applied SChart to reconstruct cellular spatial structures in existing datasets from normal mouse brain and kidney tissues to validate our approach. We also performed scRNA-seq and ST experiments on two ductal carcinoma in situ (DCIS) tissues and applied SChart to identify subclones that were restricted to different ducts, and specific T cell states adjacent to the tumor areas. Our data shows that SChart can accurately map single cells in diverse tissue types to resolve their spatial organization into cellular neighborhoods and tissue structures.

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Multiplexed Single-Cell in situ RNA Profiling

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.775410 ◽

2021 ◽

Vol 8 ◽

Author(s):

Yu-Sheng Wang ◽

Jia Guo

Keyword(s):

Single Cell ◽

Single Cells ◽

Disease Diagnosis ◽

Tissue Architecture ◽

Rna Profiling ◽

Rna Analysis ◽

Technological Advances ◽

Health And Disease ◽

Type Classification

The ability to quantify a large number of varied transcripts in single cells in their native spatial context is crucial to accelerate our understanding of health and disease. Bulk cell RNA analysis masks the heterogeneity in the cell population, while the conventional RNA imaging approaches suffer from low multiplexing capacity. Recent advances in multiplexed fluorescence in situ hybridization (FISH) methods enable comprehensive RNA profiling in individual cells in situ. These technologies will have wide applications in many biological and biomedical fields, including cell type classification, signaling network analysis, tissue architecture, disease diagnosis and patient stratification, etc. In this minireview, we will present the recent technological advances of multiplexed single-cell in situ RNA profiling assays, discuss their advantages and limitations, describe their biological applications, highlight the current challenges, and propose potential solutions.

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